U.S. patent application number 14/076001 was filed with the patent office on 2014-04-03 for amphiphile prodrugs.
The applicant listed for this patent is Commonwealth Scientific and Industrial Research Organisation. Invention is credited to Calum John Drummond, Xiaojuan Gong, Minoo Jalili Moghaddam, Sharon Marie Sagnella.
Application Number | 20140094426 14/076001 |
Document ID | / |
Family ID | 42232824 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140094426 |
Kind Code |
A1 |
Drummond; Calum John ; et
al. |
April 3, 2014 |
AMPHIPHILE PRODRUGS
Abstract
Amphiphilic prodrugs of general formula A-X are disclosed,
wherein A is a biologically active agent or may be metabolized to a
biologically active agent; and X is R, or up to three R moieties
attached to a linker, Y.sub.1, Y.sub.2 or Y.sub.3. Self-assembly of
the amphiphilic prodrugs into reverse lyotropic phases,
particularly hexagonal, cubic and sponge, is disclosed.
Inventors: |
Drummond; Calum John;
(Elwood, AU) ; Sagnella; Sharon Marie; (Ryde,
AU) ; Moghaddam; Minoo Jalili; (Pymble, AU) ;
Gong; Xiaojuan; (Marsfield, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Commonwealth Scientific and Industrial Research
Organisation |
Campbell |
|
AU |
|
|
Family ID: |
42232824 |
Appl. No.: |
14/076001 |
Filed: |
November 8, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13132880 |
Aug 29, 2011 |
8603999 |
|
|
PCT/AU2009/001586 |
Dec 4, 2009 |
|
|
|
14076001 |
|
|
|
|
Current U.S.
Class: |
514/49 ; 514/630;
536/28.51; 564/223 |
Current CPC
Class: |
C07C 233/18 20130101;
A61K 9/107 20130101; A61P 35/00 20180101; C07H 19/02 20130101; C07H
19/067 20130101; A61K 9/146 20130101; A61K 47/54 20170801 |
Class at
Publication: |
514/49 ;
536/28.51; 514/630; 564/223 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07C 233/18 20060101 C07C233/18; C07H 19/02 20060101
C07H019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 2008 |
AU |
2008906311 |
Claims
1-12. (canceled)
13. A prodrug of a general formula (I): A-X I wherein A is a
biologically active agent or an agent capable of being metabolised
to a biologically active agent; and X is selected from the group
consisting of a substituent according to formula (a), a substituent
according to formula (b), a substituent according to formula (c)
and a substituent according to formula (d): ##STR00033## wherein R
is selected from a group consisting of alkyl, alkenyl, alkynyl,
branched alkyl, branched alkenyl, branched alkynyl, substituted
alkyl, substituted alkenyl and substituted alkynyl groups and their
analogues; Y.sub.1 is a linker group that is covalently attached to
the group R at one attachment site and to A at a second attachment
site; Y.sub.2 is a linker group that is covalently attached to two
R groups (which may be identical or different) at two independent
attachment sites and is attached to A at a third attachment site;
Y.sub.3 is a linker group that is covalently attached to three R
groups (which may be identical or different) at three independent
attachment sites and is attached to A at a fourth attachment site;
and wherein A is selected from the group consisting of a compound
according to formula (II) ##STR00034## cyclosporine, paclitaxel,
indomethacin, fenofibrate, progesterone, amphotericin B (AMB) and
dopamine.
14. A prodrug according to claim 13 wherein A is a compound
according to formula (II).
15. A prodrug according to claim 14, wherein X is represented by
formula (a) and R has a chain length equivalent to 10 to 30 carbon
atoms.
16. A prodrug according to claim 13, wherein A is selected from the
group consisting of cyclosporine, paclitaxel, indomethacin,
fenofibrate, progesterone and amphotericin B (AMB).
17. A prodrug according to claim 13, wherein A is dopamine.
18. A self assembled structure formed from the prodrug of claim 16,
wherein the structure exhibits a phase under physiological
conditions selected from the group consisting of lamellar,
hexagonal phase, cubic phase, and sponge phase.
19. The self assembled structure of claim 18, wherein the phase is
an inverse phase.
20. A self assembled structure formed from the prodrug of claim 17,
wherein the structure exhibits a phase under physiological
conditions selected from the group consisting of lamellar,
hexagonal phase, cubic phase, and sponge phase.
21. The self assembled structure of claim 20, wherein the phase is
an inverse phase.
22. A self assembled structure formed from the prodrug of claim 16,
wherein the structure is a colloidal particle selected from the
group consisting of colloidosome and solid lipid particle.
23. A self assembled structure formed from the prodrug of claim 17,
wherein the structure is a colloidal particle selected from the
group consisting of colloidosome and solid lipid particle.
24. A prodrug according to claim 13, wherein A itself is a prodrug
that is converted by hydrolytic, oxidative, reductive or enzymatic
cleavage to the biologically active agent.
25. A prodrug according to claim 24, wherein the biologically
active agent is dopamine.
26. A method of modulating the bioavailability of a biologically
active agent or an agent capable of being metabolised to a
biologically active agent, A, the method including covalently
linking A to at least one tail component, X, to form an amphiphile,
wherein the link is cleavable in vivo to release the biologically
active agent or an agent capable of being metabolised to a
biologically active agent from the self-assembled structure; and
administering the amphiphile to a patient such that the amphiphile
self-assembles into a self-assembled structure; wherein X is
selected from the group consisting of a substituent according to
formula (a), a substituent according to formula (b), a substituent
according to formula (c) and a substituent according to formula
(d): ##STR00035## wherein R is selected from a group consisting of
alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched
alkynyl, substituted alkyl, substituted alkenyl and substituted
alkynyl groups and their analogues; Y.sub.1 is a linker group that
is covalently attached to the group R at one attachment site and to
A at a second attachment site; Y.sub.2 is a linker group that is
covalently attached to two R groups (which may be identical or
different) at two independent attachment sites and is attached to A
at a third attachment site; and Y.sub.3 is a linker group that is
covalently attached to three R groups (which may be identical or
different) at three independent attachment sites and is attached to
A at a fourth attachment site.
27. The method of claim 26, wherein the self-assembled structure
exhibits a phase under physiological conditions selected from the
group consisting of lamellar, hexagonal phase, cubic phase, and
sponge phase.
28. The method of claim 26, wherein the phase is an inverse
phase.
29. The method of claim 26, wherein the self-assembled structure is
a colloidal particle selected from the group consisting of
colloidosome and solid lipid particle.
30. The method of claim 26, wherein the amphiphile is an enzymatic
substrate for an enzyme that promotes formation of A from the
amphiphile.
31. The method of claim 26, wherein R has a chain length equivalent
to 10 to 30 carbon atoms.
32. The method of claim 26, wherein A is selected from the group
consisting of cyclosporine, paclitaxel, indomethacin, fenofibrate,
progesterone and amphotericin B (AMB).
33. The method of claim 26, wherein A is dopamine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to improved prodrugs, and
compositions thereof. In particular, it relates to amphiphilic
prodrugs.
BACKGROUND OF THE INVENTION
[0002] It is important that the dosage of any drug fall within a
therapeutic window. The therapeutic window is defined at its lower
boundary by the minimum concentration required to exert a
therapeutic effect, and at its upper boundary by the concentration
at which unacceptable toxicity effects are observed. A difficulty
with some therapeutic agents, including chemotherapeutic agents
such as 5-fluorouracil, is that they possess high toxicity and/or a
fast clearance rate. This results in difficulties dosing within the
therapeutic window. The dosing method generally employed for drugs
that possess these properties often results in administration of a
supra-optimal dose that rapidly falls to a sub-optimal level
between administrations.
[0003] Several divergent approaches have been employed in an
attempt to improve dosage regimes of therapeutic agents.
[0004] One approach is to chemically modify the active therapeutic
agent and generate a prodrug. In vivo the prodrug is converted, for
example, by hydrolytic, oxidative, reductive or enzymatic cleavage
to the biologically active agent.
[0005] One such drug that has been successful converted into a
prodrug is the 5-fluorouracil (5-FU) prodrug, such as Capecitabine
or its analogues. Compounds of this nature are disclosed in a
general sense in U.S. Pat. No. 4,966,891 and equivalent application
EP 0316704 (F. Hoffmann-La Roche AG). Capecitabine undergoes three
chemical conversions in vivo to generate 5-fluorouracil, namely:
carboxylesterase-catalysed hydrolysis to generate
5'-deoxy-5-fluorocytidine; conversion of 5'-deoxy-5-fluorocytidine
to 5'-deoxy-5-fluorouridine catalysed by cytidine deaminase,
followed by conversion of 5'-deoxy-5-fluorouridine to active 5-FU
preferentially at tumour sites catalysed by the angiogenic factor
thymidine phosphorylase. In spite of being less toxic than 5-FU,
Capecitabine and its analogues still possess substantial drawbacks;
namely, they still possess an undesirable rapid clearance rate.
[0006] Another approach to prolong clearance is to encapsulate or
otherwise non-covalently incorporate the biologically active drug
or prodrug into a drug delivery vehicle or matrix. One investigated
material is a biologically inert amphiphilic matrix. Amphiphiles
are compounds that possess a hydrophilic portion and a hydrophobic
portion. Under certain conditions, amphiphiles spontaneously
aggregate, or self-assemble, into structures that possess at least
some degree of internal order. The self-assembly behaviour of
amphiphiles in solvent arises because of the preferential
interaction between the solvent and either the hydrophilic or
hydrophobic portion of the amphiphilic molecule. When an amphiphile
is exposed to a polar solvent, the hydrophilic portion of the
amphiphile tends to preferentially interact with the polar solvent,
resulting in the formation of hydrophilic domains (`solvent
domain`). The hydrophobic portion of the amphiphile molecules tend
to be excluded from this domain, resulting in the de facto
formation of a hydrophobic domain (`amphiphile domain`). Such
self-generated aggregates are referred to throughout the
specification as self-assembled structures. When being used as a
drug delivery vehicle, the amphiphile self-assembled structure acts
as an inert carrier of the biologically active agent. Amphiphile
self-assembled structures represent promising drug-delivery
vehicles, because the presence of both hydrophilic and hydrophobic
domains potentially allows for the incorporation of both polar and
non-polar active agents into the structure.
[0007] As self-assembled structures may exhibit a variety of
orientational orders, these will be discussed here for clarity. If
long-range orientational order is observed within the
self-assembled structure at equilibrium, the self-assembled
structure is termed a `mesophase`, a `lyotropic liquid crystalline
phase`, a `lyotropic phase` or, as used herein, simply a `phase` of
`bulk phase`. Note that as well as the lyotropic liquid crystalline
phase, there is another principal type of liquid crystalline phase,
namely, the: thermotropic liquid crystalline phase. Thermotropic
liquid crystals can be formed by heating a crystalline solid or by
cooling an isotropic melt of an appropriate solute. Lyotropic
liquid crystals may be formed by addition of a solvent to an
appropriate solid or liquid amphiphile. The manipulation of
parameters such as amphiphile concentration and chemical structure,
solvent composition, temperature and pressure may result in the
amphiphile-solvent mixture adopting lyotropic phases with
distinctive characteristics.
[0008] Lyotropic phases may be classified in terms of the curvature
of the interface between the hydrophilic and hydrophobic domains.
The curvature between these domains is dependent upon several
factors, including the concentration and molecular structure of the
amphiphile. When the interface displays net curvature towards the
hydrophobic domain, the phase is termed `normal`. When the
interface displays net curvature towards the hydrophilic domain,
the phase is termed `reverse` or `inverse` (used interchangeably
herein). If the net curvature of the system approaches zero, then
the resulting phase may possess a lamellar-type structure that
consists of planar amphiphile bilayers separated by solvent
domains. Alternatively, the net curvature may approach zero if each
point on the surface is as convex in one dimension as it is concave
in another dimension; such phases are referred to as `minimal
surface` phases. Examples of particular phases that can be formed
by self-assembled structures include but are not limited to:
micellar (normal and reversed), hexagonal (normal and reversed),
lamellar, cubic (normal, reversed and bicontinuous), and other
intermediate phases such as reverse micellar cubic, the ribbon,
mesh, or non-cubic `sponge` bicontinuous phases.
[0009] Also, as well as the bulk phases described above, amphiphile
self-assembled structure may be dispersed to form colloidal
particles (so-called `colloidosomes`) that retain the internal
structure of the non-dispersed bulk phase. When these particles
possess the internal structure of a reversed bicontinuous cubic
phase, the particles are colloquially referred to as cubosomes.
Similarly, when the particles possess the internal structure of a
reversed hexagonal phase, they are referred to as hexosomes. When
the particles possess the internal structure of a lamellar phase,
they are referred to as liposomes. Colloidal particles may also be
formed from `sponge` phases.
[0010] Another form of amphiphile self-assembled structure that has
been utilised for drug delivery applications are solid lipid
particles. Solid lipid particles are comprised of a solid lipid
core stabilised by a surfactant surface layer, such as polysorbate
80.
[0011] As mentioned above, certain of these amphiphile
self-assembled structures comprising biologically inert amphiphiles
have been investigated for drug-delivery applications. These
self-assembled structures are intended to act as an inert matrix or
carrier into which biologically active molecules may be
non-covalently incorporated. For instance, EP 0 126 751B2 discloses
the use of bulk cubic and reversed hexagonal phases for drug
delivery applications. Certain of the colloidal particles have also
been investigated for their application as drug delivery vehicles.
For instance, U.S. Pat. No. 5,531,925 discloses colloidal particles
comprising an interior of an amphiphilic-based phase, surrounded by
a surface phase anchored to the bi- or mono-layer of the interior
phase. The interior phase of the particles of U.S. Pat. No.
5,531,925 may be selected from reversed cubic, hexagonal or
intermediate, or L.sub.3 (`sponge`) phases, or mixtures thereof.
Certain solid lipid particles have been used as carriers for
hydrophobic drugs. For example Campothecin, an anticancer agent
which was mixed with an amphiphile, stabilised by poloxamer and
then dispersed by homogenisation into solid lipid particles
demonstrated increased drug levels in the brain tissues (Yang
1999).
[0012] Unfortunately, the self-assembled structures/drug delivery
vehicles described above possess properties that make them
unsuitable for their intended application, the undesirable
properties including (i) toxicity, (ii) inappropriate absorption,
distribution, metabolism and excretion profiles, and (iii)
inappropriate biodegradability properties. Moreover, it is often
difficult to achieve sufficient drug loadings into the structure
such that a therapeutic effect is observed when the drug delivery
vehicle is administered.
[0013] In an effort to increase drug loadings, the "pharmacosome"
approach has been employed. This approach involves generating a
prodrug that is capable of assembling into a micelle or liposome.
Jin et al., identify some lipid-nucleoside analogues that can form
normal lamellar vesicles (Jin 2005, Zhang 2006). However, micelles
and liposomes also possess substantial drawbacks as phases suitable
for drug delivery. For instance, micellar systems can disintegrate
under dilution and below the critical micelle concentration (CMC).
Additionally, oral application of liposomes is limited due to the
fast uptake of the liposomes by phagocytes of the immune systems in
stomach and duodenum.
[0014] All of the above-described approaches suffer from
substantial drawbacks. Accordingly, there remains a need to
generate better methods of drug delivery.
[0015] Reference to any prior art in the specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
Australia or any other jurisdiction or that this prior art could
reasonably be expected to be ascertained, understood and regarded
as relevant by a person skilled in the art.
SUMMARY OF THE INVENTION
[0016] The current invention seeks to provide prodrugs capable of
self-assembly into higher order hexagonal, cubic and/or sponge
phase. Two forms of self-assembled structures have been identified
as being particularly suitable to act as drug delivery vehicles,
namely lyotropic liquid crystals and solid lipid particles. The
invention also provides pharmaceutical compositions thereof. These
higher order phases provide a modified release profile for the drug
when compared with the lower order micellar and liposomal
pharmacosomes of the prior art.
[0017] Accordingly, in a first aspect of the present invention
there is provided a prodrug of a general formula (I):
A-X I
wherein A is a biologically active agent or an agent capable of
being metabolised to a biologically active agent; and [0018] X is
selected from the group consisting of a substituent according to
formula (a), a substituent according to formula (b), a substituent
according to formula (c), and a substituent according to formula
(d):
##STR00001##
[0018] wherein R is selected from a group consisting of alkyl,
alkenyl, alkynyl, branched alkyl, branched alkenyl, branched
alkynyl, substituted alkyl, substituted alkenyl and substituted
alkynyl groups and their analogues; Y.sub.1 is a linker group that
is covalently attached to one R group at one attachment site and to
A at a second attachment site; Y.sub.2 is a linker group that is
covalently attached to two R groups (which may be identical or
different) at two independent attachment sites and is attached to A
at a third attachment site; and Y.sub.3 is a linker group that is
covalently attached to three R groups (which may be identical or
different) at three independent attachment sites and is attached to
A at a fourth attachment site.
[0019] The biologically active agent may be a drug, in which case
A-X represents a prodrug. The agent capable of being metabolised to
a biologically active agent may be a prodrug, in which case A-X
represents a pre-prodrug. The biologically active agent is
preferably a therapeutically active agent.
[0020] Accordingly, in an embodiment of this aspect there is
provided a prodrug of a general formula (I):
A-X I
wherein A is a therapeutically active agent or an agent capable of
being metabolised to a therapeutically active agent; [0021] X is
selected from the group consisting of a substituent according to
formula (a), a substituent according to formula (b), a substituent
according to formula (c) and a substituent according to formula
(d):
##STR00002##
[0021] wherein Y.sub.1 is a linker group that is covalently
attached to the group R at one attachment site and to the
therapeutically active agent A at a second attachment site; Y.sub.2
is a linker group that is covalently attached to two R groups
(which may be identical or different) at two independent attachment
sites and is attached to the therapeutically active agent A at a
third attachment site; Y.sub.3 is a linker group that is covalently
attached to three R groups (which may be identical or different) at
three independent attachment sites and is attached to the
therapeutically active agent A at a fourth attachment site; and R
is selected from a group consisting of alkyl, alkenyl, alkynyl,
branched alkyl, branched alkenyl, branched alkynyl, substituted
alkyl, substituted alkenyl and substituted alkynyl groups and their
analogues.
[0022] In another embodiment of this aspect there is provided a
prodrug of a general formula (I):
A-X I
wherein A is a biologically active agent or an agent capable of
being metabolised to a biologically active agent; [0023] X is
selected from the group consisting of a substituent according to
formula (a), a substituent according to formula (b), a substituent
according to formula (c) and a substituent according to formula
(d):
##STR00003##
[0023] wherein R is selected from a group consisting of alkyl,
alkenyl, alkynyl, branched alkyl, branched alkenyl, branched
alkynyl, substituted alkyl, substituted alkenyl and substituted
alkynyl groups and their analogues; Y.sub.1 is a linker group that
is covalently attached to one R group at one attachment site and to
A at a second attachment site; Y.sub.2 is a linker group that is
covalently attached to two R groups (which may be identical or
different) at two independent attachment sites and is attached to A
at a third attachment site; and Y.sub.3 is a linker group that is
covalently attached to three R groups (which may be identical or
different) at three independent attachment sites and is attached to
A at a fourth attachment site.
[0024] Optionally, R has a linear chain length equivalent to 8 to
30 carbon atoms. R is generally hydrophobic. In one embodiment, R
is alpha-tocopherol. In another embodiment, R is an isoprenoid
group. In other embodiments, R is an hydroxylated alkyl or
hydroxylated alkenyl group. Preferred embodiments of R are: alkyl,
alkenyl, branched alkyl and alkenyl(isoprenoid) and their analogues
such as alpha-tocopherol, hydroxylated alkyl or alkenyl groups. In
preferred embodiments, R has a chain length equivalent to 10 to 30
carbon atoms. Preferably, the chain length is equivalent to 10 to
24 carbon atoms, and more preferably equivalent to 14 to 24 carbon
atoms. When X is a substituent according to formula (c) or formula
(d), each R may be independently selected from the group consisting
of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl,
branched alkynyl, substituted alkyl, substituted alkenyl and
substituted alkynyl groups and their analogues. Alternatively, both
R groups may be identical. Generally, R is intended to confer self
assembling properties to A.
[0025] In some embodiments according to the current invention,
Y.sub.1, Y.sub.2 and Y.sub.3 are linker groups. A "linker" refers
to a group that acts as a spacer between the biologically active
agent A and the group R. Linkers are at least bifunctional in the
case of Y.sub.1, are at least trifunctional in the case of Y.sub.2,
and are at least tetrafunctional in the case of Y.sub.3, containing
at least one functional group (an "attachment site") to anchor the
group R at one site in the molecule, and another selectively
cleavable functional group at another attachment site to anchor the
drug A. Examples of functional groups, including selectively
cleavable functional groups, include but are not limited to:
ethers, esters, amides, carbamates, imides, imines, carbonates,
thioethers, thioesters, and disulfides.
[0026] For instance, Y.sub.1, Y.sub.2, Y.sub.3 may be at least one
functional group attached to at least one selectively cleavable
functional group. Preferably, Y.sub.1, Y.sub.2, Y.sub.3 includes a
moiety that links at least one functional group and at least one
selectively cleavable functional group. The moiety may be, for
example, selected from the group consisting of heteroatoms, alkyl,
alkenyl, alkyne, where these may be cyclic and/or include further
heteroatoms and functional substituents (such as carbonyl,
carboxylic, amide, hydroxyl, ether, amine), or a combination of any
of these.
[0027] Y.sub.1 includes a selectively cleavable functional group,
and typically will consist of a selectively cleavable functional
group. Examples of Y.sub.2 include: diethanolamine,
propane-1,2,3-tricarboxylic acid, cysteine, aspartic acid,
asparagine, serine, tyrosine, arginine, histidine, threonine,
lysine, glutamic acid and glutamine. Examples of Y.sub.3 include:
citric acid and tris(hydroxymethyl)aminomethane (Tris). The
examples provided for Y.sub.1, Y.sub.2 and Y.sub.3 are not intended
to be an exhaustive list and the current invention contemplates
other embodiments of Y.sub.1, Y.sub.2 and Y.sub.3.
[0028] The skilled person would understand which compounds and
methods are suitable for attaching A to X.
[0029] Preferably, A is a hydrophilic biologically active agent.
For example, A is a biologically active agent with a log P value of
less than 0. In other embodiments, A is an agent capable of being
metabolised to a biologically active agent, the biologically active
agent being hydrophilic with a log P value of less than 0. In one
embodiment, A is itself a prodrug that is converted, for example,
by hydrolytic, oxidative, reductive or enzymatic cleavage to the
biologically active agent by one or more reactions or steps. When A
is itself a prodrug, the general formula (I) may be considered to
describe a compound referred to as a pre-prodrug.
[0030] In one preferred embodiment, the general formula (I)
represents a compound according to the formula (II):
##STR00004##
where R is as defined as in Formula (I), and is a functional group
capable of conferring self-assembly properties to the compound.
[0031] The compound according to the formula (II) can be made as
described in Scheme 1 below.
[0032] Particularly preferred embodiments of the self-assembled
structures of the present invention comprise at least one compound
selected from the following group:
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-tetramethyl-hexadecyloxycarbonyl)cyt-
idine, 5'-deoxy-5-fluoro-N.sup.4-(hexadecyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N.sup.4-(cis-9-octadecenyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine, and
5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecenyl-1-oxycarbonyl)cytidine. In other embodiments,
the self-assembled structures of the present invention consist
essentially of at least one compound selected from the following
group:
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-tetramethyl-hexadecyloxycarbonyl)cyt-
idine, 5'-deoxy-5-fluoro-N.sup.4-(hexadecyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N.sup.4-(cis-9-octadecenyloxycarbonyl)cytidine,
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine, and
5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecenyl-1-oxycarbonyl)cytidine.
[0033] In another preferred embodiment, A is dopamine.
[0034] Preferably, a prodrug of a general formula (I) is capable of
forming a self-assembled structure having a lyotropic phase that
displays lamellar, hexagonal, cubic or sponge morphologies. More
preferably, the phase is a cubic, hexagonal, or sponge phase. More
preferably still, the phase is an inverse phase.
[0035] In a second aspect of the invention there is provided
self-assembled structures of the prodrugs of the general formula
(I) of the above aspect.
[0036] Preferably, the self-assembled structure is a lyotropic
phase that displays lamellar, hexagonal, cubic or sponge
morphologies. More preferably, the phase is a cubic, hexagonal, or
sponge phase. More preferably still, the self-assembled structure
of the prodrug displays inverse phase morphologies. Generally
inverse phases are advantageous as drug delivery vehicles because
of their thermodynamic stability in excess water, greater surface
area and controlled channel dimensions, the latter property being
particularly important for release of active embedded within a
self-assembled matrix. Accordingly, there is provided prodrugs that
are capable of self-assembly into inverse lamellar, inverse cubic,
inverse sponge or inverse hexagonal phases. The self-assembled
structure may also be a solid lipid particle. The self-assembled
structure of the prodrugs according to the current invention may be
a bulk phase, or may be colloidal particles derived therefrom.
Particularly preferred colloidal particles may be selected from the
following group: cubosomes, hexosomes and "sponge" particles.
Depending on conditions, more than one phase may be present in a
self-assembled structure.
[0037] In a particularly preferred embodiment the self-assembled
structures are of compounds of 5-fluorouracil prodrugs of the
formula (II). Preferably, the self-assembled structures are solid
lipid particles. Such particles may be suitably stabilised for
pharmaceutical use by a surfactant stabiliser, such as polysorbate
or poloxamer.
[0038] In a third aspect of the present invention there is provided
a pharmaceutical composition for the treatment of a disease state
comprising as an active ingredient self-assembled structures of
Formula (I) or (II). In some embodiments, the pharmaceutical
composition for the treatment of a disease state consists
essentially of an active ingredient that is a self-assembled
structures of Formula (I) or (II). In some embodiments, the
self-assembled structures display a hexagonal, cubic or sponge
phase. Preferably, the active ingredient is self-assembled
structures of Formula (II) comprising a lamellar bulk phase or
liposomal colloidal particles. In other embodiments, the
self-assembled structures are solid lipid particles.
[0039] In some embodiments, the disease state is that of the
presence of a tumor, and the pharmaceutical composition comprises
as an active ingredient solid lipid particles or self-assembled
structures of Formula (II).
[0040] The self-assembled structure/active ingredient is preferably
present in the pharmaceutical composition in a therapeutically
effective amount.
[0041] In a fourth aspect of the present invention there is
provided a method for treatment of a disease state comprising
administering a therapeutically effective amount of a
pharmaceutical composition for the treatment of a disease state
comprising as an active ingredient self-assembled structures of
Formula (I) or (II) to a patient. In some embodiments, the
self-assembled structures display a hexagonal, cubic or sponge
phase. Preferably, the active ingredient is self-assembled
structures of Formula (II), more preferably comprising a lamellar
bulk phase or being liposomal colloidal particles. In other
embodiments, the self-assembled structures are solid lipid
particles.
[0042] In some embodiments, the disease state is that of the
presence of a tumor, and in this case it is preferable that the
pharmaceutical composition comprises an active ingredient of a
self-assembled structure of Formula (II). The self-assembled
structure may be a solid lipid particle.
[0043] In a fifth aspect of the present invention there is provided
a self-assembled structure according to the current invention for
the manufacture of a medicament for the treatment of a disease
state. In some embodiments, the self-assembled structures display a
hexagonal, cubic or sponge phase. Preferably, the prodrug or
pre-prodrug forming the self-assembled structure is of Formula
(II), in which case it is more that the self-assembled structure
comprises a lamellar bulk phase or liposomal colloidal particles.
In other embodiments, the self-assembled structures are solid lipid
particles.
[0044] In some embodiments, the disease state is that of the
presence of a tumor, and the self-assembled structure is of Formula
(II). The self-assembled structure may be a solid lipid
particle.
[0045] In a sixth aspect of the present invention there is provided
a method of modulating the release of a biologically active agent
or an agent capable of being metabolised to a biologically active
agent, the method including covalently linking the biologically
active agent or an agent capable of being metabolised to a
biologically active agent to at least one tail component to form an
amphiphile capable of self-assembling into a self-assembled
structure stable under physiological conditions, and wherein the
amphiphile is cleavable in vivo to release the biologically active
agent or an agent capable of being metabolised to a biologically
active agent in a biologically active form.
[0046] In one embodiment of this aspect there is provided a method
of modulating the bioavailability of a biologically active agent or
an agent capable of being metabolised to a biologically active
agent, the method including covalently linking the biologically
active agent or an agent capable of being metabolised to a
biologically active agent to at least one tail component to form an
amphiphile, wherein the link is cleavable in vivo to release the
biologically active agent or an agent capable of being metabolised
to a biologically active agent from the self-assembled structure;
administering the amphiphile to a patient such that the amphiphile
self-assembles into a self-assembled structure.
[0047] Preferably, the amphiphile self-assembles to form a
self-assembled structure of a lyotropic phase that displays
lamellar, hexagonal, cubic and/or sponge morphologies. More
preferably, the amphiphile self-assembles into lamellar, inverse
hexagonal or inverse cubic phases.
[0048] In one embodiment, the biologically active agent or an agent
capable of being metabolised to a biologically active agent is a
compound A as described for the above aspects.
[0049] In one embodiment, the tail component is an R group as
described for the above aspects.
[0050] In some embodiments, the tail component is connected to the
biologically active agent using a cleavable linker Y.sub.1, Y.sub.2
or Y.sub.3 as described for the above aspects.
[0051] Preferably, the amphiphile is an enzymatic substrate for an
enzyme that promotes formation of the biologically active form of
the biologically active agent present in the amphiphile. That is,
it is preferable that the amphiphile is predetermined to be one
which may be acted upon by an enzyme present in the patient. More
preferably, the enzyme acts on the cleavable linker. In embodiments
where the biologically active agent is itself a prodrug, at least
one further chemical modification step may then be necessary before
the amphiphile is converted to the biologically active form.
[0052] A further aspect of the present invention relates to a
process for preparing the bulk phase according to the current
invention. There is further provided a bulk phase according to the
current invention prepared by the process of this aspect.
[0053] A further aspect of the present invention relates to a
process for preparing colloidal particles from a bulk phase
according to the current invention. There is further provided
colloidal particles prepared from a bulk phase according to the
current invention by the process of this aspect.
[0054] It will be understood that the term "comprises" (or its
grammatical variants) as used in this specification is equivalent
to the term "includes" and should not be taken as excluding the
presence of other elements or features.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1: Schematic picture of the different phases that can
occur upon hydration of different amphiphiles. Abbreviations for
different mesophases are micellar (L.sub.1); micellar cubic
(I.sub.1), normal hexagonal (H.sub.1), bicontinuous cubic
(V.sub.1), Lamellar (L.sub..alpha.), reversed bicontinuous cubic
(V.sub.2), reversed hexagonal (H.sub.2), reversed micellar cubic
(I.sub.2), and reversed micellar (L.sub.2), where subscripts 1 and
2 refer to "normal" and "reversed" phases, respectively.
[0056] FIG. 2: Particle size distribution (diameter in nm) as
determined by DLS of solid lipid particles of 5-FCPal at 25.degree.
C. The average size distribution taken from 3 separate measurements
is shown.
[0057] FIG. 3: Particle size distribution (diameter in nm) as
determined by DLS of 5-FCPhy/F127/ethanol dispersions. Upper curve
represents the coarse dispersions formed directly using ethanol
method. Bottom curve is the size distribution after size controlled
membrane extrusion.
[0058] FIG. 4: Particle size distribution (diameter in nm) as
determined by DLS of dispersions formed from 5-FCOle/F127/ethanol
water solution. Top curve represents the coarse dispersions formed
directly using ethanol method. Bottom curve is the size
distribution after size controlled membrane extrusion.
[0059] FIG. 5: Cryo-TEM image of 5-FCPal solid lipid particles.
Scale bar is 2 microns.
[0060] FIG. 6: Cryo-TEM images of 5-FCPhy hexosome particles
(dispersion consisting 4.74% of 5-FCPhy, 0.45% F127, 9% ethanol and
85.7% of water). Scale bar is 50 nm.
[0061] FIG. 7: Cryo-TEM images of dispersions containing 2.3%
5-FCOle, 0.2% F127, 4.87% ethanol and 92.6% water. Top image shows
a cubosome which form at physiological temperature. Bottom image
shows unilamellar liposomes present at 25.degree. C. Scale bar in
both images in 200 nm.
[0062] FIG. 8: Plot of the hydrolysis of Capecitabine by porcine
liver carboxylesterase (CES) as a function of reaction time. The
enzyme concentration for Capecitabine is 0.005 mg/ml.
[0063] FIG. 9: Plot of the hydrolysis of 5-FCPal by porcine liver
CES as a function of reaction time. The enzyme concentration for
5-FCPal is 0.5 mg/ml.
[0064] FIG. 10: Plot of the hydrolysis of 5-FCPhy by porcine liver
CES as a function of reaction time. The enzyme concentration for
5-FCPhy is 0.5 mg/ml.
[0065] FIG. 11: Plot of the hydrolysis of 5-FCOle by porcine liver
CES as a function of reaction time. The enzyme concentration for
5-FCOle is 0.5 mg/ml.
[0066] FIG. 12: Effect of administration of 5-FU on mouse breast
tumour volume versus time. Volume (length.times.breadth) of tumour
was measured and recorded on day 1, 4, 7, 14, and 21 of the
drug/pro-drug administration
[0067] FIG. 13: Effect of administration of capecitabine on mouse
breast tumour volume versus time. Volume (length.times.breadth) of
tumour was measured and recorded on day 1, 4, 7, 14, and 21 of the
drug/pro-drug administration.
[0068] FIG. 14: Effect of administration of 5-FCPal on mouse breast
tumour volume versus time. Volume (length.times.breadth) of tumour
was measured and recorded on day 1, 4, 7, 14, and 21 of the
drug/pro-drug administration.
[0069] FIG. 15: Effect of administration of 5-FCOle on mouse breast
tumour volume versus time. Volume (length.times.breadth) of tumour
was measured and recorded on day 1, 4, 7, 14, and 21 of the
drug/pro-drug administration.
[0070] FIG. 16: Effect of administration of 5-FCPhy on mouse breast
tumour volume versus time. Volume (length.times.breadth) of tumour
was measured and recorded on day 1, 4, 7, 14, and 21 of the
drug/pro-drug administration.
[0071] FIG. 17: SAXS of Neat Farnesoyl Dopamine at 25.degree.
C.
[0072] FIG. 18: The average 4T1 tumour volume for the four
different treatment groups (control, capecitabine, 5-FCPhy, and
5-FCOle administered daily for 17 days).
[0073] FIG. 19: Images of the tumours and spleens from animals
sacrificed at 17 days for the four treatment groups of Example 7
(shown from left to right are 5-FCOle, 5-FCPhy, capecitabine and
control).
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0074] It will be noted that various terms employed in the
specification, examples and claims have meanings that will be
understood by one of ordinary skill in the art. However, for
clarity of meaning intended in this document, certain terms are
defined below.
[0075] The term "prodrug" as used throughout the specification
refers to a biologically active agent including structural
modifications thereto, such that in vivo the prodrug is converted,
for example, by hydrolytic, oxidative, reductive or enzymatic
cleavage to the biologically active agent by one or more reactions
or steps. It includes an agent that requires one or more chemical
conversion steps or steps of metabolism to produce the active
molecule--that is, this term is also understood to encompass
"pre-prodrugs".
[0076] The term `5-fluorouracil prodrug` as used throughout the
specification refers to a compound of the general formula (II) that
is capable of being converted to 5-FU in vivo by, for instance,
means of chemical and/or enzymatic modification.
##STR00005##
wherein R is as herein defined and R.sub.3 and R.sub.4
independently represent either hydrogen or easily hydrolysable
radicals known to those skilled in the art. Such radicals include,
but are not limited to, acetate, methyl ether, methoxymethyl ether,
and silyl ethers.
[0077] The term "self-assembled structure" as used throughout the
specification is meant to refer to an aggregate of amphiphiles that
possess some degree of internal organisational order. The
self-assembled structures may be formed by contacting the
amphiphile with solvent. The self-assembled structure may refer to
either a bulk lyotropic phase, a colloidal particle derived
therefrom (a so-called "colloidosome"), or a solid lipid
particle.
[0078] The term "bulk phase" as used throughout the specification
is understood to mean a lyotropic phase that includes but is not
limited to: micellar cubic (I.sub.1); normal hexagonal (H.sub.1);
bicontinuous cubic (V.sub.1); lamellar (L.sub..quadrature.);
reversed bicontinuous cubic (V.sub.2); reversed hexagonal
(H.sub.2); reversed micellar cubic (I.sub.2) and sponge (L.sub.3)
phases.
[0079] The term "colloidal particle" as used throughout the
specification is to be understood to refer to "colloidosomes" and
solid lipid particles. The term "colloidosome" as used throughout
the specification is to be understood to refer to a colloidal
particle that possesses the same internal nanostructure of a bulk
lyotropic phase. The term solid lipid particle as used throughout
the specification is understood to mean a colloidal particle of the
prodrug of the current invention, wherein the colloidal particle
comprises a core of the neat prodrug and usually will be stabilised
by a surface layer of surfactant. The neat prodrug core may be in a
crystalline, microcrystalline, liquid crystalline or a
non-crystalline form. It will be understood that the term
"particle" refers to particles that may be nanoparticles or
microparticles based on their average size. Often such particles
are referred to as "solid lipid nanoparticles" although they may in
fact be in a size range of microparticles. This form of
self-assembled structure does not swell upon contact with excess
solvent.
[0080] The term "hexagonal phase" as used throughout the
specification is to be understood to mean an amphiphile phase
consisting of long, rod-like micelles packed into a hexagonal
array. A "normal hexagonal phase" is a hexagonal phase consisting
of long, rod-like normal micelles, whilst an "inverse hexagonal
phase" is a hexagonal phase consisting of long, rod-like inverse
micelles. The normal hexagonal phase may be referred to as the
"H.sub.I phase" and the inverse hexagonal phase may be referred to
as the "H.sub.II phase". When a colloidosome possesses the internal
structure of a bulk hexagonal phase the colloidosome may be
referred to as a "hexosome".
[0081] The term "lamellar phase" as used throughout the
specification is to be understood to mean a stacked bilayer
arrangement, where opposing monolayers of the hydrophilic portion
of amphiphile molecules are separated by a polar solvent domain,
while the hydrophobic portion of the amphiphile molecule of the
back-to-back layers are in intimate contact to form a hydrophobic
layer. The planar lamellar phase is referred to as the
"L.sub..quadrature. phase".
[0082] The term "cubic phase" as used throughout the specification
refers to two main classes of phases: micellar cubic and
bicontinuous cubic. "Micellar cubic phase" refers to a phase
consisting of spherical micelles arranged in a cubic array. A
"normal micellar cubic phase" or "I.sub.I phase" consists of
spherical normal micelles arranged in a cubic array, whilst an
"inverse micellar cubic phase" or "I.sub.II phase" consists of
spherical inverse micelles arranged in a cubic array. "Bicontinuous
cubic phase" refers to a family of closely related phases that
consist of a single curved lipid bilayer that forms a complex
network that separates the polar solvent space into two continuous,
but non-intersecting volumes. Bicontinuous cubic phases possess
long range order based upon a cubic unit cell. Bicontinuous cubic
phases have zero mean curvature; that is, at all points on surface
of the amphiphile bilayer, the surface is as convex as it is
concave. Bicontinuous cubic phases may be of the normal ("v.sub.I
phase") or reverse ("v.sub.II phase") type. Several types of long
range orientational orders have been observed for bicontinuous
cubic phases; the orientational order in these phases correspond to
space groups Ia3d, Pn3m, and Im3m. When a colloidosome possesses
the internal structure of a bulk cubic phase the colloidosome may
be referred to as a "cubosome".
[0083] The term "sponge phase" or "L.sub.3 phase" as used
throughout the specification refers to a phase that resembles a
bicontinuous cubic phase, in that it possesses an amphiphile
bilayer that separates the polar solvent space into two unconnected
volumes, but it does not possess long range order. Accordingly,
these phases are analogous to a "melted cubic phase".
[0084] The term "lattice parameter" as used throughout the
specification means a set of lattice constants that define a unit
cell of a crystalline solid or liquid crystal, and may include
values such as the length of the unit cell.
[0085] The term "isoprenoid" as used throughout the specification
is to mean an alkyl chain consisting of isoprene
(2-methyl-1,3-butadiene)monomers or subunits. The use of the term
"isoprenoid" as used herein is intended to encompass unsaturated,
partially saturated or fully saturated isoprene analogues and
derivatives.
[0086] The term "pharmaceutical composition" as used throughout the
specification means a composition comprising a therapeutically
effective amount of at least one prodrug according to the current
invention and at least one pharmaceutically acceptable carrier,
excipient, diluent, additive or vehicle selected based upon the
intended form of administration, and consistent with conventional
pharmaceutical practices.
[0087] The terms "biologically active agent", "therapeutically
active agent", "pharmaceutically active agent", "active agent" and
"active ingredient" as used throughout the specification to refer
to substances that are intended for, without limitation, the
diagnosis, cure, mitigation, treatment, prevention and/or
modification of a state in a biological system. Reference to a
"biologically active agent" is broader than reference to a
"therapeutically active agent". The terms "drug" and therapeutic
agent are used interchangeably throughout this specification.
[0088] As used herein, "therapeutically effective amount" relates
to the amount or dose of a drug such as a 5-fluorouracil prodrug or
composition thereof that will lead to one or more desired effects,
in particular the inhibition or cessation of tumour growth. A
therapeutically effective amount of a substance will vary according
to factors such as the disease state, age, sex, and weight of a
subject, and the ability of the substance to elicit a desired
response in the subject.
[0089] The abbreviation "5-FCPhy" as used throughout the
specification refers to
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-tetramethyl-hexadecyl-1-ox-
ycarbonyl)cytidine.
[0090] The abbreviation "5-FCPal" as used throughout the
specification refers to
5'-deoxy-5-fluoro-N.sup.4-(hexadecyl-1-oxycarbonyl)cytidine.
[0091] The abbreviation "5-FCOle" as used throughout the
specification refers to
5'-deoxy-5-fluoro-N.sup.4-(cis-9-octadecenyl-1-oxycarbonyl)cyti-
dine.
[0092] The abbreviation "5-FCSte" as used throughout the
specification refers to
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine.
[0093] The abbreviation "5-FCLle" as used throughout the
specification refers to 5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecadien-1-oxycarbonyl)cytidine.
[0094] The abbreviation "5-FCLln" as used throughout the
specification refers to 5'-deoxy-5-fluoro-N.sup.4-(cis-6, cis-9,
cis-12-octadecatrien-1-oxycarbonyl)cytidine.
[0095] The abbreviation "5-FCSte" as used throughout the
specification refers to
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine.
[0096] The abbreviation "5-FCLeo" as used throughout the
specification refers to 5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecenyl-1-oxycarbonyl)cytidine.
[0097] The abbreviation "CES" as used throughout the specification
refers to an enzyme with carboxylesterase function.
[0098] It will be recognised by one skilled in the art that the
formation of the desired lyotropic liquid crystalline phases of the
current invention require a stringent balance between the specific
hydrophilic and hydrophobic domains. Accordingly, the person of
ordinary skill in the art will recognise that the selection of X in
relation to A will dictate whether the prodrug of the current
invention will form either the lyotropic phases and/or the solid
lipid particles according to the current invention.
[0099] In general, the interplay between surfactant head group,
tail and volume is very important in determining lyotropic phase
behaviour. The relationship between the molecular geometry and
phase behaviour can be described by the critical packing parameter
(CPP). CPP is defined as CPP=v/a.sub.0l.sub.c, where v is molecular
volume, a.sub.o is the cross-sectional area of the surfactant head
group, and l.sub.e corresponds to the hydrophobic tail length.
Since the development of this formula, CPP has been used widely in
predicting the mesophase behaviour based on the curvature of the
molecule. For a molecule with a small head group and a bulky
hydrophobe, the CPP value would be greater than 1, thereby inducing
a mean negative interfacial curvature and potentially formation of
an inverse mesophase.
[0100] The cleavable tail according to the current invention is
selected based upon formation of a CPP greater than one when
considered in context of the head group according to the current
invention. FIG. 1 illustrates this interplay between the head and
tail groups. The phases to the left of the lamellar phases have a
critical packing density of less than 1 and often they happen at
lower concentrations of the amphiphiles. The phases to the right of
the lamellar phases have a CPP of more than 1 and usually occur at
higher concentration of the amphiphiles. The CPP is not constant
for an amphiphile molecule and changes with external factors such
as temperature, pressure, concentration of the amphiphile and pH,
as well as some additional solvents and additives. However, still
this parameter can be used as a simple speculation of the phases
that may occur upon hydration of the amphiphiles at room
temperature or physiological temperature and at physiological pHs
and pressure.
[0101] In addition to the phases shown in FIG. 1, less common
phases can also occur upon hydration of amphiphiles such as sponge
phase (L.sub.3). This phase has a bicontinuous sponge-like
structure with a lipid bilayer separating the polar solvent space
into two unconnected sections similar to bicontinuous cubic phases.
However, unlike cubic phases, sponge phases do not possess long
range orders and their internal structure can be envisioned as a
melted cubic phase.
[0102] A preferred embodiment according to the current invention is
a self-assembled structure comprising the compounds according to
formula (II) above.
[0103] The current invention contemplates that the biologically
active agent A may itself be a prodrug instead of a drug or active.
It will be recognised by the skilled addressee that in the
compounds according to formula (II) above, A is a prodrug that
undergoes modification in vivo to the biologically active agent,
5-fluorouracil. That is, A is a precursor to the biologically
active agent formed in vivo after cleavage of the prodrug by, for
instance, an enzyme.
[0104] Preferred embodiments of R include myristyl, palmityl,
stearyl, oleyl, linoleyl, linolenyl, arachidonyl, phytanyl and
H-farnasyl chains.
[0105] Particularly preferred embodiments of the compounds of the
present invention are
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-tetramethyl-hexadecyl-1-oxycarbonyl)-
cytidine (5-FCPhy),
5'-deoxy-5-fluoro-N.sup.4-(hexadecyl-1-oxycarbonyl)cytidine
(5-FCPal),
5'-deoxy-5-fluoro-N.sup.4-(cis-9-octadecenyl-1-oxycarbonyl)cytidine
(5-FCOle),
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine
(5-FCSte), 5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecadien-1-oxycarbonyl)cytidine (5-FCLle),
5'-deoxy-5-fluoro-N.sup.4-(cis-6, cis-9,
cis-12-octadecatrien-1-oxycarbonyl)cytidine (5-FCLln), as used
throughout the specification is understood to mean
5'-deoxy-5-fluoro-N.sup.4-(octadecyl-1-oxycarbonyl)cytidine
(5-FCSte), 5'-deoxy-5-fluoro-N.sup.4-(cis-9,
cis-12-octadecenyl-1-oxycarbonyl)cytidine (5-FCLeo), farnesoyl
dopamine, or their pharmaceutically acceptable forms including
solvates, hydrates, and salts.
[0106] The synthesis of the preferred compounds of the current
invention may be carried out according to general methods known to
those skilled in the art, for instance those disclosed in U.S. Pat.
No. 4,966,891. In a particularly preferred embodiment, the
compounds are prepared according to scheme 1:
##STR00006##
wherein R is defined as herein described; Lg is a leaving group
that is preferably a halide or pseudohalide, and is most preferably
chloride; and Pg is a protecting group that is preferably acetyl.
The selection of the identity of the protecting group will readily
be determined by one of ordinary skill in the art with a minimum
amount of experimentation, and is also exemplified in the
accompanying examples. The synthesis of starting material (E) is
described in various publications such as, for example Shimma
(Shimma 2000). Deprotection of the intermediate (F) to yield the
5-FU prodrug according to formula (II) may be carried out by
methods known to those skilled in the art, following procedures
described in references, such as, for example Wuts and Greene (Wuts
2007).
[0107] Reaction conditions for the synthesis of compounds according
to the current invention would be readily determined by one of
ordinary skill in the art with a minimum amount of experimentation,
and are also exemplified in the accompanying examples.
[0108] The starting materials and reagents used to synthesise the
compounds according to the current invention are either available
from commercial suppliers such as, for example, the Aldrich
Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.),
Sigma Chemical Company (St. Louis, Mo.), Lancaster Synthesis (Ward
Hill, Mass.), or are prepared by methods known to those of ordinary
skill in the art, following procedures described in references such
as Fieser and Fieser's Reagents for Organic Synthesis (Fieser
1991), March's Advanced Organic Chemistry (Smith 2001) and
Comprehensive Organic Transformations (Larock 1999).
[0109] Another preferred embodiment according to the current
invention is a self-assembled structure comprising according to
formula (I), wherein A is dopamine. Accordingly, in this embodiment
the structure of the compounds of the self-assembled structure may
be described by formula (III):
##STR00007##
wherein X is defined for formula (I).
[0110] Preferred embodiments of R for the compounds according to
formula (III) above include oleate and arachidonate.
[0111] The self-assembled structures of the current invention
represent a desirable prodrug delivery system, owing to their
modified release properties relative to prodrugs that do no undergo
self-assembly into lamellar, inverse cubic, inverse hexagonal and
sponge phases or alternatively solid lipid particles Without
wishing to be bound by theory or mode of action, it is believed
that the self-assembled structures of the current invention possess
modified release properties firstly, due to the differences of the
hydrolytic effect on the self-assembled amphiphile molecules and
the complexity of access to single molecules in a self-assembly
system compared with that of the isolated single molecules in
non-assembled systems. Secondly, in the case of the preferred
compounds it is believed that the hydrophobic tail R of the
preferred prodrugs result in compounds with less favourable
substrate activity for the first enzyme required to convert the
prodrug, Capecitabine and its analogues to 5-FU, and thus resulting
in a modified release profile for the compounds according to the
current invention. Lastly, it is also believed that the hydrolysis
of the compounds of the current invention releases fatty chain
moieties that may, in themselves form self-assembled structures
which in turn may alter the local environment of the enzymatic
reaction and consequently affect enzymatic behaviour.
[0112] It is further similarly believed that the self-assembled
structures according to the current invention are more desirable
than prodrug self-assembled lyotropic structures that display
micellar morphologies. The prodrug lyotropic hexagonal, cubic and
sponge phases according to the current invention possess much
greater amphiphile: solvent interface area than any of the
previously disclosed prodrug self-assembled structures.
Furthermore, unlike micelles, the inverse phases according to the
current invention are stable in excess aqueous solvent.
[0113] In one embodiment, the self-assembled structures of the
current invention comprise at least one solvent domain and at least
one amphiphile domain, wherein the amphiphile domain comprises at
least one of the compounds of according to formula (II), where R is
defined as any functional group capable of conferring self-assembly
properties to the prodrug.
[0114] The solvent domain of the current invention comprises at
least one polar solvent. Examples of suitable solvents include
solvents conventionally used for amphiphile self-assembly, such as,
for example, but are not limited to the following: water,
formamide, N-methylformamide, glycerol, ethylene glycol, propylene
glycol, butylene glycol, N-methylacetamide, hydrazine and select
ionic liquids such as ethylammonium nitrate; and mixtures
thereof.
[0115] The solvent may also comprise other components, including
e.g. salts, pH buffering agents, sugars such as glucose and
sucrose. In addition to the amphiphilic prodrug the composition of
the current invention may also comprise at least one other
amphiphile that is capable of self-assembly behaviour. Amphiphiles
capable of self-assembly behaviour are known to those skilled in
the art and are described in various publications, such as, for
example, Drummond and Fong (Drummond 1999) Laughlin (Laughlin 1996,
2000) the Handbook of Lipid Research (Small 1986). Examples of
amphiphiles that are capable of self-assembly include, but are not
limited to: surfactants, lipids, and block copolymers.
[0116] In another aspect according to the present invention, the
self-assembled structure may include at least one other
pharmaceutically active agent that is capable of being incorporated
into the self-assembled structure. Pharmaceutically active agents
that are capable of being incorporated into an amphiphile drug
delivery vehicle are known to a person skilled in the art. See, for
example, WO 2005/0210046 (DBL Australia Pty Ltd) and WO9830206.
Examples of pharmaceutically or biologically active agents that may
be incorporated into the vehicle include but are not limited to:
globular proteins and glyoproteins, highly reactive lipids such as
prostaglandins, bioactive large drug molecules such as proteins,
polysaccharides, DNA and RNA and smaller drug molecules such as
cyclosporine, paclitaxel, indomethacin, fenofibrate, progesterone,
amphotericin B (AMB).
[0117] The self-assembled structures of the current invention may
also comprise at least one other component intended to stabilise
the self-assembled structure. Examples of stabilising reagents are
triblock copolymers of PEG-PPO-PEG of different building blocks and
more specifically poloxamer 407, as well as PEG lipid stabilising
reagents such as polysorbate (for example, polysorbate 80).
Bulk Phases
[0118] In one aspect, the self-assembled structure of the current
invention comprises at least one bulk lyotropic phase.
[0119] The bulk lyotropic phase of the current invention comprises
at least one phase selected from the following group: lamellar,
normal hexagonal, normal micellar cubic, normal bicontinuous cubic,
inverse bicontinuous cubic, L.sub.3 `sponge`, and inverse
hexagonal. Preferably, the bulk phase comprises at least one phase
selected from the group consisting of inverse hexagonal, inverse
cubic phase, L3 `sponge` phase and lamellar phases. Most
preferably, the bulk phase comprises inverse hexagonal and inverse
cubic phase.
[0120] In a preferred embodiment, the bulk lyotropic phases
according to the current invention may be readily produced at a
temperature range of about room temperature to about 50.degree. C.
and be stable within this temperature range for at least several
months.
[0121] A preferred embodiment according to the current invention
are bulk lyotropic inverse phases. The thermodynamic stability of
the lyotropic phases to dilution in excess aqueous solvent means
that the bulk phase maintains its primary higher ordered structure,
although the lattice parameter might be changed due to the swelling
of the amphiphile in water. Most preferably, the lyotropic phase
according to the current invention is an inverse bicontinuous cubic
phase.
[0122] It will be recognised by one skilled in the art that the
observed lyotropic phase is dependent upon temperature. The bulk
phases according to the current invention are stable between room
temperature and physiological temperature, are preferably stable at
temperatures from about 35 to about 40.degree. C. and are most
preferably stable from about 35 to about 37.degree. C.
[0123] Processes for preparing bulk phases according to the current
invention are known to those skilled in the art. In one embodiment,
bulk phases according to the present invention may be prepared by
mixing each amphiphile in an appropriate buffer to the appropriate
concentration. Examples of appropriate buffers include but are not
limited to physiologically acceptable buffers, such as, for
example, phosphate, phosphate buffered saline (PBS),
tris(hydroxymethyl)aminomethane (Tris),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
Tris-sucrose, Tris-glycine, and glycine buffers.
[0124] In another embodiment, the preferred inverse cubic phases
according to the current invention are prepared by mechanically
mixing molten lipid between room temperature and 50.degree. C.
until an optically clear and visually homogenous sample are
obtained. Optionally, addition of a co-solvent such as, for
example, ethanol in the range of 10-20% by weight may assist the
homogenisation process.
Colloidal Particles: Colloidosomes
[0125] A further aspect of the invention relates to self-assembled
structures of the current invention that comprise one or more
particles that retain the internal structure of the bulk phase.
Such particles are referred to as "colloidosomes".
[0126] In one embodiment, the self-assembled structures of the
current invention comprise colloidosomes selected from the
following group: cubosomes, hexosomes and "sponge` type particles.
In a preferred embodiment, the colloidal particles are selected
from the following group: cubosomes and hexosomes; most preferably,
the colloidal particles are cubosomes. The invention also includes
liposomes of compounds according to Formula (II).
[0127] In a particularly preferred embodiment according to the
current invention, the colloidosomes are derived from an inverse
phase. The thermodynamic stability of the lyotropic phases
according to the present invention means that the bulk phases can
progressively be diluted in excess aqueous solvent and dispersed
into colloidosomes while maintaining the same liquid crystalline
structures as that of bulk phases.
[0128] The colloidosomes according to the current invention may be
prepared according to processes known to those skilled in the art.
For example, colloidosomes may be prepared by hydration of a thin
lipid film in water or saline solution (e.g., phosphate buffered
saline). In addition sugars such as glucose and dextrose might be
added to the media. Reverse phase colloidosomes such as inverse
cubosomes and hexosomes may be hydrated in water to form gel like
bulk phases that can be consequently dispersed into particles by
using shear forces such as sonication and high pressure
homogenisation in the presence of stabilising agents.
[0129] It will be recognised by one of ordinary skill in the art
that in order to prepare stable colloidosomes it is necessary to
add a stabilisation agent or fragmentation agent. Suitable
fragmentation agents are known to those skilled in the art and
include, for example, poloxamer or polysorbate. Poloxamer is the
most widely used stabilising agents for inverse phase colloidosomes
and is a block copolymer of polyethylene glycol (PEG) and
polypropylene oxide (PPO). In a preferred embodiment according to
the current invention, the stabilising agent are triblock
copolymers of PEG-PPO-PEG of different building blocks. In a
particularly preferred embodiment according to the current
invention, the stabilisation agent is poloxamer 407. In another
embodiment, the stabilisation agent is a non-ionic block copolymer
surfactant terminated with primary hydroxyl groups, sold under the
trade name Pluronic.RTM. F127 by BASF AG. This stabilisation agent
is referred to simply as "F127" hereinafter.
[0130] In one embodiment, colloidal particles are prepared by
dispersing a bulk phase. The bulk phases of the current invention
may be dispersed by dropwise addition of an ethanolic solution of
the bulk phases into water containing a stabilising reagent.
Alternatively, the bulk phase may be dispersed by adding water
containing at least one stabilising reagent to the bulk phases. The
size of these particles can be controlled by means of vortexing,
sonication, filtration, extrusion and homogenisation, techniques
well known to one skilled in the art.
[0131] In a preferred embodiment, colloidosome dispersions
according to the current invention are prepared by dissolving an
appropriate amount of the neat amphiphile prodrug and a surfactant
in a water miscible solvent. The water miscible solvent may be one
or more solvents selected from the group consisting of ethanol,
propanol, and butanol; is preferably ethanol and propanol and is
most preferably ethanol. The prodrug-surfactant mixture is well
mixed under vortex until the solvent-surfactant-prodrug mixture is
homogeneous. Optionally, the mixture may be heated to facilitate
dissolution of the prodrug and surfactant into the water miscible
solvent in temperatures <50.degree. C. The dissolved mixture is
then added in a controlled manner to an aqueous solution.
Preferably, the aqueous solution is water. Preferably, the
prodrug-surfactant mixture is added dropwise to water. Preferably,
the water to which the mixture is being added is agitated; most
preferably, the water is being agitated by means of a vortex.
[0132] The coarse colloidosome prepared according to this
embodiment may optionally be subject to one or more additional
processing steps. Such processing methods are known to those
skilled in the art and include, for example, sonication, probe
sonication, high pressure homogenisation, and stepwise extrusion
through membranes. The membranes employed for stepwise extrusion
may possess pore sizes including, for example, 0.8, 0.4, 0.2, 0.1
and 0.05 .mu.m. In one embodiment, the processing step is a size
selection process.
[0133] In a preferred embodiment, the course colloidosome
preparation is further processed by means of passing through a
series of polycarbonate (PC) membranes. The size range of the
membranes will be selected by a person skilled in the art according
to the desired particle size of the final product. The equipment
which may be used for this processing step are known to those
skilled in the art, but may include, for example, a
mini-extruder.
[0134] It will be recognised by the skilled addressee that the size
of the colloidosomes of the current invention will depend upon the
intended use. For example, for intravenous administration the
preferred colloidosome size range is commonly between about 30 nm
and about 10 .mu.m. More preferably, the size range is between
about 30 nm and about 1 .mu.m for intravenous application.
[0135] For delivery of colloidosomes into specific organs such as
liver and passive targeting to tumours, particle sizes of between
about 30 nm to about 1000 nm are contemplated. More preferably
particle sizes are about 30 nm to less than about 500 nm.
Particularly preferred are colloidal particles of sizes between
about 30 nm to about 300 nm. Without wishing to be bound by theory,
it is believed that particles of the size between 30-300 nm are
passively targeted to cancer cells, owing to their enhanced
permeation and retention time in the leakier and chaotic
neovasculature of solid tumours. See, for example Brannon-Peppas L.
et al (Brannon-Peppas 2004).
[0136] In one embodiment depicted in FIG. 3, a coarse (unprocessed)
dispersion of 5-FCPhy/F127/ethanol particles according to the
current invention displays a bimodal average size distribution. In
another embodiment depicted in FIG. 3, particles of
5-FCPhy/F127/ethanol after treatment with a controlled membrane
extrusion process have the illustrated size distribution of 100-300
nm, averaging at 164 nm. In an alternative embodiment depicted in
FIG. 4, a coarse dispersion of 5-FCOle/F127/ethanol particles
according to the current invention have a trimodal size
distribution. In another embodiment depicted in FIG. 4, particles
of 5-FCOle/F127/ethanol after treatment with a controlled membrane
extrusion process have a size distribution of 150-500 nm, average
size of 255 nm as illustrated.
Colloidal Particles: Solid Lipid Particles
[0137] In another embodiment, the self-assembled structure
according to the present invention is a solid lipid particle.
[0138] A preferred aspect of the current invention seeks to provide
solid lipid particles comprised of at least one 5-fluorouracil
prodrug. Solid lipid particles according to the current invention
may be manufactured by processes known to those skilled in the art.
See, for example, Mehnert and Mader. (Mehnert 2001)
[0139] The appropriate process used to manufacture solid lipid
particles according to the current invention may be selected
according to the physicochemical properties of the prodrug of the
current invention. It will be recognised by one skilled in the art
that some of the typical methods to manufacture solid lipid
particles, for example those methods that require the lipid to be
melted whilst in an aqueous solution, are not applicable to the
prodrugs according to the current invention that possess a melting
point higher than 100.degree. C.
[0140] In one embodiment, the solid lipid particles of the current
invention are prepared according to mechanical methods. According
to this embodiment, one or more stabilisers are added to the neat
amphiphile. Examples of stabilisers include, but are not limited
to: triblock polymers (for example, poloxamer 407). The amount of
stabiliser added to the neat amphiphile may be between about 1-10%
(w/w). Depending on the nature of the amphiphile and the
stabiliser, the amount of stabiliser added may be between about
5-10% (w/w). Optionally, other additives may be added to the
amphiphile. Other additives are known to those skilled in the art
and may include, for example ethanol, propanol and butanol to ease
the high viscosity of the bulk phases. The amphiphile mix is then
melted, and water is added to the melted amphiphile mixture. To
prepare the initial bulk phases, usually 20-30% of water by weight
is added to the amphiphile, usually at room temperature (about 22
to about 25.degree. C.). The amphiphile-water mixture is then
sheared using methods known to those skilled in the art. In a
preferred embodiment, the amphiphile-water mixture is sheared using
rough homogenization. The mixture may then undergo further
processing to produce particles of desirable size and
polydispersity. Methods of further processing are known to those
skilled in the art and may include, for example, high pressure
homogenization, ultrasonication, and filtration through different
membranes with known pore sizes.
[0141] The average size and size distribution of the solid lipid
particles according to the current invention are similar to those
described for the colloidosomes according to the current invention.
In one embodiment, depicted in FIG. 2, the particles display the
size distribution of 350-1100 nm as illustrated.
Pharmaceutical Compositions
[0142] A further aspect of this invention relates to pharmaceutical
compositions of the current invention. In one embodiment, the
pharmaceutical composition according to the present invention
comprises at least one of compounds according to formula (I) or
formula (II). In another embodiment, the pharmaceutical composition
comprises at least one self-assembled structure according to The
current invention. In a further embodiment, the composition
comprises at least one of the solid lipid particles of the current
invention.
[0143] In one embodiment, the pharmaceutical composition according
to the current invention may be freeze-dried, spray freeze dried,
lyophilised or spray-dried powder.
[0144] Pharmaceutical compositions according to the present
invention may include pharmaceutically acceptable carriers,
excipients, diluents, additives and vehicles selected based upon
the intended form of administration, and consistent with
conventional pharmaceutical practices. Suitable pharmaceutical
carriers, excipients, diluents, additives and vehicles are known to
those skilled in the art and are described in publications, such
as, for example Remington: The Science and Practice of
Pharmacy.
[0145] The pharmaceutical compositions according to the present
invention may further include adjuvants that include, but are not
limited to: preservatives, wetting agents or antimicrobial agents.
Other adjuvants include but are not limited to: cryoprotectants,
spray drying adjuvants, buffers, isotonically adjusting agents, and
pH adjusting materials.
[0146] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. Dosage unit forms will generally contain between
from about 1 mg to about 5,000 mg of an active ingredient,
preferably contain between 20 and 1,000 mg of an active ingredient,
and most preferably between 100 and 750 mg of an active
ingredient.
[0147] It will be understood that reference to the mass of the
active ingredient refers to the mass of the prodrug, and not the
mass of self-assembled structures or solid lipid particles
thereof.
Methods of Treatment
[0148] Another aspect of this invention relates to use of a
self-assembled structure, solid lipid particle or pharmaceutical
compositions thereof according to the present invention for the
inhibition of tumour growth. In yet another aspect there is
provided a method comprising administering to a subject in need
thereof a therapeutically effective amount of a composition
including a prodrug according to formula (I), (II) or (III). In yet
another aspect there is provided a use of a composition including a
prodrug according to formula (I), (II) or (III) in the manufacture
of a medicament for administration to a subject in need thereof in
a therapeutically effective amount.
[0149] In a preferred embodiment, a pharmaceutical composition of
the current invention is used to inhibit growth of solid and
metastatic tumours. In a particularly preferred embodiment, a
pharmaceutical composition according to the current invention is
used to inhibit growth of solid or metastatic tumours associated
with colon cancer, colorectal cancer or breast cancer.
[0150] It will be recognised that the intended form of
administration of the self-assembled structure will be as either
its bulk phase, as colloidal particles derived therefrom or as
solid lipid particles.
[0151] The dosage regimen of a self-assembled structure, solid
lipid particle or pharmaceutical compositions thereof according to
the current invention will vary depending upon known factors such
as the pharmacodynamic characteristics of the compounds,
self-assembled structures, colloid particles and compositions
thereof of the current invention, and their mode and route of
administration; the age, sex, health, medical condition, and weight
of the patient, the nature and extent of symptoms, the kind of
concurrent treatment, the frequency of treatment, the renal,
hepatic and cardiovascular and otherwise general health status of
the patient in need of such treatment, and can readily be
determined by standard clinical techniques.
[0152] It will be understood that the invention disclosed and
defined in this specification extends to all alternative
combinations of two or more of the individual features mentioned or
evident from the text or drawings. All of these different
combinations constitute various alternative aspects of the
invention.
[0153] The examples that follow are intended to illustrate but in
no way limit the present invention.
Example 1
Synthesis of 5-FU Prodrugs
[0154] The general scheme for the synthesis of various prodrug
amphiphiles with varying hydrophobic chains used in this invention
are shown in Scheme 2.
##STR00008## ##STR00009##
[0155] Materials:
[0156] Materials and solvents were supplied from Sigma-Aldrich with
analytical or spectroscopic grade and used without further
modification.
[0157] Nuclear Magnetic Resonance (NMR):
[0158] The .sup.1H NMR spectra (200 MHz) were recorded on a Bruker
AC200 spectrometer in deuterated solvent with Tetramethylsilane
((CH.sub.3).sub.4Si, TMS) as internal standard unless otherwise
stated. Solute concentrations were approximately 10 mg/ml in
standard 5 mm NMR tubes. The .sup.13C NMR spectra (500 MHz) were
obtained from Bruker AC400 in CDCl.sub.3. The spectra were analysed
using MestRe-C 2.3a software. The chemical shift values (.delta.)
were expressed in ppm, coupling constants were expressed as J
values, in Hertz units.
[0159] High Performance Liquid Chromatography (HPLC) and Ultra
Performance Liquid Chromatography (UPLC):
[0160] Analytical HPLC was performed on Waters HPLC equipment
(Waters Corporation, Milford, Mass., USA), comprising of a 600
solvent delivery system with a 600 automated gradient controller
using a Phenomenex Gemini C18 column (5 .mu.M, 4.6.times.150 mm)
and an Altech 2000 Evaporative Light scattering Detector (ELSD).
The mobile phases consisted of an isocratic 70% methanol and 30%
water solvent system with 1.00 mL/min pumping rate. UPLC was
carried out on Waters Acquity UPLC BEH.TM. equipped with a C18
column (1.7 micron) 50 mm.times.2.1 mm. The mobile solvent system
was ethanol water with 0.4 ml/min flow rate, solvent A was
water/ethanol 90/10, and solvent B was 100% ethanol. At first 2
min, the gradient run from 100% A to 100% B, then the gradient
remained as 100% A. The total running time was 5 min for UPLC. The
results for HPLC and UPLC were recorded on both ELSD and UV-Vis
(.lamda.=260 nm) detectors.
[0161] Flash Column Chromatography:
[0162] Flash column chromatography was used for purification of
most synthesized compounds. Columns was prepacked with SiOH (40-63
.mu.m) purchased from Buchi. The eluting fractions were tested on
thin layer chromatography (TLC) aluminium plates precoated with
silica gel 60 containing fluorescent indicator (F.sub.254).
Compounds on the TLC plates were visualized by dipping into 3%
phosphomolybdic acid in ethanol solution, followed by charring on a
hot spot. Mass spectra were recorded on Thermo Finnigan LC-MS with
atmospheric pressure chemical ionization (APCI) source in the
positive (+) ion mode. Samples were usually introduced dissolved in
DCM. Solvents were removed using a rotary evaporator under reduced
pressure with water bath temperature below 50.degree. C.
Synthesis of 5-deoxy-1,2,3-tri-O-Acetyl-.beta.-D-ribofuranoside
(4)
##STR00010##
[0163] Methyl-2,3-O-isopropylidene-.beta.-D-ribofuranoside (1)
##STR00011##
[0165] Powdered D-ribose (60 g, 400 mmol) and SnCl.sub.2.2H.sub.2O
(90 g, 400 mmol) were suspended in a mixture of acetone (600 ml)
and methanol (156 ml). A catalytic amount of concentrated
H.sub.2SO.sub.4 (4.24 g, 2.3 ml) was added dropwise into the
solution. Then the mixture was heated and stirred at 40-45.degree.
C. overnight. After the reaction completed, the resulting mixture
was filtered through filter paper and the filter cake was washed
with a mixture of acetone and methanol (1:1 mixture, 100 ml). Then
the filtrate was neutralized (pH 6-7) with saturated NaHCO.sub.3
aqueous solution. The resulting milky solution was once again
filtered through filter paper. Both acetone and methanol in the
filtrate were then removed under reduced pressure. The aqueous
solution thus obtained was extracted with ethyl acetate (EtOAc),
the combined organic layers were washed with brine (saturated NaCl
solution), dried with Na.sub.2SO.sub.4 and evaporated in vacuo to
yield methyl-2,3-O-isopropylidene-.beta.-D-ribofuranoside as yellow
oil (54.20 g, 66.4% yield). .sup.1H NMR (CDCl.sub.3): .delta.1.31
and 1.48 (2s, each 3H, CMe.sub.2), 3.25 (br s, 1H, OH), 3.44 (s,
3H, --OMe), 3.62 (dd, 1H, J=3.2 Hz, CH.sub.2), 3.68 (dd, 1H, J=2.8
Hz, CH.sub.2), 4.44 (t, 1H, J=2.6 Hz, H-4), 4.60 (d, 1H, J=5.7 Hz,
H-2), 4.83 (d, 1H, J=5.6 Hz, H-3), 4.97 (s, 1H, H-1).
Methyl-2,3-O-isopropylidene-5-O-tosyl-.beta.-D-ribofuranoside
(2)
##STR00012##
[0167] To a cold solution containing
Methyl-2,3-O-isopropylidene-.beta.-D-ribofuranoside (1) (108.4 g,
531 mmol) in CH.sub.2Cl.sub.2 (1000 ml) dropwise adding 300 ml of
toluene-4-sulfonyl chloride (140 g, 734 mmol) in anhydrous pyridine
solution. With vigorous stirring, the reaction was carried out at
0-5.degree. C. for 20 h. The resulting solution was washed with
NaHCO.sub.3 aqueous solution, brine and evaporated to yield a
syrupy mass (185 g, 97.2%), which can be further crystallized from
hexane and dried under high vacuum to give pure
methyl-2,3-O-isopropylidene-5-O-tosyl-.beta.-D-ribofuranoside
(138.8 g, 72.86% yield) as a white solid crystals. .sup.1H NMR
(CDCl.sub.3): .delta.1.28 and 1.45 (2s, each 3H, CMe.sub.2), 2.46
(s, 3H, aromatic Me), 3.24 (s, 3H, --OMe), 4.01 (d, 2H, J=7.2 Hz,
H-5), 4.31 (dt, 1H, J=7.3 Hz, H-4), 4.53 (d, 1H, J=6.0 Hz, H-2),
4.60 (dd, 1H, J=5.6 Hz, H-3), 4.93 (s, 1H, H-1), 7.36 (d, 1H, J=8.0
Hz, aromatic H), 7.81 (d, 2H, J=8.2 Hz, aromatic H).
Methyl-2,3-O-isopropylidene-5-deoxy-.beta.-D-ribofuranoside (3)
##STR00013##
[0169] 20 g of sodium borohydride (NaBH.sub.4) was reacted with
crude methyl-2,3-O-isopropylidene-5-O-tosyl-.beta.-D-ribofuranoside
(2) (50 g, 140 mmol) in 400 ml of dimethyl sulfoxide solution for
20 h at 80-85.degree. C. After cooling the flask to room
temperature, the reaction mixture was poured into 400 ml of 1%
aqueous acetic acid solution and stirred for 30 min. The residue
was extracted with chloroform and the collected organic layer was
washed with sufficient amount of water. After dried with anhydrous
magnesium sulfate, chloroform was removed under reduced pressure to
give a crude compound with light yellow colour. The main biproduct
of this reaction was
methyl-2,3-O-isopropylidene-.beta.-D-ribofuranoside (1) due to the
hydrolysis of sulfonyl group. The crude compound was further
purified and yielded 20.2 g of
methyl-2,3-O-isopropylidene-5-deoxy-.beta.-D-ribofuranoside (3)
(71.8%) as a clear colourless liquid upon distillation. .sup.1H NMR
(CDCl.sub.3): 1.27 and 1.48 (2s, each 3H, CMe.sub.2), 61.31 (d, 3H,
J=1.5 Hz, H-5), 3.33 (s, 3H, OMe), 4.35 (q, 1H, J=7.7 Hz, H-4),
4.51 (d, 1H, J=5.4 Hz, H-2), 4.64 (d, 1H, J=5.8 Hz, H-3), 4.94 (s,
1H, H-1).
1,2,3-tri-O-acetyl-5-deoxy-fi-D-ribofuranose (4)
##STR00014##
[0171] To compound 3 (30 g, 148 mmol), added sulfuric acid solution
(0.04 N, 360 mL), and heated to 80-90.degree. C. for 17 h. When the
mixture was cooled to room temperature, solid Na.sub.2CO.sub.3 was
added to neutralize the solution to pH 7.0, and then evaporated to
dryness. The residue was then dissolved in anhydrous pyridine (350
ml), treated with acetic anhydride (150 ml, 1.58 mol), and stirred
at room temperature for 16 h. Saturated NaHCO.sub.3 (10 was poured
into the reaction mixture and stirred at room temperature for 1
hour to remove the excess amount of acetic anhydride. Then the
mixture was extracted with CH.sub.2Cl.sub.2 three times, and the
combined organic layer was washed with water, dried over
Na.sub.2SO.sub.4, and evaporated in vacuo to provide a mixture of
the two anomers of 1,2,3-tri-O-acetyl-5-deoxy-D-ribofuranose (27.9
g, 72.4% yield) as a brown liquid. The anomeric mixture was
purified by flash column chromatography on silica gel with
cyclohexane-ethyl acetate (70:30), then the pure white crystals of
.beta. anomer (4.65 g, 12.1% yield) were obtained from
crystallization with ethyl acetate-hexane. .sup.1H NMR (CDCl.sub.3)
of pure 1,2,3-tri-O-acetyl-5-deoxy-.beta.-D-ribofuranose (4):
.delta.1.37 (d, 3H, J=6.3 Hz, H-5), 2.08, 2.10, 2.12 (3s, each 3H,
OMe), 4.28 (m, 1H, J=6.5 Hz, H-4), 5.10 (dd, 1H, J=6.7 Hz, H-2),
5.34 (dd, 1H, J=4.8 Hz, H-3), 6.11 (d, 1H, J=1.0 Hz, H-1).
Synthesis of 2',3'-Di-O-acetyl-5'-deoxy-5-fluorocytidine (5)
##STR00015##
[0173] 5-Fluorocytosine (2.42 g, 18.8 mmol) was suspended in
toluene (10 ml) and hexyamethyl-disilazane (3.03 g, 18.8 mmol). The
mixture was heated at 100.degree. C. overnight. After concentrating
the reaction mixture under reduced pressure, methylene chloride (30
ml) and 5-deoxy-1,2,3-tri-O-Acetyl-.beta.-D-ribofuranoside (4)
(5.16 g, 18.8 mmol) were added to the residue. Then, anhydrous
stannic chloride (4.90 g, 18.8 mmol) was added dropwise to the
ice-cooled reaction mixture over a period of 20 min. After stirring
the mixture at room temperature for an additional 2 h, sodium
bicarbonate (10 g) was added, followed by the dropwise addition of
water (20 ml). After stirring the resulting mixture at room
temperature overnight, 4% sodium bicarbonate solution was poured
into the reaction mixture and stirred for additional 1 h. The
mixture was then extracted with water-methylene chloride. The
organic layer was collected, dried with Na.sub.2SO.sub.4, the
solvent was removed under reduced pressure to give brown syrup. The
residue was then partially purified by silica gel chromatography
using CH.sub.2Cl.sub.2:MeOH (5:1) as an eluent to yield crude
target compound (5) (4.455 g, 13.5 mmol, 72%). .sup.1H NMR in
DMSO-d.sub.6: .delta.1.34 (d, 3H, J=6.4 Hz, H-5), 2.05, 2.06 (2s,
each 3H, OMe), 4.14 (q, 1H, J=5.0 Hz, H-4), 5.10 (t, 1H, J=6.4 Hz,
H-2), 5.43 (dd, 1H, J=6.31 Hz, H-3), 5.77 (dd, 1H, J=1.0 Hz, H-1),
7.71 (br s, 1H, N--CH.dbd.C--F), 7.97 (br s, 1H, NH), 8.02 (d, 1H,
J=7.0 Hz, NH).
Synthesis and characterisation of
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-Tetramethyl-hexadecyloxycarbonyl)cyt-
idine (8a)--5FCPhy
##STR00016##
[0174] 3,7,11,15-Tetramethyl-hexadecyl chloroformate (6a)
##STR00017##
[0175] 3,7,11,15-Tetramethyl-hexadecane-1-ol
##STR00018##
[0177] To a solution containing 50 g (169 mmol) of phytol
(3,7,11,15-tetramethyl-hexadec-2-en-1-01, 97% mixture of isomers)
and 200 ml ethanol, reaction catalysis Raney nickel (5 g, 50%
slurry in water) was added. After stirring the mixture under
hydrogen atmosphere (15 psi) for 2 days, the catalyst was removed
by vacuum filtration several times through the combination layers
of silica and Celite bed on the top. The filtrate was concentrated
under reduced pressure to give phytanol (48.8 g, 97.0%) as a
colourless oil. .sup.1H NMR in CDCl.sub.3: .delta.0.83, 0.85, 0.86,
0.88, 0.91 (5s, each 3H, CH.sub.3), 0.94-1.71 (m, 25H,
10CH.sub.2+4CH+OH), 3.58-3.78 (m, 2H, CH.sub.2OH).
3,7,11,15-Tetramethyl-hexadecyl chloroformate (6a)
##STR00019##
[0179] Phytanol (48.8 g, 163.5 mmol), and triphosgene (16.17 g,
54.66 mmol) were dissolve in 300 ml of dichloromethane, stirred and
cooled on an ice bath. Anhydrous pyridine (12.93 g, 163.5 mmol) was
added dropwise over a period of 1 hour. The reaction mixture was
stirred for an additional 1 h at room temperature, and then quickly
extracted with extra methylene chloride and ice water. The organic
layer was pooled, dried over Na.sub.2SO.sub.4 and evaporated to
dryness to yield 52.22 g (88.7%) of phytanyl chloroformate as
yellow liquid. .sup.1H NMR in CDCl.sub.3: .delta.0.83, 0.85, 0.86,
0.88, (4s, each 3H, CH.sub.3), 0.91 (d, 3H, J=6.0 Hz, CH.sub.3),
0.95-1.40 (m, 20H, CH.sub.2), 1.39-1.68 (m, 4H, CH), 1.68-1.88 (m,
1H, OH), 4.27-4.47 (m, 2H, CH.sub.2OH).
2',3'-Di-O-acetyl-5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-Tetramethyl-hexadec-
yl oxycarbonyl)cytidine (7a)
##STR00020##
[0181] To the ice-cooled solution containing
2',3'-Di-O-acetyl-5'-deoxy-5-fluoroocytidine (5) (9.76 g, 29.64
mmol) in dry CH.sub.2Cl.sub.2 (40 ml) and anhydrous pyridine (5
ml), phytanyl chloroformate (12 g, 33 mmol) (6a) was added dropwise
in order to keep the low reaction temperature. After stirring the
mixture for overnight at room temperature, MeOH (1.3 ml) was added
in one portion to stop the reaction. The mixture was evaporated to
a syrup mass under reduced pressure. To this residue, diethyl ether
(50 ml) was added and the suspension was stirred for 30 min at room
temperature. The insoluble precipitate was filtered through a glass
filter. The precipitate was washed with 30 ml of diethyl ether. The
filtrate and the washings were collected, dried over anhydrous
Na.sub.2SO.sub.4, and evaporated to dryness. The crude target
compound was further purified using silica column chromatography
CH.sub.2Cl.sub.2:MeOH=5:1 to give
2',3'-Di-O-acetyl-5'-deoxy-5-fluoro-N.sup.4-(phytanyloxycarbonyl)cytidine
(16.7 g, 84.1%) (7a). .sup.1H NMR in CDCl.sub.3: .delta.50.83,
0.85, 0.86, 0.88 (4s, each 3H, CH.sub.3), 0.91 (d, 3H, J=6.6 Hz,
CH.sub.3), 0.96-1.88 (m, 24H, CH.sub.2+CH), 1.47 (d, 3H, J=6.6 Hz,
H-5), 2.10, 2.12 (2s, each 3H, OMe), 4.13-4.37 (m, 3H,
H-4+CH.sub.2OC.dbd.ONH), 5.01 (t, 1H, J=5.4 Hz, H-2), 5.29 (t, 1H,
J=5.8 Hz, H-3), 5.96 (d, 1H, J=3.8 Hz, H-1), 7.39 (br s, 1H,
N--CH.dbd.C--F).
5'-deoxy-5-fluoro-N.sup.4-(3,7,11,15-Tetramethyl-hexadecyloxycarbonyl)cyti-
dine (8a)-5FCPhy
##STR00021##
[0183] The obtained crude compound 7a (16.70 g, 24.89 mmol) was
dissolved in MeOH (50 ml) and cooled down on an ice bath. NaOH (24
ml, 8M) solution was added dropwise to maintain the reaction
temperature at 4.degree. C. Then the pH of the reaction mixture was
immediately adjusted to 7 by dropwise addition of HCl solution
(2.3M). The organic layer was separated and washed with water,
dried over anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate
was evaporated to dryness under reduced pressure. The residue was
further purified by silica column chromatography using hexane-ethyl
acetate from 60:40 to 0:100 with 10% increment as the eluent. The
pure title compound (9.6 g, 67.7% yield) was obtained as a waxy
solid with light yellow colour. .sup.1H NMR (CDCl.sub.3):
.delta.0.83, 0.85, 0.86, 0.88 (4s, 12H, CH.sub.3), 0.91 (d, 3H,
J=6.1 Hz, CH.sub.3), 0.95-1.84 (m, 28H, CH.sub.2+CH+OH), 1.40 (d,
3H, J=6.4 Hz, H-5), 3.91 (t, 1H, H-3), 4.15-4.38 (m, 4H,
COOCH.sub.2+H-2+H-4), 5.69 (d, 1H, J=4.2 Hz, H-1), 7.79 (br s, 1H,
N--CH.dbd.C--F). MS (APCI): MW: 570.21. .sup.13C NMR
(CDCl.sub.3,100 MHz): 3 [19.64, 19.71, 22.59, 22.68, 24.27, 24.30,
24.45, 24, 77, 27.93, 29.68, 32.76, 35.45, 35.54, 37.25, 37.30,
37.38, 37.42, 37.48, 39.33](CH.sub.3+CH.sub.2+CH+C5), 65.33
(OCH.sub.2), [74.94, 75.12 (br), 80.75](C2, C3, C4), 92.00 (br)
(C1), 124.3-128.0 (br), 135.0-138.0 (br), 153.11 (NHCOO). Elemental
Analysis: calculated: C, 63.24; H, 9.20; N, 7.38; F, 3.30. Found:
C, 63.59; H, 9.32; N, 7.11; F, 3.29.
[0184] TGA:
[0185] 5-FC-Phy decomposed in a multi-step process which started at
95.degree. C. and continued to 500.degree. C. The first
decomposition with peak at 105.degree. C. attributed to only 0.71%
of 75.39% total weight loss up to 500.degree. C. The second
degradation step, from 123.degree. C. to 174.degree. C., attributed
5.9%. The major degradation happened between 174.degree.
C.-330.degree. C. accounted for over 60% of total weight loss. The
degradation continued to 500.degree. C. with two more steps of
decomposition. This result revealed that the safe operation
temperature for 5-FCPhy is less than 95.degree. C.
[0186] DSC: DSC scans were performed on the neat 5-FCPhy at two
heating rates, 10 and 2.5.degree. C./min. Thermal properties of
5-FCPhy observed by DSC included a glass transition near minus 70
deg C. Melting points obtained from visual observation are listed
in Table 1.
TABLE-US-00001 TABLE 1 Melting points obtained from visual
observation Temperature Visual Melting scan rate Point .degree. C.
10.degree. C./min 50-70 2.5.degree. C./min 53-68
Synthesis and characterisation of
5'-deoxy-5-fluoro-N.sup.4-(hexadecyloxycarbonyl)cytidine
(8b)--5FCPal
##STR00022##
[0188] 2',3'-Di-O-acetyl-5'-deoxy-5-fluorocytidine (compound 5) was
synthesised by the same procedures described in example 1.
Cetyl(hexadecyl)chloroformate was purchased from Sigma-Aldrich with
96% purity. Compound 5 (3.97 g, 12.06 mmol) was dissolved in a
mixture of dry CH.sub.2Cl.sub.2 (20 ml) and anhydrous pyridine (2.5
ml). To the ice-cooled solution, cetyl chloroformate (4.41 g, 14.47
mmol) was added dropwise and stirred overnight at room temperature.
After small portion of MeOH (600 .mu.l) was added, the mixture was
evaporated to a syrup mass under reduced pressure using rotary
evaporator. To this syrup residue, diethyl ether (50 ml) was added
and the suspension was stirred for 30 min at room temperature. The
insoluble precipitate was filtered through a glass filter. The
precipitate was washed further with diethyl ether. The filtrate and
the washings were collected, dried over anhydrous Na.sub.2SO.sub.4,
and evaporated to dryness to give crude compound (7b). MS (APCI):
598.38. The crude compound 7 was dissolved in 50 ml of MeOH and
stirred on an ice bath. NaOH solution (20 ml, 8M) was added
dropwise to maintain the reaction temperature at 4.degree. C. After
immediate neutralization of the reaction mixture with dropwise
addition of HCl (2.3M) solution, the organic layer was combined and
washed with water, dried over anhydrous Na.sub.2SO.sub.4 and
filtered. The filtrate was evaporated to dryness under reduced
pressure. The pure white crystal compound (8b) (3.5 g, 56.5%) was
obtained by crystallization from acetone.
[0189] The purity of the final compound was greater than 99%
confirmed by UPLC and TLC. The molecular formula is
C.sub.26H.sub.44FN.sub.3O.sub.6 and the molecular weight is 513.64.
.sup.1H NMR (CDCl.sub.3, 200 MHz): .delta.0.88 (t, 3H, J=6.1 Hz,
CH.sub.3), 1.26 (s, 24H, CH.sub.2), 1.39 (d, 3H, J=6.9 Hz, H-5),
1.62 (s, 2H, CH.sub.2CH.sub.3), 1.63-1.80 (m, 2H, 9CH.sub.2),
3.89-4.01 (m, 1H, H-3), 4.11-4.42 (m, 4H, .alpha.CH.sub.2+H-2+H-4),
5.65 (d, 1H, J=4.1 Hz, H-1). .sup.13C NMR (CDCl.sub.3, 400 MHz):
.delta.14.07 (CH.sub.3), 18.52 (C5), [22.65, 25.76, 28.55, 29.26,
29.32, 29.51, 29.58, 29.63, 29.67, 31.89](CH.sub.2), 66.80
(OCH.sub.2), [74.95, 75.12 (br), 80.68](C2, C3, C4), 92.00 (br)
(C1), 124.3-128.0 (br), 135.0-138.0 (br), 153.19 (NHCO). MS (APCI):
514.15.
[0190] Elemental analysis: calculated: C, 60.80; H, 8.63; N, 8.18;
F, 3.70. found: C, 60.98; H, 8.74; N, 8.08; F, 3.70.
[0191] TGA:
[0192] The result for the 5-FCPal revealed that the decomposition
process for this prodrug began at approximately 120.degree. C. and
continued to occur at higher temperatures. Thermal decomposition
took place in three steps. The first step occurred between
120.degree. C. to 180.degree. C. with a mass loss of 7.21%. This
loss is accompanied by a colour change of the sample from white
crystal to yellow solid. The UPLC analysis of a 130.degree. C.
preheated sample showed two hydrophilic degradation peaks in
addition to the main compound peak indicating that the first
degradation occurred at two positions within the hydrophilic head
group and at the drug-lipid linkage. Mass spectroscopy with an APCI
probe partially confirmed this result, but no further attempts were
made to determine the exact cleavage position and degradation
product. The second degradation step occurred between 130.degree.
C. and 350.degree. C. resulting in a loss of 65% of the total mass.
At the highest temperature examined (500.degree. C.), 81% of the
total mass was lost due to degradation. The TGA result suggests
that 5-FCPal becomes thermally unstable above 120.degree. C., and
thus the temperature should be kept below the above temperature at
all times during purification or formulation in order to avoid
degradation of the conjugate.
[0193] DSC:
[0194] DSC scans were performed on the neat 5-FCPal at three
heating rates, 10, 2.5 and 0.2.degree. C./min. The characteristic
phase transition temperatures and their corresponding enthalpy
together with melting points obtained from visual observation are
listed in Table 2.
[0195] The phase transition temperatures were recorded at the
maxima of the endothermic peaks. Enthalpies were obtained by
integration of the transition peaks. A single endothermic peak
present around 115.degree. C. corresponding to the melting
transition that was similar to those observed by visual inspection.
Given that this compound starts to undergo degradation at
temperatures above 120.degree. C. as indicated in the TGA data, the
broad peak that is present after the melting transition can be
attributed to the degradation process. To increase resolution and
separate thermal effects more clearly, a heating rate of
0.2.degree. C./min was employed. At this heating rate, the melting
temperature decreased to 108.degree. C. with a slightly decreased
enthalpy. Moreover, the temperature of the first degradation peak
also decreased dramatically from over 125.degree. C. to 113.degree.
C. Taking peak associated enthalpy information into consideration,
it is clear that the shift in melting point and degradation
temperature to lower temperatures is derived from the differences
in the heating rate. In general, with decreased heating rate in DSC
measurement, the resolution is increased, the minor effects, such
as glass transition, are minimized or even hidden, thus the thermal
behaviour, for example melting point, become much sharper and are
able to be separated from merged peaks. Furthermore, at slower
heating rate, the sample starts to absorb heat to induce the
thermal behaviour at relatively lower temperatures in the slow
heating process, a shift towards lower temperature of peak maxima
compared to faster heating rates is usually seen. This trend can be
seen in Table 2.
TABLE-US-00002 TABLE 2 Thermal properties of 5-FCPal determined by
DSC. Pre transition Pre transition Pre transition Melting Point
T.sub.max .degree. C. T.sub.max .degree. C. T.sub.max .degree. C.
Visual Tm .degree. C. [transition [transition [transition Melting
[transition DSC enthalpy enthalpy enthalpy Point enthalpy scan rate
KJ mol.sup.-1] KJ mol.sup.-1] KJ mol.sup.-1] .degree. C. KJ
mol.sup.-1] 10.degree. C./min -- 48.16 [-0.68] 93.49 [-1.70]
113-119 118.32 [-32.86] 2.5.degree. C./min -- 47.32 [-1.58] 92.40
[-1.18] 110-119 114.94 [-32.29] 0.2.degree. C./min 19.88 [-1.19]
46.27 [-0.91] 90.54 [-0.73] -- 108.35 [-29.34]
Synthesis and characterisation of
5'-deoxy-5-fluoro-N.sup.4-(cis-9-Octadecenyl
oxycarbonyl)cytidine--5-FCole
##STR00023##
[0196] cis-9-Octadecenyl chloroformate (6c)
##STR00024##
[0198] Oleyl alcohol (17.58 g, 65.40 mmol), and triphosgene (6.47
g, 21.80 mmol) were dissolved in 180 ml dichloromethane, stirred
and cooled on an ice bath. Anhydrous pyridine (5.17 g of, 65.40
mmol) was added dropwise into the pre-cooled solution. The reaction
mixture was stirred for an additional 2 h at room temperature, and
then quickly extracted with ice water. The organic layer was
combined, dried over Na.sub.2SO.sub.4 and evaporated to giving
crude 18.25 g (84.3% yield) of oleyl chloroformate as liquid.
.sup.1H NMR in CDCl.sub.3: .delta.0.88 (t, 3H, J=6.3 Hz, CH.sub.3),
1.29 (d, 22H, J=7.7 Hz, CH.sub.2), 1.73 (t, 2H, J=6.6 Hz,
.beta.CH.sub.2), 2.02 (d, 4H, J=5.4 Hz,
--CH.sub.2--CH.dbd.CH--CH.sub.2--), 4.31 (t, 2H, J=6.6 Hz,
.alpha.CH.sub.2), 5.35 (m, 2H, --CH.dbd.CH--).
5'-deoxy-5-fluoro-N.sup.4-(cis-9-Octadecenyl-1-oxycarbonyl)cytidine
(8c)--5FCOle
##STR00025##
[0200] 2',3'-Di-O-acetyl-5'-deoxy-5-fluorocytidine (compound 5) was
synthesized following the procedures described previously. Compound
5 (3.53 g, 10.72 mmol) was dissolved in a mixture of dry
CH.sub.2Cl.sub.2 (20 ml) and anhydrous pyridine (2.3 ml). To the
ice-cooled solution, oleyl chloroformate (4.26 g, 12.86 mmol) was
added dropwise and stirred overnight at room temperature. After
small portion of MeOH (500 .mu.l) was added, the mixture was
evaporated to a syrup mass under reduced pressure using rotary
evaporator. To this residue, diethyl ether (60 ml) was added and
the suspension was stirred for 30 min at room temperature. The
insoluble precipitate was filtered through a filter paper. The
precipitate was washed further with diethyl ether. The filtrate and
the washings were collected, dried over anhydrous Na.sub.2SO.sub.4,
and evaporated to dryness to give crude compound (7c). MS (APCI):
624.00. This crude sample was dissolved in 50 ml of MeOH and
stirred on an ice bath. NaOH solution (20 ml, 8M) was added
dropwise to ensure the low temperature reaction condition
maintained. After immediate neutralization of the reaction mixture
with dropwise addition of HCl solution (2.3 M), the mixture was
then partitioned between CH.sub.2Cl.sub.2 and water. The organic
layer was combined and washed with water, dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was evaporated to
dryness under reduced pressure. The pure compound (8c) was obtained
by flash column chromatography purification using CHCl.sub.2 and
MeOH eluent, starting from 100% CH.sub.2Cl.sub.2 and gradually
increased the concentration to 10%. MeOH. The title compound was
obtained as a yellow solid (3.46 g, 59.8% yield). .sup.1H NMR
(CDCl.sub.3): .delta.0.88 (t, 3H, J=6.0 Hz, CH.sub.3), 1.28 (d,
22H, J=4.0 Hz, CH.sub.2), 1.42 (d, 3H, J=6.5 Hz, H-5), 1.58-1.77
(m, 2H, .beta.CH.sub.2), 2.01 (d, 4H, J=5.1 Hz,
--CH.sub.2--CH.dbd.CH--CH.sub.2--), 3.73 (br s, 1H, H-3), 3.87 (t,
1H, J=4.7 Hz, H-4), 4.18 (t, 2H, J=6.4 Hz, .alpha.CH.sub.2), 4.27
(d, 1H, J=4.5 Hz, H-2), 5.35 (t, 2H, J=5.7 Hz, --CH.dbd.CH--), 5.71
(d, 1H, J=2.9 Hz, H-1), 7.79 (br s, 1H, N--CH.dbd.C--F). .sup.13C
NMR (CDCl.sub.3,100 MHz): .delta.14.07 (CH.sub.3), 18.54 (C5),
[22.64, 25.76, 27.16, 27.18, 28.54, 29.21, 29.28, 29.41, 29.48,
29.72, 31.86](CH.sub.2), 66.85 (OCH.sub.2), [74.94, 75.12 (br),
80.70](C2, C3, C4), 92.00 (br) (C1), [129.71, 129.95] (CH),
124.3-128.0 (br), 135.0-138.0 (br), 153.15 (NHCOO). MS (APCI):
540.28.
[0201] TGA:
[0202] The TGA results for the prodrug amphiphile 5-FCOle showed a
three step thermal degradation process. The first step accounted
for 80.3% total mass loss of the original sample up to 500.degree.
C. The degradation starting at 123.degree. C. indicates that the
temperature should be well below such temperature to retain the
intact structure of 5-FCOle. A successive mass loss occurred
between 123.degree. C. to 400.degree. C., accounting for over 70%
of weight loss. A further 7% weight loss happened when temperature
reached 500.degree. C.
[0203] DSC:
[0204] DSC scans were performed on the neat 5-FCOle at three
different heating rates, 10, 2.5 and 0.2.degree. C./min. The
characteristic phase transition temperatures and their
corresponding enthalpies together with melting points obtained from
visual observation are listed in Table 3.
TABLE-US-00003 TABLE 3 Thermal properties of 5-FCOIe determined by
DSC. Pre Pre Pre transition transition transition Melting point
T.sub.max .degree. C. T.sub.max .degree. C. T.sub.max .degree. C.
Glass Visual T.sub.max .degree. C. [transition [transition
[transition Transition Melting [transition DSC enthalpy enthalpy
enthalpy Tg Onset .degree. C. Point enthalpy scan rate KJ
mol.sup.-1] KJ mol.sup.-1] KJ mol.sup.-1] [Midpoint .degree. C.]
.degree. C. KJ mol.sup.-1] 10.degree. C./min -15.67 [-0.91] -5.83
[-0.72] -- -- 66-75 72.00 [-12.00] 2.5.degree. C./min -15.84
[-0.53] -6.71 [-0.73] 36.37 [-0.08] -- 65-73 71.41 [-12.10]
0.2.degree. C./min -15.42 [-2.17] -6.55 [-1.71] -- -- 63-77 74.62
[-12.78]
Synthesis and characterisation of
5'-deoxy-5-fluoro-N.sup.4-(Octadecyl-1-oxycarbonyl)cytidine--5-FCste
##STR00026##
[0205] Octadecyl chloroformate (6d)
##STR00027##
[0207] Stearyl alcohol (2.7 g, 0.01 mol), and triphosgene (0.99 g,
0.0067 mol) were dissolved in 20 ml methylene chloride, stirred and
cooled to 10-15.degree. C. Anhydrous pyridine (0.8 g of, 0.02 mol)
was added dropwise into the pre-cooled solution over a 1 hr period.
The reaction mixture was stirred for an additional 1 h, then heated
in a water bath at 65.degree. C. for 15 min until all the methylene
chloride evaporated. The residue was washed 3 times with cold
water, dried over Na.sub.2SO.sub.4 and evaporated to give stearyl
chloroformate (1.88 g, 83% yield), also known as octadecyl
chloroformate.
[0208] .sup.1H NMR (CDCl.sub.3): .delta.0.88 (t, 3H, J=6.4 Hz,
CH.sub.3), 1.26 (m, 30 CH.sub.2), 1.62 (m, 2H, .alpha.-CH.sub.2),
4.31 (t, 2H, J=6.7 Hz, .beta.-CH2)
5'-deoxy-5-fluoro-N.sup.4-(Octadecyl-1-oxycarbonyl)cytidine
(8d)--5FCste
##STR00028##
[0210] 2',3'-Di-O-acetyl-5'-deoxy-5-fluorocytidine (compound 5) was
synthesized following the procedures described previously. Compound
5 (1.5 g, 0.0046 mol) was dissolved in a mixture of dry
CH.sub.2Cl.sub.2 (20 ml) and anhydrous pyridine (1 ml) over an ice
bath. To the ice-cooled solution, compound 6d (1.82 g, 0.0055 mol,
1.2 equiv.) was added dropwise and stirred overnight at room
temperature. After a small portion of MeOH (250 .mu.l) was added,
the mixture was evaporated to dryness. To this residue, diethyl
ether (50 ml) was added and the suspension was stirred for 60 min
at room temperature. The insoluble precipitate was filtered through
a filter paper. The precipitate was washed further with diethyl
ether. The filtrate and the washings were collected, dried over
anhydrous Na.sub.2SO.sub.4, and evaporated to dryness to give crude
compound (7d). This crude sample was dissolved in 20 ml of MeOH and
stirred on an ice bath. NaOH solution (10 ml, 8M) was added
dropwise to ensure the low temperature reaction condition
maintained. After immediate neutralization of the reaction mixture
with dropwise addition of HCl solution (4 M), the mixture was then
partitioned between CH.sub.2Cl.sub.2 and water. The organic layer
was combined and washed with water, dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was evaporated to
dryness under reduced pressure. The pure compound (8d) was obtained
by flash column chromatography purification using CHCl.sub.2 and
MeOH eluent, starting from 100% CH.sub.2Cl.sub.2 and gradually
increasing the concentration to 10% MeOH. The title compound 8d was
obtained as a white solid (1.97 g, 78% yield).
[0211] .sup.1H NMR (CDCl.sub.3): .delta.0.9 (t, 3H, J=6.4 Hz,
--CH.sub.3), 1.25 (m, 28H, CH.sub.2), 1.39 (d, 3H, J=6.6 Hz,
ribose-CH.sub.3 (H-5)), 1.7 (t, 2H, J=7 Hz. .beta.CH.sub.2), 3.9
(dd, 1H, J=3.7, 5.1, H-4), 4.17-4.27 (m, 1H, H-2), 4.20 (s, 2H,
.alpha.CH2), 4.29-4.40 (m, 1H, H-3), 5.64 (d, 1H, J=4 Hz, H-1), 7.8
(b.s., 1H, N--CH.dbd.C--F).
[0212] .sup.13C NMR (CDCl.sub.3): .delta.14.1 (CH.sub.3), 15.9
(ribose-CH.sub.3) [22.7, 25.8, 29.2, 29.3, 29.5, 29.6, 29.7, 29.7,
31.9] CH.sub.2, 67.0 OCH.sub.2) [76.4, 77.0, 77.6] (C2, C3, C4),
95.0br (C1), 120.3 (CF), 153.4 (NHCOO) 157-160br (Aromatic C)
[0213] DSC:
[0214] DSC scans were performed on the neat 5-FCste at 2.5.degree.
C./min. The characteristic phase transition temperatures and their
corresponding enthalpies together with melting points obtained from
visual observation are listed in Table 4.
TABLE-US-00004 TABLE 4 Thermal properties of 5-FCste determined by
DSC. Peak Peak Peak Peak T .degree. C. T .degree. C. T .degree. C.
T .degree. C. [transition [transition [transition [transition DSC
enthalpy enthalpy enthalpy enthalpy scan rate KJ mol.sup.-1] KJ
mol.sup.-1] KJ mol.sup.-1] KJ mol.sup.-1] 12.5.degree. C./min 8.59
[38.84] 15.29 [-3.26] 117.37 [-75.49] 135.08 [-6.41]
Synthesis and characterisation of
5'-deoxy-5-fluoro-N.sup.4-(cis-9,12-Octadecenyl
oxycarbonyl)cytidine--5-FCIeo
##STR00029##
[0215] cis-9,12-Octadecenyl chloroformate (6e)
##STR00030##
[0217] First, linoleic acid was converted to linoleoyl. 4.5 mmol
LiAlH4 (0.17 g--1.25 equivalent of acid to reduce) was suspended in
20 ml dry diethyl ether. 1 g (3.57 mmol) of linoleic acid was added
dropwise in 10 ml diethyl ether, and it was ensured that the
addition created a gentle reflux. The reaction vessel was protected
from light to prevent degradation. The reaction was continually
stirred for 3 hours and left overnight to ensure completion as
determined by TLC. The flask was placed in an ice water bath and 4
ml of 10% sulphuric acid was added with care. The organic layer was
decanted, and aqueous suspension washed with ether (20 ml.times.3).
The ether fractions were combined and washed with water twice and
dried over Na.sub.2SO.sub.4, filtered and evaporated to obtain a
white-yellowish wax, linoleoyl (85% yield).
[0218] Then, linoleoyl (20 g, 0.0075 mol), and triphosgene (1.3 g,
0.0044 mol) were dissolved in 20 ml methylene chloride, stirred and
cooled to 10-15.degree. C. Anhydrous pyridine (0.8 g of, 0.013 mol)
was added dropwise into the pre-cooled solution over a 1 hr period.
The reaction mixture was stirred for an additional 1 h, then heated
in a water bath at 65.degree. C. for 15 min until all the methylene
chloride evaporated. The residue was washed 3 times with cold
water, dried over Na.sub.2SO.sub.4 and evaporated to give linoleoyl
chloroformate (2.14 g, 87% yield), also known as
cis-9,12-Octadecenyl chloroformate.
[0219] .sup.1H NMR (CDCl.sub.3): .delta.0.88 (t, 3H, J=6.2 Hz,
CH.sub.3), 1.20-1.4 (m, 14H, CH.sub.2), 1.63-1.73 (m, 2H,
.beta.CH.sub.2), 2.01 (d, 4H, J=5.1 Hz, --CH.sub.2--CH.dbd.), 2.77
(t, 2H, J=5.52 Hz, .dbd.CHCH.sub.2CH.dbd.), 4.35 (t, 2H, J=6.4 Hz,
.alpha.CH.sub.2), 5.37 (m, 4H, --CH.dbd.CH--), 5.41 (m, 4H,
.dbd.CH).
5'-deoxy-5-fluoro-N.sup.4-(cis-9,12-Octadecenyl-1-oxycarbonyl)cytidine
(8e)--5FCIeo
##STR00031##
[0221] 2',3'-Di-O-acetyl-5'-deoxy-5-fluorocytidine (compound 5) was
synthesized following the procedures described previously. Compound
5 (2.43 g, 0.0074 mol) was dissolved in a mixture of dry
CH.sub.2Cl.sub.2 (20 ml) and anhydrous pyridine (1 ml) over an ice
bath. To the ice-cooled solution, compound 6e (2.43 g, 0.0074 mol,
1 equiv.) was added dropwise and stirred overnight at room
temperature. Due to the light sensitive nature of the unsaturated
chloroformate the reaction was kept insulated from ambient light.
After a small portion of MeOH (250 .mu.l) was added, the mixture
was evaporated to dryness. To this residue, diethyl ether (100 ml)
was added and the suspension was stirred for 10 min at room
temperature. The insoluble precipitate was filtered through a
filter paper. The precipitate was washed further with diethyl
ether. The filtrate and the washings were collected, dried over
anhydrous Na.sub.2SO.sub.4, and evaporated to dryness to give crude
compound (7e). This crude sample was dissolved in 20 ml of MeOH and
stirred on an ice bath. NaOH solution (10 ml, 8M) was added
dropwise to ensure the low temperature reaction condition
maintained. After immediate neutralization of the reaction mixture
with dropwise addition of HCl solution (4 M), the mixture was then
partitioned between CH.sub.2Cl.sub.2 and water. The organic layer
was combined and washed with water, dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was evaporated to
dryness under reduced pressure. The pure compound (8e) was obtained
by flash column chromatography purification using CHCl.sub.2 and
MeOH eluent, starting from 100% CH.sub.2Cl.sub.2 and gradually
increasing the concentration to 10% MeOH. The title compound 8e was
obtained as a yellowish wax (3.1 g, 75% yield).
[0222] .sup.1H NMR (CDCl.sub.3): .delta.0.88 (t, 3H, J=6.2 Hz,
CH.sub.3), 1.20-1.4 (m, 14H, CH.sub.2), 1.39 (d, 3H, J=6.4 Hz,
H-5), 1.63-1.73 (m, 2H, .beta.CH.sub.2), 2.01 (d, 4H, J=5.1 Hz,
--CH.sub.2--CH.dbd.), 2.77 (t, 2H, J=5.52 Hz,
.dbd.CHCH.sub.2CH.dbd.), 3.9 (dd, 1H, J=3.8 Hz, 5.3 Hz, H-3), 4.20
(s, 1H, H-2), 4.17-4.24 (t, 2H, .alpha.-CH.sub.2), 4.33-4.38 (m,
1H, H-3), 5.34 (m, 4H, .dbd.CH), 5.63 (d, 1H, J=4 Hz, H-1) (t, 1H,
J=4.7 Hz, H-4), 4.18 (t, 2H, J=6.4 Hz, .alpha.CH.sub.2), 4.27 (d,
1H, J=4.5 Hz, H-2), 5.37 (m, 4H, --CH.dbd.CH--), 5.71 (d, 1H, J=2.9
Hz, H-1), 7.8 (br s, 1H, N--CH.dbd.C--F).
[0223] DSC:
[0224] DSC scans were performed on the neat 5-FCIeo at 2.5.degree.
C./min. The characteristic phase transition temperatures and their
corresponding enthalpies together with melting points obtained from
visual observation are listed in Table 5.
TABLE-US-00005 TABLE 5 Thermal properties of 5-FCleo determined by
DSC. Peak Peak Peak T .degree. C. T .degree. C. T .degree. C.
[transition [transition [transition DSC enthalpy enthalpy enthalpy
scan rate KJ mol.sup.-1] KJ mol.sup.-1] KJ mol.sup.-1] 2.5.degree.
C./min -35.24 [0.60] 97.33 [2.41] 146.01 [0.13]
Synthesis and Characterisation of Hexahydrofarnesoyl Dopamine
Hexahydrofarnesol
[0225] 3,7,11-Trimethyl-dodecan-1-ol (hexahydrofarnesol) was made
in-house in the same manner as previously published (D. Wells, C.
Fong, I. Krodkiewska, C. J. Drummond, J. Phys. Chem. B., 110,
5112-5119, 2006).
H-Farnesoic Acid
[0226] Hexahydrofarnesol (12 g, 53 mmol) was diluted with 150 ml of
glacial acetic acid and cooled in an ice bath. Chromium trioxide
(13.13 g 131.34 mmol) was dissolved in 15 ml of water and added
over 2 hours to the reaction mixture and was left overnight
allowing the reaction mixture to return to room temperature. An
aliquot was taken out, quenched with water and a few crystals of
sodium metabisulphite, followed by extraction with petroleum spirit
40/60. The sample was analysed by NMR. If the alcohol was converted
completely, the whole mixture was extracted twice with petroleum
spirit 40/60. The extract was then washed with brine and a small
addition of sodium metabisulphite. A greenish extract was filtered
through a 1 cm pad of silica. The filtrate was evaporated to
dryness under reduced pressure. The crude acid was subsequently
vacuum distilled using Buchi Kugelrohr. The main fraction,
collected at 145.degree. C./1.6.times.10.sup.-2 mm Hg was a
colourless oil confirmed by .sup.1HNMR.
Hexahydrofarnesoyl Chloride
[0227] Hexahydrofarnesoic acid (7.785 g, 32.11 mmol) was diluted
with 35 ml of dry DCM, a catalytic amount of dry DMF was added to
the mixture and the solution cooled to .about.0.degree. C. The
reaction flask was kept under Argon atmosphere. Oxalyl chloride
4.89 g (38.54 mmols, 1.2 eq.), dissolved in 20 ml of dry DCM, was
added dropwise and the cooling bath was removed. The reaction
mixture remained colourless when cold, then became strongly
coloured at RT. NMR confirmed the complete conversion of the acid
to its chloride. The solvent was removed under vacuum to yield
7.947 g of orange-brown oil (94% yield). The crude acid chloride
was obtained by vacuum distillation; the main fraction was
collected at 125.degree. C./7.9.times.10.sup.-3 mm Hg. Yield
calculated for the pure product was 84.9%.
Hexahydrofarnesoyl Dopamine
##STR00032##
[0229] Dopamine hydrochloride (10.07 g, 53.135 mmol) was dissolved
in 60 ml of dry DMF and maintained under Argon atmosphere. It
formed colourless solution. Dry triethylamine (8.06 g, 79.70 mmol)
was added and cooled to -20.degree. C. Hexahydrofarnesoyl chloride
(6.93 g, 26.57 mmol) was diluted with 50 ml of dry DCM and added
dropwise to the reaction mixture over 2 hours, while maintaining
the temperature under -20.degree. C. The RM was heterogenous during
the acid chloride addition. After 30 min, the RM was allowed to
return slowly to RT and stirred for a further 2 hours. NMR of an
aliquot showed no unreacted acid chloride. 100 ml of DCM and 75 ml
of brine was added and the mixture was acidified with diluted
hydrochloric acid to pH 3. The separated aqueous layer was
back-washed with DCM and the combined DCM phase was washed twice
with 75 ml of brine. DCM extract, still containing DMF was
concentrated under vacuum, resulting in a brown heavy oil, 10.5
g.
[0230] This material was preadsorbed on 20 g of silica and
chromatographed using chloroform as eluent.
[0231] That first purification gave the product of .about.95%
purity. The product was redissolved in ethanol and purified on
prepHPLC using a Gemini-NX 10 micrometer C.sub.18 250.times.50 mm
Phenomenex column to obtain product with purity more than 99%.
[0232] MS (APCI, positive ion): 378.17, .sup.1HNMR: 6.66, d, J 8.0
Hz, 1H, Ar--H5; 6.64, d, J 2.0 Hz, 1H, Ar--H2; 6.52, dd, J 8.0, 2.0
Hz, 1H, Ar--H6; 3.33, t, J 7.2 Hz, 2H, NCH.sub.2; 2.62, t, J 7.2
Hz, 2H, CH.sub.2Ar; 2.15-2.09, m, 1H, C(O)CH.sub.2; 1.95-1.89, m,
1H, C(O)CH.sub.2; 1.59-1.45, m, 1H, CHMe.sub.2; 1.44-1.01, m, 14H,
6.times.CH.sub.2, 2.times.CH; 0.90-0.84, m, 12H,
4.times.CH.sub.3.
Example 2
Demonstration of Self-Assembly Behaviour
[0233] Cross-Polarised Light Microscopy (Water Penetration
Scans):
[0234] Lyotropic mesophase behaviour was studied using water
penetration experiments. Neat amphiphile was placed on a glass
slide, melted and cooled to facilitate sample homogenization and
then covered with a glass cover slip. A few drops of Milli-Q water
were then introduced to the edge of the cover slip which was drawn
in by capillary action resulting in a concentration gradient from
pure amphiphile in the centre to pure water around the edges of the
cover slip. The generated mesophases were identified by their
characteristic birefringent textures. Samples were then heated at
2.degree. C./min from room temperature to .about.99.degree. C.
using an FP90 Linkam hotstage (Linkam Scientific Instruments,
England). Thermotropic phase behaviour was also examined in the
similar method but in the absence of water. The phases were viewed
using an Olympus GX51 inverted polarising microscope (Olympus
Australia Pty. Ltd, Australia) equipped with an Olympus C-5060
digital camera for image capture. All images were taken with
100.times. magnification unless elsewhere stated. Visual melting
point was also determined on the same system with a heating rate of
10.degree. C./min and 2.5.degree. C./min.
[0235] Small Angle X-Ray Scattering (SAXS):
[0236] The bulk phase of the binary amphiphile/water system was
prepared by weighing the appropriate amount of sample and water in
a sample vial. For those samples requiring pre-treatment, the
samples were quickly melted in a pre-heated temperature controlled
silicon-oil heating bath prior to addition of water. Homogenization
of the amphiphile/water system was achieved through vigorous
agitation using a mechanical vortex with subsequent standing at
room temperature for a minimum of 7 days to equilibrate before
taking measurements. The prepared samples were analysed using an
Anton-Paar SAXSess (Graz, Austria) with PANalytical PW3830
stand-alone X-ray generator operating at 40 kv, 50 mA with a
sealed-tube Cu anode (.lamda..sub.Cu-K/.alpha.=0.154 nm). The
samples were loaded into a paste cell sample holder at room
temperature. All measurements were performed in the heating
direction and equilibrated at each temperature for at least 10 min
prior to data collection. Temperature control was via a TCU
Temperature Control Unit (Anton Paar). All samples were measured
using line collimation equipped with an advanced CCD detector
(Anton Paar GmbH, 24.times.24 .mu.m pixel size; .DELTA.q=0.0037
nm.sup.-1). The scattering results were recorded and analysed by
the accompanied Anton Paar software.
Results
[0237] The results of SAXS are summarised in Tables 6, 7 and 8 and
indicate that self-assembled lyotropic liquid crystalline phases
were formed for the prodrugs 5-FCPhy and 5-FCOle and a lamellar
non-swelling crystalline phase was present. The results of water
penetration scans are discussed below.
[0238] The textures obtained from water penetration scans along the
established concentration gradient from neat amphiphile to pure
water provide a quick insight into the lyotropic phase behaviour of
a surfactant/water system. Melted neat amphiphile was placed on a
microscopic slide and water was then introduced around the edge
5-FCPhy has the ability to form 3D inversed bicontinous cubic phase
(Pn3m space group) initially and then transform to 2D hexagonal
phase with elevated temperature or with prolonged equilibration
time. Likewise, prodrug 5-FCOle was able to form traditional
lamellar (L.quadrature.) phase at room temperature and 3D cubic
(Pn3m) phase at elevated temperatures above 33.degree. C. 5FC-Pal
did not swell in the presence of aqueous solutions.
TABLE-US-00006 TABLE 6 The phase behaviour of equilibrated 5-FCPhy,
at various temperatures. H.sub.2 = hexagonal, Pn3m = 3D inverse
cubic, RT = 25.degree. C. Measured Composition Temperature Phases
at RT Lattice parameter 5-FCPhy RT 37.degree. C. Amorphous --
without water 5-FCPhy RT, 37.degree. C. H.sub.2 RT: H.sub.2(5.82
nm) in Excess water 37.degree. C.: H.sub.2(5.60 nm)
TABLE-US-00007 TABLE 7 The phase behaviour of equilibrated 5-FCOle,
at various temperatures. Lc = smectic crystalline phase. L.alpha. =
Lamellar liquid crystalline, and Pn3m (D) inverse cubic phases Mea-
Mea- suring sured Lattice tech- Temper- parameter nique Composition
ature Phases (nm) Error XRD neat 25.degree. C. Smectic 2.977 0.624
crystal (Lc) SAXS neat 25, 37.degree. C. Smectic 25.degree. C.:
2.99 25.degree. C.: crystal 37.degree. C.: 3.00 0.673 (Lc)
37.degree. C.: 0.845 In excess water RT, RT: L.alpha. 25.degree. C.
25.degree. C.: 37.degree. C. 37.degree. C. L.alpha.: 6.01 0.650
Pn3m 37.degree. C. 37.degree. C.: Pn3m: 10.40 0.432 In excess water
37.degree. C. Pn3m 37.degree. C. 37.degree. C.: with 2.3% F127
Pn3m: 9.10 0.005 and 4.87% Ethanol
TABLE-US-00008 TABLE 8 The phase behaviour of 5-FCPal measured with
SAXS and confirmed with XRD Equilibration Measured Composition Time
Temperature Phases d-spacing without water -- RT, 37, 50, 70,
Lamellar RT: Lc 90.degree. C. (crystal) (2.87 nm) In excess @RT 7
Days RT, 37, 50.degree. C. Lamellar RT: Lc water (crystal) (2.86
nm)
[0239] The 5-FCSte amphiphile was shown to form lamellar phases in
excess water at physiological temperatures, similar to those
observed by the 5-FCPal compound. The aliphatic chain of 5-FCSte
has no unsaturation and therefore is not likely to induce
sufficient curvature to form inverse lyotropic liquid crystalline
phases. The 5-FCLeo derivative was shown to form inverse phases at
limited hydration and physiological temperatures including the
inverse hexagonal phase, and a fluid isotropic phase has been
observed under excess water conditions.
[0240] For the dopamine prodrug amphiphile, SAXS at 25.degree. C.
of the neat and excess water amphiphile shows the following
results: Lamellar 1.sup.st and 2.sup.nd order peaks at d=23.37A
with error=0.06 A. Two additional peaks of an unidentified phase in
the ratio 6:7 to each other are also present at d=59.09 A with
error =0.17 A. FIG. 17 shows a SAXS pattern of the neat material at
room temperature.
Example 3
Preparation of Colloidal Particles or Dispersions
[0241] The preferred prodrugs according to the current invention
can be dispersed into aqueous solution and form colloidal particles
with very fine internal nanostructures and in the size range of
100-1500 nm, by using the following procedure.
[0242] Colloidosome Dispersions:
[0243] Typical colloidosome dispersions were prepared for 5-FCPhy
and 5-FCOle according to the following method: an appropriate
amount of 5-FCPhy or 5-FCOle, Poloxamer 407
(PEO.sub.98PPO.sub.67PEO.sub.98 with average formula weight of
12,500; BASF) equivalent to 10% (w/w) of neat amphiphile and
absolute ethanol were weighed in a sample vial. The dissolved
mixture was added dropwise into milli-Q water under vigorous
vortexing. The final composition for 5-FCPhy particle dispersion
was: 4.74% 5-FCPhy, 0.45% F127, 9% ethanol, 85.7% water while for
5-FCOle the composition was: 2.3% of 5-FCOle, 0.2% F127, 4.87%
ethanol and 92.6% of water. These crude dispersions were then
passed through a series of PC membranes from the size range from 1
um to 100 nm to obtain a more uniform size distribution for the
particles. The equipment used for size control is a mini-extruder
(Avanti Polar Lipids, USA). Polycarbonate filters of the sizes 1
.quadrature.m, 800 nm, 400 nm, 200 nm and 100 nm were used
consecutively to reduce the size of the particles to 100-200 nm.
The final concentration of colloidosomes were determined by ultra
performance liquid chromatography (UPLC) and then diluted to 1
mg/mL for use in the enzyme hydrolysis study The particle size
distribution and morphology of the above suspensions were
determined using the method as described herein by using zetasizer
and cryo-TEM characterisation methods.
[0244] Solid Lipid Particles:
[0245] Solid lipid particles of prodrug 5-FCPal were prepared using
mechanical methods. Approximately 700 mg of 5-FCPal crystal with
10% (w/w) of poloxamer F127 were weighed in a vial and heated to
just above the melting point (120.degree. C.). The melted prodrug
and poloxamer mixture was then transferred to 40 mL of pre-warmed
water upon vigorous vortexing. The mixture was then sheared with a
rough homogenizer (Ultra Turrax.RTM., T18 Basic, IKA.RTM. Werke
GmbH & Co. KG, Germany) at 14,000 rpm for 10 min. The resultant
dispersion was further transferred to the ultrasonic benchtop
cleaner for a few hours of ultra sonication. High pressure
homogenization (EmulsiFlex-C3 Homogenizer, Avestin Inc., Canada) at
50 psi with elevated temperature at approximately 50.degree. C. was
applied to homogenize the dispersions for 20 min and reduce the
particle size.
[0246] The final concentration of solid lipid particles was
determined by ultra performance liquid chromatography (UPLC) and
then diluted to 1 mg/mL for the enzymatic hydrolysis study. The
particle size distribution and morphology of above solution were
determined using methods as described herein.
[0247] Cryo-TEM
[0248] Cryo-TEM images of lyotropric liquid crystalline particles
and solid lipid particles were obtained using a laboratory-built
vitrification system allowing humidity to be kept close to 90%
during sample plunging and vitrification. 4-5 .mu.l of sample
solution was applied to a 300 mesh copper TEM grid coated with a
lacey carbon film (ProSciTech, Thuringowa Qld 4817Australia) and
allowed to settle for 30 s. The grid was manually blotted for 10-15
s, and the resulting thin film was then vitrified by plunging into
liquid ethane. Grids were stored in liquid nitrogen before
transferring into a Gatan 626-DH Cryo-holder. Imaging was carried
out using an FEI Tecnai 12 TEM, operating at 120 kV, equipped with
a MegaView III CCD camera and AnalySis imaging software (Olympus
Soft Imaging Solutions). The sample was kept at a temperature of
-180.degree. C. and standard low-dose procedures were used to
minimize radiation damage.
[0249] Dispersions Characterization: Particle Size
Distribution:
[0250] Determination of the particle size distribution of the
colloidosome and solid lipid particle dispersions were carried out
using a Zetasizer (nano zs, Malvern, England) equipped with a
He--Ne Laser (4 mw, 633 nm) and an avalanche photodiode detector.
Dynamic light scattering (DLS) analysis was performed on the
dispersion in a disposable sizing cuvette with the scattering angle
of .theta.=90.degree. at 25.degree. C. Each measurement was
repeated at least two times and the measurement time for every
individual run was 60 s. The viscosity and RI value of 0.8872 cp
and 1.330 were used respectively in the data calculation. The size
distribution was recorded by intensity.
[0251] The results of the particle dispersions manufactured from
the three different prodrugs are shown in FIGS. 2-4.
[0252] The 5-FCPhy and 5-FCOle, which demonstrated inverse
hexagonal and cubic phases at bulk phases, at physiological
temperature, dispersed into smaller particles (mean of 164 and 255
nm respectively) with a relatively narrow size distribution
(100-300 nm and 150-500 nm respectively). The 5-FCPal had a
crystalline structure and formed solid lipid particles with
somewhat larger particles (mean 700 nm), compared to other two
prodrug amphiphiles 5-FCPhy and 5-FCOle.
[0253] Typical cryo-TEM micrographs of the three different particle
dispersions are shown in FIGS. 5-7.
Example 4
In Vitro Results--Enzyme Assay
[0254] Materials.
[0255] High purity Capecitabine (as solid crystals) was purchased
from Xingcheng Chemphar Co., Ltd, P.R. China. Surfactant drug
conjugates, 5-FCPal, 5-FCPhy and 5-FCOle were synthesized according
to the methods in Example 1. Carboxylesterase from porcine liver
with 131 Units/mg (pH 8.0, 25.degree. C.) was purchased from
Sigma-Aldrich. Millipore water was used to prepare the prodrugs
solutions with appropriate concentrations, while phosphate buffered
saline (pH 7.4, PBS) was used in the preparation of different
concentrations of enzyme. 1 mg/ml for prodrugs 5-FCPal, 5-FCOle and
5-FCPhy, 0.01 mg/ml for Capecitabine
[0256] Prodrugs Solution Preparation and Characterization.
[0257] Stock solution of Capecitabine was prepared by dissolving
pure white Capecitabine into warm water. 1 mg/mL of Capecitabine
solution was then further obtained by diluting the stock solution
with milli-Q water. Colloidosome dispersions were prepared for
5-FCPhy and 5-FCOle according to the method described by Examples
2-3. The ready solution for hydrolysis study was prepared via
mixing the same volume of prodrug 1 mg/ml solution with enzyme
solution, the final concentration of Capecitabine, 5-FCPhy, 5-FCPal
and 5-FCOle for the hydrolysis study were 1.39 mM, 0.878 mM, 0.973
mM and 0.926 mM respectively. Prior to the experiments, prodrug
particles were incubated at 37.degree. C. for at least two weeks to
achieve the equilibrium state of certain phases.
[0258] Enzyme Solution Preparation.
[0259] Stock solution of 10 mg/mL carboxylesterase solution was
prepared by weighting 10 mg of powder enzyme in 1 mL of PBS buffer
and shaking gently by hand. The concentration of the enzyme
solution was adjusted to 1 mg/mL and 0.01 mg/mL by dilution of the
stock solution. These solutions were used for the enzymatic
hydrolysis of the prodrugs and Capecitabine respectively. The final
enzyme concentration for this study was 0.5 mg/mL for the three
prodrugs and 0.005 mg/ml for Capecitabine.
[0260] Hydrolysis Conditions.
[0261] To measure the rate of the enzymatic reaction precisely, the
starting substrate and the enzyme solution were mixed for a defined
time by rapidly mixing together the two stock solutions and
shaking. The time at which enzyme solution was added was taken as
time zero for the hydrolysis reaction. The mixture was maintained
at 37.degree. C. throughout the experiment. The concentration of
the hydrolysis product was then measured at various times using
LC/MS (Finnigan LCQ Series, Thermo Scientific, USA). The time at
which the sample was injected into the LC column was taken to be
the reaction time for such sample. The hydrolysis progress curve
showing the decrease in the concentration of original substrate was
subsequently plotted.
[0262] LC/MS Conditions.
[0263] The concentrations of prodrugs were determined using Thermo
Finnigan LC/MS equipped with an atmospheric pressure chemical
ionization (APCI) probe in positive ion mode. 10 .mu.L of sample
withdrawn from the reaction solution was injected directly into a
Platinum EPS C18 100 .ANG. 5u LC column with the length of 150 mm
and internal diameter of 4.6 mm (Alltech, Australia). A mixture of
30% LCMS grade water and 70% Methanol was employed as mobile phase
with the flow rate of 1 ml/min. The sample after the column
separation was eluted to APCI source (MS full positive scan) to
determine the molecular weight of the substrates. The vaporizer
temperature of the APCI probe was set at 450.degree. C. and the
capillary temperature was operated at 200.degree. C. The sample
temperature was well maintained at 37.degree. C. during the entire
measurement by incubating the sample in the temperature controlled
auto-sampler. Data were acquired and processed with Xcalibur Quan
chromatography software.
Prodrug Hydrolysis.
[0264] The decreased concentration of substrate and the increased
hydrolysis product 5'-dFCyd were detected based on the different
retention time on chromatogram and m/z value in the MS positive
scan. The more hydrophilic compound at the retention time around
1.8 min with m/z of 246.3 was accountable for the hydrolysed
5'-dFCyd, the retention time of 2.14 min with m/z of 360.0 was
assigned as prodrug Capecitabine. The synthesized prodrugs had a
retention time of 7.50 min, 7.02 min, 7.07 min with m/z of 570.3,
514.2, 540.2 corresponding to 5-FCPhy, 5-FCPal and 5-FCOle
respectively. The decreased/increased ratio of reactant/5' dFCyd
was determined by comparing the integration areas of the
corresponding peaks.
[0265] The reactions curves of the four prodrugs with the CES
hydrolysis are plotted and presented in FIGS. 8-11. The enzyme
hydrolysis reaction was plotted as the logarithm of the ratio of
original prodrug concentration to the decreased concentration of
prodrug at a certain incubation time as a function of reaction time
(Ln [S].sub.0/[S] vs T).
[0266] In order to compare the enzymatic activity for each
individual substrate to CES, we calculate a global reaction rate
expressed as a specific activity (SA) (.mu.mol/min/mg enzyme):
SA=.DELTA.[S]/(.DELTA.t[C.sub.CES])
[0267] Here, as is common when discussing the SA, [C.sub.CES] is
the enzyme concentration in mg/ml, .DELTA.[S] is the substrate
concentration change in .mu.mol/ml during .DELTA.t time. Specific
activity values, SA, at various times, t, for each prodrug upon CES
hydrolysis are presented in Table 9 (below).
TABLE-US-00009 TABLE 9 The physicochemical properties and
hydrolysis profile of each prodrug at physiological temperature.
Lyotropic Average phase Particle Specific Activity (SA)
(.mu.mol/ml) at time t (h) Prodrug behaviour size (nm) t = 0.5 t =
1 t = 2 t = 6 t = 12 t = 24 Capecitabine Dissolved in -- 933 733
717 598 -- -- water 5-FCPhy Hexosome 219 -- 1.03 1.30 0.67 0.89
1.15 (H.sub.2) 5-FCPal Solid lipid 661 -- 0.30 0.38 0.33 -- 0.27
particle 5-FCOIe Cubosome 261 -- 4.97 3.70 2.24 2.37 -- (Pn3m)
[0268] It was found from the experiment that Capecitabine was
extremely susceptible to this enzyme. With 5 mg/ml and 0.5 mg/ml
enzyme concentration, Capecitabine hydrolyses to 5-FC within 30
min, so the enzyme concentration was adjusted to 100 times less
than that for prodrugs (50 pg/ml vs 0.5 mg/ml) in order to obtain
sufficient data points for comparison. The enzyme activities for
Capecitabine obtained from three enzymatic concentrations were of
similar value at around .about.600-900 .mu.mol/min/mg enzyme. In
the following, the enzyme activity of 50 pg/ml concentration was
chosen for comparing with other prodrugs. 5-FCPal, having an SA
value of 0.30, is least sensitive to CES hydrolysis. Prodrugs
5-FCPhy and 5-FCOle were about 4 and 10 times higher than 5-FCPal,
respectively.
[0269] The results obtained here clearly indicate that there is
considerable variation in hydrolysis rate between the prodrugs with
different colloidosome nanostructure, and that of Capecitabine. In
general, self-assembled amphiphiles according to the current
invention underwent a much more sluggish hydrolysis than that of
the Capecitabine. Amphiphile prodrugs 5-FCPhy and 5-FCOle showed a
sustained hydrolysis profile, and fully converted to 5'-dFCyd
within 24 hours. 5-FCPal hydrolysed with a very slow rate which
continued towards full conversion for more than 7 days.
[0270] As the four types of prodrugs experience different
microenvironments in water, it is deduced that the hydrolysis
profiles have been influenced by the particle structure
[0271] Liver CES plays an important role in drug and lipid
metabolism. It is noteworthy that different nanostructured
particles of prodrugs showed considerable different enzyme
catalytic properties. Unlike Capecitabine, the present prodrug
particles showed a prolonged and sustained hydrolysis rate upon CES
addition, lasting for days or a week. The main reason for such
sustained hydrolysis is due to the unique physicochemical
properties of the prodrugs. The particle size and surface areas may
also contribute, at least in part, to such different prodrug
hydrolysis properties.
Example 5
In Vivo Toxicity Assessment of Prodrugs in Healthy Mice
[0272] Capecitabine solution and prodrug particle solutions were
administered direct to the stomach of female BALB/c mice (6 weeks
of age) daily. The vehicle containing the stabiliser Poloxamer 407
in water was employed as negative control group. The highest dose,
0.5 mmol/prodrug/mouse/day was used to test if this dosage is toxic
to the healthy mice. Except 5-FCOle which was incubated at
37.degree. C. and injected with pre-warmed syringes throughout the
experiment, the other prodrugs were administered at room
temperature. All test groups contained 6 mice. After 20 days, mice
were sacrificed, and the organs were weighed and prepared for
histological evaluation. No mice died during the study period and
there were no obvious adverse effects on the mice from the
administration of the pro-drugs.
Example 6
In Vivo Experiments with 5-FU and Pro-Drugs
[0273] 90 female BALB/c were injected with 5.times.10.sup.4 4T1
cells (mouse breast cancer cells) in the second mammary fat pad on
the right hand side. The tumour was allowed to form for 6 days
prior to drug administration. On day 6, tumours were palpable in
most of the mice. Approximately 5% of animals did not form tumours
throughout the course of the experiment. Mice were placed into
groups of 6, and 15 different treatments were administered orally
on a daily basis for 21 days. Dosages administered are displayed in
the Table 10 below.
TABLE-US-00010 TABLE 10 Dosage of each animal group (6 mice per
group) High dose Medium dose Low dose Control 360 microL -- -- (1.5
mg/mL) 5-FU 200 microL 100 microL -- (3 mg/mL) (3 mg/mL) Capeci-
240 microL 120 microL 50 microL tabine (15 mg/mL) (15 mg/mL) (15
mg/mL) 5-FCPal 450 microL(225 .times. 2) 225 microL 90 microL (12.6
mg/mL) (12.6 mg/mL) (12.6 mg/mL) 5-FCOle 500 microL(250 .times. 2)
250 microL 100 microL (10.75 mg/mL) (10.75 mg/mL) (10.75 mg/mL)
5-FCPhy 590 microL(295 .times. 2) 295 microL 118 microL (9.6 mg/mL)
(9.6 mg/mL) (9.6 mg/mL)
[0274] The length and breadth of the tumours were measured and
recorded on day 1, 4, 7, 14, and 21 of the drug administration. On
Day 22, mice were euthanized. Blood was collected via cardiac
puncture, followed by removal of the tumour, liver, spleen and
kidney for histological analysis. Lungs were injected with indium
ink prior to removal to allow for quantification of the number of
lung metastasis in each animal.
[0275] The average tumour volume for each treatment group was
calculated and plotted over time and can be seen in the graphs
displayed in FIGS. 12-16. Only data for mice that developed tumours
are included in FIGS. 12-16. No real difference in tumour size was
seen at the early time points, however, by day 14, a dose dependant
trend was visible for all drugs tested. By day 21, the animals
receiving the highest doses of each drug displayed the smallest
overall tumour volume. While the 5FU showed the largest effect, the
toxicity of this drug was apparent in the behaviour of the mice
receiving this drug. Mice were lethargic and appeared generally ill
with significant weight loss, while mice receiving the other
treatments were still moving around quite well despite the large
size of the developing tumours. The 5FC phytanoyl pro-drug showed
the second smallest overall tumour volume at day 21, and animals
appeared active and healthy. Measurements significantly less than
the control are marked by * on the graphs. No significant
difference was seen between different pro-drug treatment groups,
however trends indicate that the highest dose of each drug was the
most effective.
Example 7
In Vivo Experiments with 5-FCPhy and 5-FCOle
[0276] Results of a second in vivo study conducted as in Example 6
are shown in the above graph. In this experiment, only 5-FCPhy and
5-FCOle were administered as these had demonstrated the best
results from the initial study of Example 6. Also, the dosage of
both compounds was increased to 1.5 mmol for each treatment. The
graph in FIG. 18 shows the average 4T1 tumour volume for the four
different treatment groups (control, capecitabine, 5-FCPhy, and
5-FCOle) over the course of the experiment. By day 17, the control
group had the largest tumour, while the 5-FCOle group showed
significantly smaller tumour volumes by day 17. On day 17, animals
from the control group, capecitabine group, 5-FCPhy, and half of
the 5-FCOle group were sacrificed due to tumour size, and treatment
was stopped on the 5-FCOle treatment group. It took a full week for
the 5-FCOle tumours to grow and reach a similar size as the other
treatment groups.
[0277] Images of the tumours and spleens from animals sacrificed at
17 days for the four treatment groups are presented in FIG. 19. The
5-FCOle and 5-FCPhy groups, particularly the 5-FCole group, shows
significantly smaller tumours and normal spleens. Tumours increase
in size with the average control tumour being larger than the other
treatment groups. In addition, spleens are enlarged 2-3 times in
all other groups.
REFERENCE LIST
[0278] Yang, S. C.; Lu, L. F.; Cai, Y.; Zhu, J. B.; Liang, B. W.;
Yang, C. Z., Body distribution in mice of intravenously injected
camptothecin solid lipid nanoparticles and targeting effect on
brain. Journal of Controlled Release 1999, 59, (3), 299-307. [0279]
Jin; Y; et al. Self-assembled drug delivery systems. 1. Properties
and in vitro/in vivo behaviour of acylovir self-assembled
nanoparticles (SAN). International Journal of Pharmaceutics, 2006,
309, 199-207 [0280] Zhang, J. X.; Qiu, L. Y.; Wu, X. L.; Jin, Y.;
Zu, K. J., Temperature-triggered nanosphere formation through
self-assembly of amphiphilic polyphosphazene. Macromolecular
Chemistry and Physics 2006, 207, (14) 1289-1296. [0281] Shimma, N.;
Umeda, I.; Arasaki, M.; Murasaki, C.; Masubuchi, K.; Kohchi, Y.;
Miwa, M.; Ura, M.; Sawada, N.; Tahara, H.; Kuruma, I.; Horii, I.;
Ishitsuka, H., The design and synthesis of a new tumor-selective
fluoropyrimidine carbamate, capecitabine. Bioorganic &
Medicinal Chemistry 2000, 8, (7), 1697-1706. [0282] Wuts, P. G. M.
Greene, T. W. Protective Groups in Organic Synthesis. John Wiley
& Sons, Inc. Hoboken, N.J.: 2007. [0283] Fieser, M; Fieser, L.
F.; Smith, J. G; Reagents for Organic Synthesis, Vols 1-17, John
Wiley and Sons, New York, N.Y., 1991 [0284] Smith, M. B.; March J.;
March's Advanced Organic Chemistry, 5th Ed.; John Wiley and Sons,
New York, N.Y., 2001 [0285] Larock, R. C; Comprehensive Organic
Transformations, 2nd Ed.; John Wiley and Sons, New York, N.Y., 1999
[0286] Drummond, C. J.; Fong, C., Surfactant self-assembly objects
as novel drug delivery vehicles. Current Opinion in Colloid and
Interface Science 2000, 4, (6), 449-456. [0287] Laughlin, R. G.;
Lynch, M. L.; Marcott, C.; Munyon, R. L.; Marrer, A. M.; Kochvar,
K. A., Phase studies by Diffusive Interfacial Transport using
near-infrared analysis for water (DIT-NIR). Journal of Physical
Chemistry B 2000, 104, (31), 7354-7362. [0288] Laughlin, R. G.; The
Aqeuous Phase Behaviour of Surfactants, Academic Press, San Diego,
Calif., 1996. [0289] Small, D., Handbook of Lipid Research. ed. D.
J. Hanahan ed.; Plenum Press, New York: 1986; Vol. 4. [0290]
Cherezov, V.; Fersi, H.; Caffrey, M., Crystallization Screens:
Compatibility with the Lipidic Cubic Phase for in Meso
Crystallization of Membrane Proteins. Biophys J, 2001; 81, 225-242.
[0291] Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng,
A.; Burns, J. L., Novel Process for Producing Cubic Liquid
Crystalline Nanoparticles (Cubosomes). Langmuir, 2001; 17(19),
5748-5756. [0292] Brannon-Peppas L.; Blanchette, J. O; Nanoparticle
and targeted systems for cancer therapy, Advanced Drug Delivery
Reviews, 2004, 56(11), 1649-1659 [0293] Remington: The Science and
Practice of Pharmacy, 21st Ed, University of the Sciences in
Philadelphia (eds), Lippincott Williams & Wilkins,
Philadelphia, Pa., 2005 [0294] Mehnert, W.; Mader, K., Solid lipid
nanoparticles--Production, characterization and applications.
Advanced Drug Delivery Reviews 2001, 47, (2-3), 165-196. [0295] D.
Wells, C. Fong, I. Krodkiewska, C. J. Drummond, J. Phys. Chem. B.,
110, 5112-5119, 2006
[0296] It will be understood that the invention disclosed and
defined in this specification extends to all alternative
combinations of two or more of the individual features mentioned or
evident from the text or drawings. All of these different
combinations constitute various alternative aspects of the
invention.
* * * * *