U.S. patent application number 12/581644 was filed with the patent office on 2010-02-25 for polyaminoacids functionalized wtih alpha-tocopherol, and uses thereof, especially therapeutic uses.
This patent application is currently assigned to Flamel Technologies, S.A.. Invention is credited to Stephanie Angot, Olivier Breyne, You-Ping Chan.
Application Number | 20100048735 12/581644 |
Document ID | / |
Family ID | 29559029 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100048735 |
Kind Code |
A1 |
Chan; You-Ping ; et
al. |
February 25, 2010 |
POLYAMINOACIDS FUNCTIONALIZED WTIH ALPHA-TOCOPHEROL, AND USES
THEREOF, ESPECIALLY THERAPEUTIC USES
Abstract
The invention concerns novel biodegradable polyaminoacid
materials, useful in particular for vectoring active principles(s).
The invention also concerns novel pharmaceutical, cosmetic dietetic
or phytosanitary compositions based on said polyaminoacids. The
invention aims at providing a novel polymer raw material, capable
of being used for vectoring active principles and enabling optimal
fulfillment of all specified requirements: biocompatibility,
biodegradability, easy and inexpensive transformation into
particles vectoring active principles, said particles being
themselves capable of forming colloidal suspensions, of being
easily associated with numerous active principles, and of releasing
said active principles in vivo. Therefor, the present invention
concerns first of all amphiphilic polyaminoacids comprising
aspartic acid units and/or glutamic acid units, characterized in
that at least part of said units carry grafts, including at least
one alph.alpha.-tocopherol motif, for example (polyglutamate or
polyaspartate grafted with alph.alpha.-tocopherol of synthetic or
natural origin).
Inventors: |
Chan; You-Ping; (Ternay,
FR) ; Angot; Stephanie; (Gradignan, FR) ;
Breyne; Olivier; (Lyon, FR) |
Correspondence
Address: |
PATTON BOGGS LLP
8484 WESTPARK DRIVE, SUITE 900
MCLEAN
VA
22102
US
|
Assignee: |
Flamel Technologies, S.A.
Venissieux Cedex
FR
|
Family ID: |
29559029 |
Appl. No.: |
12/581644 |
Filed: |
October 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10516733 |
Oct 3, 2005 |
|
|
|
PCT/FR03/50003 |
Jun 3, 2003 |
|
|
|
12581644 |
|
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Current U.S.
Class: |
514/772.1 ;
528/314; 528/322 |
Current CPC
Class: |
C08G 69/10 20130101;
A61K 47/34 20130101; A61K 2800/57 20130101; C08G 69/48 20130101;
A61P 43/00 20180101; A61K 8/678 20130101; A61K 9/5146 20130101;
A61Q 19/00 20130101; A61K 8/88 20130101 |
Class at
Publication: |
514/772.1 ;
528/322; 528/314 |
International
Class: |
A61K 47/34 20060101
A61K047/34; C08G 73/10 20060101 C08G073/10; A61P 43/00 20060101
A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 2002 |
FR |
02 07008 |
Claims
1. Method of making a polyamino acid where at least some polyamino
acid units bear side chains comprising at least one
.alpha.-tocopherol unit, said method comprising: i) manufacturing
polyamino acid polymer comprising at least one aspartic unit and/or
glutamic unit; ii) dissolving the polyamino acid polymer in a first
solvent to form a mixture; iii) adding .alpha.-tocopherol, at least
one nucleophilic catalyst and at least one carbodiimide to said
mixture to form a reaction mixture; iv) forming a precipitated
polymer by reacting the reaction mixture with a precipitating
agent, where at least some of the polyamino acid units have side
chains comprising at least one .alpha.-tocopherol unit.
2. The method of claim 1, wherein said method further comprises: v)
purifying the precipitated polymer by dissolving the precipitated
polymer in a second solvent, and precipitating the polymer out of
solution to form a precipitated polymer.
3. The method of claim 2, wherein said first solvent and second
solvent are the same.
4. The method of claim 1, wherein said first solvent is
dimethylformamide.
5. The method of claim 1, wherein said at least one nucleophilic
catalyst is dimethylaminopyridine.
6. The method of claim 1, wherein said at least one carbodiimide is
diisopropylcarbodiimide.
7. The method of claim 1, wherein said precipitating agent
comprises sodium chloride and hydrochloric acid.
8. The method of claim 1, wherein said manufacturing of
.alpha.-L-polyamino acid polymer comprises polymerization of
NCAGLuOMe followed by hydrolysis.
9. The method of claim 1, wherein said precipitated polymer has the
general formula (I) below: ##STR00006## in which: R.sup.1
represents H, a linear C2 to C10 or branched C3 to C10 acyl group,
or a pyroglutamate; R represents H, a C2 to C10 linear or C3 to C10
branched alkyl, benzyl or a terminal amino acid unit; R.sup.3 is H
or a cationic species; R.sup.4 represents a direct bond or a
"spacer" based on 1 to 4 amino acid units; A independently
represents a --CH.sub.2-- (aspartic unit) or --CH.sub.2--H.sub.2--
(glutamic unit) radical; and T represents an .alpha.-tocopherol
unit.
10. The method of claim 1, wherein the polyamino acid has a
.alpha.-L-configuration.
11. The method of claim 10, wherein the polamino acids are
.alpha.-L-glutamate or .alpha.-L-glutamic homopolymers.
12. The method of claim 10, wherein the polamino acids are
.alpha.-L-aspartate or .alpha.-L-aspartic homopolymers.
13. The method of claim 10, wherein the polamino acids are
.alpha.-L-aspartate .alpha.-L-glutamate or .alpha.-L-aspartic
.alpha.-L-glutamic copolymers.
14. The method of claim 1, wherein the molar grafting with
.alpha.-tocopherol is between 3% and 70%.
15. The method of claim 1, wherein the molar grafting with
.alpha.-tocopherol is between 5% and 50%.
16. Method of making a polyamino acid where at least some units of
the polyamino acids bear side chains comprising at least one
.alpha.-tocopherol unit, said method comprising: i) manufacturing
polyamino acid polymer comprising at least one aspartic unit and/or
glutamic unit; ii) forming a first mixture by adding the polyamino
acid polymer to a first solvent at a temperature of
.gtoreq.40.degree. C.; iii) reducing the temperature of the first
mixture when the polyamino acid polymer dissolves; iv) dissolving
.alpha.-tocopherol, at least one nucleophilic catalyst and at least
one carbodiimide in a second solvent; v) adding ii), iv), at least
one nucleophilic catalyst and at least one carbodiimide, to form a
reaction mixture; vi) reacting the precipitating agent after a
pre-determined amount of time to form precipitated polymer, where
at least some units of the polyamino acids bear side chains
comprising at least one .alpha.-tocopherol unit.
17. The method of claim 16, wherein said first solvent and/or
second solvent is dimethylformamide.
18. The method of claim 16, wherein said at least one nucleophilic
catalyst is dimethylaminopyridine.
19. The method of claim 16, wherein said at least one carbodiimide
is diisopropylcarbodiimide.
20. The method of claim 16, wherein said precipitating agent
comprises sodium chloride and hydrochloric acid.
21. The method of claim 16, wherein said precipitating agent has
the general formula (I) of: ##STR00007## in which: R.sup.1
represents H, a linear C2 to C10 or branched C3 to C10 acyl group,
or a pyroglutamate; R.sup.2 represents H, a C2 to C10 linear or C3
to C10 branched alkyl, benzyl or a terminal amino acid unit;
R.sup.3 is H or a cationic species selected from the group
consisting of: sodium metallic cations, potassium metallic cations,
calcium metallic cations, magnesium metallic cations, amine-based
cations, oligoamine-based cations, cations based on polyamine,
cations based on lysine, cations based on arginine, polylysine and
oligolysine; R.sup.4 represents a direct bond or a "spacer" based
on 1 to 4 amino acid units; A independently represents a
--CH.sub.2-- (aspartic unit) or --CH.sub.2--H.sub.2-- (glutamic
unit) radical; n/(n+m) ranges from 0.5 to 100 mol %; n+m ranges
from 3 to 1000; and T represents an .alpha.-tocopherol unit.
22. Method of preparing a pharmaceutical composition comprising a
polyamino acid where at least some units of the polyamino acid bear
side chains comprising at least one .alpha.-tocopherol unit, said
method comprising: i) manufacturing polyamino acid polymer
comprising at least one aspartic unit and/or glutamic unit; ii)
forming a first mixture by dissolving the polyamino acid polymer in
a first solvent; iii) forming a second mixture by dissolving
.alpha.-tocopherol, at least one nucleophilic catalyst and at least
one carbodiimide in a second solvent; iv) combining ii) with iii)
to form a reaction mixture; v) reacting iv) with a precipitating
agent to form precipitated polymer; vi) incubating the precipitated
polymer and at least one active principle together in solution
under conditions such that a fraction of the at least one active
principle associates with the precipitated polymer; vii) separating
the at least one active principle that is associated with the
precipitated polymer from the unassociated active principle and
precipitated polymer, whereby the pharmaceutical composition
comprises the at least one active principle that is associated with
the precipitated polymer, and where at least some units of the
polyamino acid bear side chains comprising at least one
.alpha.-tocopherol unit.
23. The method of claim 22, wherein the polamino acids are
.alpha.-L-glutamate or .alpha.-L-glutamic homopolymers.
24. The method of claim 22, wherein the polamino acids are
.alpha.-L-aspartate or .alpha.-L-aspartic homopolymers.
25. The method of claim 22, wherein the polamino acids are
.alpha.-L-aspartate .alpha.-L-glutamate or .alpha.-L-aspartic
.alpha.-L-glutamic copolymers.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/513,733, filed Dec. 6, 2004, which is a National Stage
Application of PCT Application No. PCT/FR2003/50003, filed Jun. 3,
2003, which claims priority to the French Application No. FR 02
07008, filed Jun. 7, 2002, both of which are herein incorporated by
reference in their entirety.
[0002] The present invention relates to novel materials based on
biodegradable polyamino acids, which are useful especially for the
vectorization of active principal(s) (AP).
[0003] The invention is also directed toward novel pharmaceutical,
cosmetic, dietetic or plant-protection compositions based on these
polyamino acids. These compositions may be of the type allowing the
vectorization of AP and preferably being in the form of emulsions,
micelles, particles, gels, implants or films.
[0004] The APs under consideration are advantageously biologically
active compounds that may be administered to an animal or human
body via the oral, parenteral, nasal, vaginal, ocular,
subcutaneous, intravenous, intramuscular, intradermal,
intraperitoneal, intracerebral, buccal, etc. route.
[0005] The APs more particularly concerned by the invention, but
not limiting the invention thereto, are proteins, glycoproteins,
peptides, polysaccharides, lipopolysaccharides, oligonucleotides,
polynucleotides and organic molecules. However, they may also be
cosmetic products or plant-protection products, such as herbicides,
insecticides, fungicides, etc.
[0006] In the field of vectorization of active principles,
especially medicinal active principles, there is a need, in many
cases: [0007] to protect them against degradation (hydrolysis,
precipitation on site, enzymatic digestion, etc.) until they reach
their site of action, [0008] and/or to control their rate of
release so as to maintain a constant level over a given period,
[0009] and/or to convey them (while protecting them) to the site of
action. To these ends, several types of polymers have been studied
and some are even commercially available. Mention may be made, for
example, of polymers of the polylactic, polylactic-glycolic,
polyoxyethyleneoxypropylene, polyamino acid or polysaccharide type.
These polymers constitute starting materials for manufacturing, for
example, bulk implants, microparticles, nanoparticles, vesicles,
micelles or gels. Besides the fact that these polymers must be
suitable for manufacturing such systems, they must also be
biocompatible, nontoxic, nonimmunogenic and cost-effective, and
they should be easy to eliminate from the body and/or
biodegradable. As regards this last aspect, it is furthermore
essential that the biodegradation in the body should generate
nontoxic products.
[0010] By way of illustration of the prior art concerning polymers
used as starting materials for making AP vectorization systems,
various patents, patent applications or scientific articles are
mentioned below.
[0011] U.S. Pat. No. 4,652,441 describes polylactide microcapsules
encapsulating the hormone LH-RH. These microcapsules are produced
by preparing a water-in-oil-in-water emulsion comprising an aqueous
inner layer containing the hormone, a substance (gelatin) for
fixing said hormone, a polylactide oily layer, and also an aqueous
outer layer (polyvinyl alcohol). The AP is released over a period
of more than 2 weeks after subcutaneous injection.
[0012] U.S. Pat. No. 6,153,193 describes compositions based on
amphiphilic poly(oxyethylene)-poly(oxypropylene)micelles, for the
vectorization of anticancer agents such as adriamycin.
[0013] Akiyoshi et al. (J. Controlled Release 1998, 54, 313-320)
describe pullulans that have been made hydrophobic by grafting
cholesterol, and which form nanoparticles in water. These
nanoparticles, which are capable of reversibly complexing with
insulin, form stable colloidal suspensions.
[0014] U.S. Pat. No. 4,351,337 describes amphiphilic copolyamino
acids based on leucine and glutamate, which may be used in the form
of implants or microparticles for the controlled release of active
principles. Said active principles are released over a very long
period that depends on the rate of degradation of the polymer.
[0015] U.S. Pat. No. 4,888,398 describes polymers based on
polyglutamate or polyaspartate, and optionally polyleucine, with
pendent groups of alkyloxycarbonylmethyl type, placed randomly on
the polyamino acid chain. These polyamino acids, grafted with side
groups, e.g. methoxycarbonylmethyl, may be used in the form of
biodegradable implants containing a sustained-release AP.
[0016] U.S. Pat. No. 5,904,936 describes nanoparticles obtained
from a polyleucine-polyglutamate block polymer, which are capable
of forming stable colloidal suspensions capable of spontaneously
associating with biologically active proteins without denaturing
them. Said proteins may then be released in vivo in a controlled
manner, over a long period.
[0017] Patent application WO 00/30618 describes nanoparticles
obtained from a poly(sodium glutamate) (polymethyl, ethyl,
hexadecyl or dodecyl glutamate) block polymer, which are capable of
forming stable colloidal suspensions capable of spontaneously
associating with biologically active proteins without denaturing
them. Said proteins may then be released in vivo in a controlled
manner, over a long period.
[0018] These amphiphilic copolyamino acids are modified by the
presence of a hydrophobic alkyl side chain.
[0019] U.S. Pat. No. 5,449,513 describes amphiphilic block
copolymers comprising a polyoxyethylene block and a polyamino acid
block, for example poly(.beta.-benzyl-L-aspartate). These
polyoxyethylene-polybenzylaspartate polymers form micelles that are
capable of encapsulating hydrophobic active molecules such as
adriamycin or indomethacin.
[0020] Patent application WO 99/61512 describes polylysines and
polyornithines functionalized with a hydrophobic group (palmitic
acid linked to polylysine or ornithine and a hydrophilic group
(polyoxyethylene). These polymers, for example polylysine grafted
with polyoxyethylene and palmitoyl chains, form, in the presence of
cholesterol, vesicles capable of encapsulating doxorubicin or
DNA.
[0021] It is moreover known practice to employ vitamin E
derivatives, and more specifically .alpha.-tocopherol, to construct
AP vectorization systems.
[0022] Natural vitamin E consists of a mixture of compounds known
as tocopherols (see Burton and Ingold, Acc. Chem. Res. 1986, 19,
194-201) and, in this mixture, the .alpha.-tocopherol derivative is
largely in majority amount. Vitamin E and some of its derivatives
are nowadays used as a source of vitamin or as antioxidant in foods
and cosmetic products. For these common uses, the vitamin E is
found in its D-.alpha.-tocopherol, form (its natural form) or in
its D-L-.alpha.-tocopherol form (racemic and synthetic form). These
two products are considered as being essentially nontoxic at doses
considerably higher than therapeutic doses. The structure of
.alpha.-tocopherol is as follows:
##STR00001##
[0023] The chiral positions are marked with an asterisk. The
natural form has the configurations R,R,R and the synthetic form is
a mixture in which the chiral carbons are independently R or S.
[0024] As regards the vitamin E derivatives used in the field of
vectorization of active principles, there is, at the present time
and to the inventors' knowledge, no .alpha.-tocopherol-based
polymer product, with the exception of polymers of polyoxyethylene
type, one end of which is grafted with .alpha.-tocopherol-succinate
groups. Polyethylene glycol grafted with .alpha.-tocopherol
succinate at the end of the chain (PEGylated vitamin E) is moreover
available on the market, sold under the name TPGS 1000 by the
company Eastman Chemical Ltd). This product, patented in 1954 (U.S.
Pat. No. 2,680,749), is nowadays used as a source of oral-route
vitamin E. This polymer, and similarly .alpha.-tocopherol-succinate
and unmodified .alpha.-tocopherol, have been proposed for the
vectorization of active principles.
[0025] U.S. Pat. No. 5,869,703 describes similar compounds in which
the polyoxyethylene chain comprise .alpha.-tocopherol at one end
and a (meth)acrylic residue at its other end. These
.alpha.-tocopherol derivatives are used to prepare stable
amphiphilic vesicles (liposomes), used directly in cosmetic
applications.
[0026] Patent application WO 00/71163 describes formulations based
on polyethylene glycol grafted with .alpha.-tocopherol-succinate at
the end of the chain (TPGS 1000) and on .alpha.-tocopherol, for the
solubilization of paclitaxel (anticancer product). At the present
time, the toxicity associated with the polyoxyethylene part is not
known, and it is known that polyoxyethylene is not degraded in
vivo. Furthermore, this compound contains only one
.alpha.-tocopherol unit per polymer chain and it has properties in
solution similar to those of surfactants. In any case, the use of
this product for vectorization would lead to sparingly stable
polymer-active principle combinations.
[0027] Patent EP 0 243 446 describes the use of .alpha.-tocopherol
(hemi) succinate (organic acid derivative of .alpha.-tocopherol)
for the manufacture of vesicles, in combination with an amine salt.
These vesicles may be used for the encapsulation of various active
principles including small molecules, peptides and proteins. In
general, it is indicated in said patent that the organic acid may
be an amino acid or a polyamino acid. However, no details are given
in this respect. Only .alpha.-tocopherol (hemi) succinates are
illustrated.
[0028] Thus, even though there are a great many technical solutions
in the prior art, developed and proposed for the vectorization of
medicinal active principles, meeting all the requirements is
difficult to achieve, and remains unsatisfactory.
[0029] In this context, one of the essential objectives of the
present invention is to provide a novel polymeric starting
material, which may be used for AP vectorization and which can
optimally satisfy all the specification details: [0030]
biocompatibility, [0031] biodegradability, [0032] ability to be
converted easily and economically into active principle
vectorization particles, [0033] these particles themselves being
capable: [0034] of forming stable aqueous colloidal suspensions,
[0035] of readily associating with many active principles, [0036]
and of releasing these active principles in vivo.
[0037] This objective, among others, is achieved by the present
invention, which relates firstly to amphiphilic polyamino acids
comprising aspartic units and/or glutamic units, characterized in
that at least some of these units bear grafts comprising at least
one .alpha.-tocopherol unit.
[0038] These novel polymers have a biodegradable skeleton based on
polaymino acids bearing side chains comprising .alpha.-tocopherol.
These polymers have association and/or encapsulation properties
that are surprising in comparison with similar products and, what
is more, they are readily degraded in the presence of enzymes.
[0039] The Applicant has, to its merit, had the idea of combining,
in an entirely judicious and advantageous manner, particular
biodegradable polyAsp and/or polyGul polyamino acids with grafts
based on .alpha.-tocopherol (vitamin E), for the vectorization of
AP.
[0040] For the purposes of the invention, the term "polyamino acid"
covers not only oligoamino acids comprising from 2 to 20 amino acid
units, but also polyamino acids comprising more than 20 amino acid
units.
[0041] Preferably, the polyamino acids according to the present
invention are oligomers or homopolymers comprising glutamic or
aspartic amino acid repeating units or copolymers comprising a
mixture of these two types of amino acid units, said units being
partially substituted with grafts comprising .alpha.-tocopherol.
The units under consideration in these polymers are amino acids
having the D, L or D,L configuration and are linked via their
.alpha.- or .gamma.-positions for the glutamate or glutamic unit
and the .alpha.- or .beta.-position for the aspartic or aspartate
unit.
[0042] The preferred amino acid units are those having the L
configuration and a bond of .alpha. type.
[0043] Even more preferably, the polyamino acids according to the
invention correspond to the general formula (I) below:
##STR00002##
in which: [0044] R.sup.1 represents H, a linear C2 to C10 or
branched C3 to C10 acyl group, or a pyroglutamate; [0045] R.sup.2
represents H, a C2 to C10 linear or C3 to C10 branched alkyl,
benzyl or a terminal amino acid unit; [0046] R.sup.3 is H or a
cationic species preferably selected from the group comprising:
[0047] metallic cations advantageously chosen from the subgroup
comprising sodium, potassium, calcium and magnesium, [0048] organic
cations advantageously chosen from the sub group comprising: [0049]
amine-based cations, [0050] oligoamine-based cations, [0051]
cations based on polyamine (polyethyleneimine being particularly
preferred), [0052] cations based on amino acid(s) advantageously
chosen from the class comprising cations based on lysine or
arginine, [0053] or cationic polyamino acids advantageously chosen
from the subgroup comprising polylysine or oligolysine; [0054]
R.sup.4 represents a direct bond or a "spacer" based on 1 to 4
amino acid units; [0055] A independently represented a --CH.sub.2--
(aspartic unit) or --CH.sub.2--CH.sub.2-- (glutamic unit) radical;
[0056] n/(n+m) is defined as the molar degree of grafting and
ranges from 0.5 to 100 mol %; [0057] n+m ranges from 3 to 1000 and
preferably between 30 and 300; [0058] T represents an
.alpha.-tocopherol unit. For these common uses, vitamin E is found
in its D-.alpha.-tocopherol form (its natural form) or in its
D,L-.alpha.-tocopherol form (racemic and synthetic form). These two
products are considered as being essentially nontoxic at doses
considerably higher than therapeutic doses. In the context of the
invention, these two forms of .alpha.-tocopherol are preferred.
[0059] The .alpha.-tocopherol is of natural or synthetic
origin.
[0060] According to a first embodiment of the invention, the
polyamino acids are .alpha.-L-glutamate or .alpha.-glutamic
homopolymers.
[0061] According to a second embodiment of the invention, the
polyamino acids are .alpha.-L-aspartate or .alpha.-L-aspartic
homopolymers.
[0062] According to a third embodiment of the invention, the
polyamino acids are .alpha.-L-aspartate .alpha.-L-glutamate or
.alpha.-L-aspartic/.alpha.-L-glutamic copolymers.
[0063] Advantageously, the distribution of the aspartic and/or
glutamic units bearing grafts comprising at least one
.alpha.-tocopherol unit is such that the polymers thus composed are
either random, or block type or of multi-block type.
[0064] According to another mode of definition, the polyamino acids
according to the invention have a molar mass of between 2000 and
100 000 g/mol and preferably between 5000 and 40 000 g/mol.
[0065] It is moreover preferable for the molar degree of grafting
with .alpha.-tocopherol of the polyamino acids according to the
invention to be between 3% and 70% and preferably between 5% and
50%.
[0066] The polyamino acids of the invention are, remarkably,
capable of being used in several ways depending on the degree of
grafting. The methods for forming a polymer for the encapsulation
of an active principle in the various forms targeted by the
invention are known to those skilled in the art. For further
details, reference may be made, for example, to these few
particularly pertinent references:
[0067] "Microspheres, Microcapsules and Liposomes, Vol. 1.
Preparation and chemical applications" Ed. R. Arshady, Citus Books
1999. ISBN: 0-9532187-1-6.
[0068] "Sustained-Release Injectable Products" Ed. J. Senior and M.
Radomsky, Interpharm Press 2000. ISBN: 1-57491-101-5.
[0069] "Colloidal Drug Delivery Systems" Ed. J. Kreuter, Marcel
Dekker, Inc. 1994. ISBN: 0-8247-9214-9.
[0070] "Handbook of Pharmaceutical Controlled Release Technology"
Ed. D. L. Wise, Marcel Dekker, Inc. 2000. ISBN: 0-8247-0369-3.
[0071] The polyamino acids are also extremely advantageous as a
result of the fact that, at a relatively low degree of grafting of
about from 3% to 10%, they form in water at pH 7.4 (for example
with a phosphate buffer) colloidal suspensions or gels depending on
the polymer concentration. Furthermore, the polyamino acid
particles forming the dispersed phase of the colloidal suspension
can readily associate with active principles such as proteins,
peptides or small molecules. The preferred forming operation is
that described in patent application WO 00/30618 by the Applicant,
which consists in dispersing the polymer in water and incubating
the solution in the presence of an AP. This solution can then be
filtered through a 0.02 .mu.m filter and then injected directly
into a patient.
[0072] Above a degree of grafting of 10%, the polymer can form
microparticles capable of associating or of encapsulating APs. In
this context, the forming of the microparticles may take place by
codissolving the AP and the polymer in a suitable organic solvent,
and the mixture is then precipitated from water. The particles are
then recovered by filtration and can then be used for an oral
administration (in the form of a gel capsule, in compacted and/or
coated form, or alternatively in a form dispensed in an oil) or
parenterally after redispersing in water.
[0073] At degrees of grafting of greater than 30%, the redispersion
of the polymer in aqueous phase becomes more difficult due to the
smaller amount of ionizable carboxylate functions, and the polymer
precipitates. In this case, the polymer can be dissolved in a
biocompatible solvent such as N-methylpyrrolidone or a suitable oil
such as Miglyol.RTM., and then injected intramuscularly or
subcutaneously or into a tumor. The diffusion of the solvent or of
the oil results in precipitation of the polymer at the site of
injection and thus forms a deposit. These deposits then provide
controlled release by diffusion and/or erosion and/or hydrolytic or
enzymatic degradation of the polymer.
[0074] In general, the polymers of the invention, in neutral or
ionized form, may be used alone or in a liquid, solid or gel
composition and in an aqueous or organic medium.
[0075] It should be understood that the polymer based on polyamino
acids contains carboxylic functions that are either neutral (COOH
form) or ionized, depending on the pH and composition. For this
reason, the solubility in an aqueous phase depends directly on the
content of free COOH (not grafted with vitamin E) and on the pH. In
aqueous solution, the counter-cation may be a metallic cation such
as sodium, calcium or magnesium or an organic cation such as
triethanolamine, tris (hydroxymethyl) aminomethane or a polyamine
such as polyethyleneimine.
[0076] The polymers of the invention are obtained via methods known
to those skilled in the art. The polyamino acids may be obtained in
at least two ways: [0077] grafting of .alpha.-tocopherol onto a
polyamino acid, or [0078] polymerization of NCA derivatives of
.alpha.-tocopherol, followed by a selective hydrolysis.
[0079] In the first case, a polyamino acid, homopolyglutamate,
homopolyaspartate or a glutamate/aspartate copolymer, in block,
multiblock or random form, is prepared, for example, according to
standard methods.
[0080] To obtain polyamino acids of .alpha. type, the technique
most commonly used is based on the polymerization of N-carboxyamino
acid anhydrides (NCA), described, for example, in the article
"Biopolymers, 1976, 15, 1869" and in the book by H. R. Kricheldorf
"alpha-Aminoacid-N-carboxy Anhydride and related Heterocycles",
Springer Verlag (1987). The NCA derivatives are preferably
NCA-O-Me, NCA-O-Et or NCA-O-Bz derivatives (Me=methyl, Et=ethyl and
Bz=benzyl). The polymers are then hydrolyzed under suitable
conditions to give the polymer in its acid form. These methods are
inspired from the description given in patent FR 2 801 226 from the
Applicant. A certain number of polymers that may be used according
to the invention, for example, of poly-(.alpha.-L-aspartic), poly
(.alpha.-L-glutamic), poly (.alpha.-D-glutamic) and poly
(.gamma.-L-glutamic) type of variable masses are commercially
available. The polyaspartic of the .alpha.-.beta. type is obtained
by condensing aspartic acid (to obtain a poly-succinimide),
followed by a basic hydrolysis (see Tomida et al. Polymer 1997, 38,
4733-36).
[0081] The coupling of .alpha.-tocopherol with an acid function is
readily performed by reacting the polyamino acid with vitamin E in
the presence of a carbodiimide as coupling agent and, preferably, a
catalyst such as 4-dimethyl-aminopyridine, and in a suitable
solvent such as dimethylformamide (DMF), N-methylpyrrolidone (NMP)
or dimethyl sulfoxide (DMSO). The carbodiimide is, for example,
dicyclohexylcarbodiimide or diisopropylcarbodiimide. The degree of
grafting is controlled chemically via the stoichiometry of the
constituents and reagents, or the reaction time.
[0082] In the second case, an NCA derivative of .alpha.-tocopherol
having the structure below is synthesized. The synthesis is
analogous to that described for stearyl glutamate
N-carboxyanhydride by Poche et al. Macromolecules 1995, 28,
6745-53.
##STR00003##
[0083] The NCA derivative of .alpha.-tocopherol-glutamate is then
copolymerized with, for example, benzylglutamate NCA and, to obtain
the glutamate or glutamic functions, a selective hydrolysis
reaction of the benzyl functions is performed in a mixture of
trifluoroacetic acid and hydrobromic acid at room temperature. It
should be noted that this second synthetic route makes it possible
easily to prepare random, block or multiblock copolymers simply by
modifying the order of addition of the monomers.
[0084] The coupling of vitamin E via a spacer consisting of 1 to 4
amino acids may be performed via successive reactions of vitamin E
with suitably protected amino acids, which are then deprotected to
have a graftable amine function on the polymer, or by reaction with
an oligopeptide. For example, the synthesis of an
.alpha.-tocopherol with a leucine unit is performed according to a
general method well known to those skilled in the art and according
to the following scheme:
##STR00004##
[0085] It should be noted that the direct grafting of
.alpha.-tocopherol onto the polymer takes place via an ester
function, whereas in the case of the presence of a spacer based on
amino acid(s), it takes place via an amide function. As for the
preparation of an ester bond, the amide bond may be formed in the
same manner using a standard coupling agent such as a
dialkylcarbodiimide.
[0086] According to one variant of the invention, the polyamino
acids with which it is concerned not only bear .alpha.-tocopherol
grafts but also, per molecule, at least one graft of polyalkylene
glycol type linked to a glutamate and/or aspartate unit, and
preferably of formula (II) below:
##STR00005## [0087] in which [0088] R'.sup.4 represents a direct
bond or a "spacer" based on 1 to 4 amino acid units; [0089] X is a
hetero atom chosen from the group comprising oxygen, nitrogen and
sulfur; [0090] R.sup.5 and R.sup.6 independently represent H or a
linear C1 to C4 alkyl. [0091] n ranges from 3 to 1000. Preferably,
the polyalkylene glycol is a polyethylene glycol.
[0092] According to another preferred characteristic of the
invention, the molar percentage of grafting of the polyalkylene
glycol ranges from 1% to 30%.
[0093] The grafting of these pendent side groups (II) is performed
in a manner that is known per se and according to techniques within
the capability of a person skilled in the art, for example by
forming amide, ester or thioester bonds with the carboxyls of
glutamate and/or aspartate monomers. These techniques may
especially be those used for the grafting of .alpha.-tocopherol
onto a polyamino acid skeleton, said techniques being described in
the present patent application.
[0094] According to another of its aspects, the invention is
directed toward a pharmaceutical, cosmetic, dietetic or
plant-protection composition comprising at least one of the
polyamino acids as defined above.
[0095] According to one advantageous arrangement of the invention,
this composition comprises, besides .alpha.-tocopherol, at least
one active principle, which may be a therapeutic, cosmetic,
dietetic or plant-protection active principle.
[0096] Preferably, the active principle is a protein, a
glycoprotein, a polysaccharide, a liposaccharide, an
oligonucleotide, a polynucleotide or a peptide.
[0097] Even more preferably, the active principle is a hydrophobic,
hydrophilic or amphiphilic organic "small molecule".
[0098] According to the present description, the term "small
molecule" especially denotes nonprotein molecules.
[0099] This composition may be in the form of nanoparticles,
microparticles, solutions, emulsions, suspensions, gels, micelles,
implants, powders or films.
[0100] According to one of its particularly preferred forms, the
composition, containing or not containing active principle(s), is a
stable colloidal suspension of nanoparticles and/or microparticles
and/or micelles of polyamino acids, in an aqueous phase.
[0101] The composition according to the invention, when it is
pharmaceutical, may be administered via the oral, parenteral,
nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular,
intradermal, interperitoneal, intracerebral or buccal route.
[0102] It may also be envisioned for the composition to be in the
form of a solution in a biocompatible solvent, capable of being
injected subcutaneously, intramuscularly or into a tumor.
[0103] According to another variant, the composition according to
the invention is formulated such that it is injectable and such
that it is capable of forming a deposit at the site of
injection.
[0104] The invention is also directed toward compositions
comprising polyamino acids according to the invention and active
principles, and which are capable of being used for the
preparation: [0105] of medicinal products, in particular for oral,
nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular,
intradermal, intraperitoneal or intracerebral administration, the
active principles of these medicinal products possibly being,
especially, proteins, glycoproteins, proteins linked to one or more
polyalkylene glycol chains {for example polyethylene glycol (PEG),
in which case they are referred to as "PEGylated" proteins},
peptides, polysaccharides, liposaccharides, oligonucleotides,
polynucleotides and hydrophobic, hydrophilic or amphiphilic organic
small molecules; [0106] and/or nutrients; [0107] and/or cosmetic or
plant-protection products.
[0108] According to yet another of its aspects, the invention is
directed toward a process for preparing: [0109] medicinal products,
in particular for oral, nasal, vaginal, ocular, subcutaneous,
intravenous, intramuscular, intradermal, intraperitoneal or
intracerebral administration, the active principles of these
medicinal products possibly being, especially, proteins,
glycoproteins, proteins linked to one or more polyalkylene glycol
chains {for example polyethylene glycol (PEG), in which case they
are referred to as "PEGylated" proteins}, peptides,
polysaccharides, liposaccharides, oligonucleotides, polynucleotides
and hydrophobic, hydrophilic or amphiphilic organic small
molecules; [0110] and/or nutrients; [0111] and/or cosmetic or
plant-protection products; this process being characterized in that
it consists essentially in using at least one polyamino acid as
defined above and/or the composition itself also described
above.
[0112] As indicated above, the techniques for associating one or
more APs with the .alpha.-tocopherol-grafted polyamino acids
according to the invention are described especially in patent
application WO 00/30618.
[0113] The invention also relates to a therapeutic treatment method
that consists essentially in administering the composition as
described in the present description, via the oral, parenteral,
nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular,
intradermal, intraperitoneal, intracerebral or buccal route.
[0114] According to one particular embodiment, the therapeutic
treatment method consists essentially in using a composition as
described above in the form of a solution in a biocompatible
solvent, and then injecting it subcutaneously, intramuscularly or
into a tumor, preferably such that is forms a deposit at the site
of injection.
[0115] As examples of APs that may be associated with the polyamino
acids according to the invention, whether or not they are in the
form of (nano or micro) particles, mention may be made of: [0116]
proteins such as insulin, interferons, growth hormones,
interleukins, erythropoietin or cytokines; [0117] peptides such as
leuprolide or cyclosporin; [0118] small molecules such as those
belonging to the anthracyclin, taxoid or camptothecin family;
[0119] and mixtures thereof.
[0120] The invention will be understood more clearly and its
advantages and implementation variants will emerge more clearly
from the examples that follow, which describe the synthesis of
.alpha.-tocopherol-grafted polyamino acids, their conversion into
an AP vectorization system (stable aqueous suspension of
nanoparticles) and demonstration of the capacity of such a system
to associate with APs (small organic molecules, proteins, etc.) to
form pharmaceutical compositions.
EXAMPLE 1
Polymer P1
[0121] Synthesis of a Polyglutamate Grafted with .alpha.-Tocopherol
of Synthetic Origin
[0122] The .alpha.-L-polyglutamate polymer, with a mass equivalent
to about 10 000 relative to a polyoxyethylene standard, is obtained
by polymerization of NCAGLuOMe, followed by a hydrolysis, as
described in patent application FR 2 801 226. 5.5 g of this
.alpha.-L-polyglutamate polymer are dissolved in 92 ml of
dimethylformamide (DMF), by heating at 40.degree. C. for 2 hours.
Once the polymer has dissolved, the temperature is allowed to
return to 25.degree. C. and 1.49 g of D,L,.alpha.-tocopherol
(>98%, obtained from Fluka.RTM.) predissolved in 6 ml of DFM,
0.09 g of 4-dimethylaminopyridine predissolved in 6 ml of DMF and
0.57 g of diisopropylcarbodiimide predissolved in 6 ml of DMF are
successively added. After stirring for 8 hours at 25.degree. C.,
the reaction medium is poured into 800 ml of water containing 15%
sodium chloride and hydrochloric acid (pH 2). The precipitated
polymer is then recovered by filtration, washed with 0.1N
hydrochloric acid and then washed with water. The polymer is then
redissolved in 75 ml of DMF and then reprecipitated from water
containing, as previously, salt and acid at pH2. After washing
twice with water, the polymer is washed several times with
diisopropyl ether. The polymer is then oven-dried under vacuum at
40.degree. C. A yield of about 85% is obtained.
[0123] The degree of grafting estimated by proton NMR is about 7.8%
and an HPLC analysis reveals a residual tocopherol content of less
than 0.3%.
[0124] Mw (measured by GPC, eluting with NMP)=17 500 g/mol (as
polymethyl methacrylate equivalent).
EXAMPLES 2, 3, 4 AND 5
Synthesis of Polymers P2, P3, P4 and P5
[0125] Polymers containing variable amounts of tocopherol are
prepared in the same manner.
TABLE-US-00001 TABLE 1 Polymer .alpha.-Tocopherol Degree of
grafting P2 Synthetic: D,L 5.2% P3 Synthetic: D,L 12.8% P4
Synthetic D,L 20.0% P5 Synthetic D,L 50.0%
[0126] In all cases, the amount of tocopherol effectively grafted
was confirmed by NMR.
EXAMPLE 6
Polymer P6
[0127] Synthesis of a Polyglutamate Grafted with .alpha.-Tocopherol
of Natural Origin
[0128] In a similar manner, the polymer P6 is synthesized with 7.3%
of D-alph.alpha.-tocopherol of natural origin (at a purity of
98.5%, obtained from the company ADM France). The molar mass is 17
400 (GPC NMP, PMMA eq).
EXAMPLE 7
Analysis of the Polymers in Aqueous Solution
[0129] The polymers are dissolved in a phosphate-buffered saline at
pH 7.4 at concentrations ranging from 10 to 40 mg/ml, and the pH is
adjusted to 7.4 by adding 0.1N sodium hydroxide. The dissolution is
observed visually.
TABLE-US-00002 TABLE 2 Solubility in saline water at pH 7.4 Degree
of Polymer grafting Concentration Appearance P1 (D,L) 7.8% 10 to 30
mg/ml soluble and clear P3 (D,L) 12% 10 mg/ml very fine precipitate
P4 (D,L) 20% 10 mg/ml very fine precipitate P6 (D) 7.3% 10 to 30
mg/ml soluble and clear
[0130] Phosphate buffer: 0.01M phosphate, 0.0027M KCl and 0.137M
NaCl.
[0131] An observation by electron transmission of the clear
solutions of the polymer P1 deposited on a support shows the
existence of nanoparticles of 15 to 25 nm. A comparative analysis
of the solutions of polymer P1, P6 and .alpha.-tocopherol succinate
at 15 mg/ml in water at pH 7.4 (phosphate buffer) reveals that only
the .alpha.-tocopherol succinate develops a milky solution
characteristic of vesicles, as described in patent EP 0 243
446.
EXAMPLE 8
Adsorption of a Dye Onto the Polymer P1
[0132] According to one of the subjects of the invention, the
polymers may be used in the form of a colloidal suspension in water
and associated with an active principle. For this application, it
is demonstrated in the experiment below that with certain polymers,
especially those with a degree of grafting of about from 5% to 10%
tocopherol, the adsorption capacity is greater than that of a
similar compound of the prior art.
[0133] For this study, the polymer P1 was compared with a similar
polymer containing a dodecanol chain grafted onto a polyglutamate.
This polymer is described in patent WO 00/30618.
[0134] The study is performed in the following manner: the polymers
are dissolved in an aqueous solution at pH 7 (phosphate buffer) and
5 mg of the dye known as Orange OT (Rn CAS: 2646-17-5) are added.
The solutions are left in an ultrasonic bath for 1 hour to achieve
the association. The solutions are then centrifuged to remove the
nonassociated dye, and the optical density is measured at the
.lamda.max of the dye, which is at 495 nm.
TABLE-US-00003 TABLE 3 Polymer Polymer Degree of grafting
concentration Normalized OD P1 7.8 mol % 13.8 mg/ml 1
(.alpha.-tocopherol) Comparative 15 mol % 17.3 mg/ml 0.45 polymer*
(dodecanol) *WO 00/30618
[0135] It is found that at a molar degree of grafting of less than
a half and at a slightly smaller mass concentration of polymer, the
polymer P1 has a much higher capacity for association of the dye
Orange OT.
EXAMPLE 9
Synthesis of the Polymer P7
[0136] Synthesis of a Polyglutamate Containing an
.alpha.-Tocopherol Leucine Graft
[0137] The .alpha.-tocopherol leucine derivative is first
synthesized, in the following manner.
[0138] D,L-.alpha.-Tocopherol (4.3 g) is reacted with BOC-leucine
(2.3 g) in 15 ml of dichloromethane in the presence of
4-dimethylaminopyridine (244 mg) and diisopropylcarbodiimide (1.5
g). After 2 hours at 30.degree. C. the product is purified by
filtration on a column of silica. 5 g of the product
.alpha.-tocopherol leucine BOC are obtained (77% yield). Its
structure is confirmed by NMR spectroscopy. The deprotection of the
product is performed in trifluoroacetic acid at a temperature of
between 5 and 10.degree. C. for 1 hour. After purification by
filtration through silica, 3.3 g of the desired product are
isolated (78% yield). Its structure is confirmed by NMR
spectroscopy.
[0139] The grafting reaction on a polyglutamic acid is then
performed under the same conditions as in example 1, with a degree
of grafting of 7%. The structure of the polymer and the degree of
grafting were confirmed by NMR spectroscopy.
EXAMPLE 10
Synthesis of Polymer P8
[0140] Synthesis of a Polyglutamate Containing an
.alpha.-Tocopherol Graft and a Polyoxyethylene Glycol Graft.
[0141] A grafting reaction is performed as in example 1, with 11
mol % of .alpha.-tocopherol and 2 mol % of an amino
methoxypolyethylene glycol of formula
MeO(CH.sub.2CH.sub.2O).sub.aCH.sub.2CH.sub.2NH.sub.2 and of molar
mass 3000 (product obtained from the company Shearwaters). The
polymer in its acid form is obtained in a yield of 72%. Proton NMR
confirms a degree of grafting with .alpha.-tocopherol of 10.9% and
with polyethylene glycol of 1.9%.
EXAMPLE 11
Adsorption of Insulin
[0142] A solution containing 1 mg of polymer P1 and 7 mg of insulin
at pH 7.0 in 1 ml of water is prepared and is left to incubate for
2 hours. The suspension is then ultrafiltered (10 000.times.G, 20
minutes with a 100 KDa threshold). The free insulin in the filtrate
is assayed by HPLC and the amount of associated insulin is deduced
by difference. A degree of association of greater than 95% relative
to the insulin employed is measured. Under the same conditions, the
comparative polymer of example 8 allows 40% association. The
adsorption capacity of polymer P1 is thus greater.
EXAMPLE 12
In-Vitro Degradation of Polymer P1 in the Presence of Enzymes
[0143] Polymer P1 is dissolved at pH 7.5 (phosphate buffer and 10
mM of calcium cation) and at a concentration of 20 mg/ml. 0.1 ml of
protease (solution of 10 mg/ml) is added and the degradation is
monitored by aqueous GPC.
[0144] A relatively rapid degradation is found, with a half-life
time of the initial polymer of about 100 minutes.
* * * * *