U.S. patent application number 11/783801 was filed with the patent office on 2007-10-25 for colloidal suspension of submicronic particles as vectors for active principles and method for preparing same.
This patent application is currently assigned to Flamel Technologies. Invention is credited to Nathan Bryson, Franck Touraud.
Application Number | 20070248686 11/783801 |
Document ID | / |
Family ID | 9552460 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070248686 |
Kind Code |
A1 |
Touraud; Franck ; et
al. |
October 25, 2007 |
Colloidal suspension of submicronic particles as vectors for active
principles and method for preparing same
Abstract
A suspension of vector particles (PV) based on polyamino acids
and have a mean hydrodynamic diameter between 30 and 120 nm, and an
insulin load factor of from 5 to 25% of associated insulin volume
relative to the polyamino acid volume forming the vector particles.
The polyamino acids are double-block polymers containing
hydrophilic and hydrophobic monomers. The suspension may be
prepared by copolymerizing N-carboxy anhydrides of hydrophobic
monomers and precursors of hydrophilic monomers, in the presence of
N-methyl pyrrolidone and methanol. The copolymer is optionally
neutralized, subjected to dialysis, concentrated and water is
eliminated to produce a solid powder, which can be suspended in a
liquid to produce the colloidal suspension. Active principles such
as insulin or vaccines are associated with the carrier particles to
prepare special pharmaceutical products.
Inventors: |
Touraud; Franck; (Lyon,
FR) ; Bryson; Nathan; (Millery, FR) |
Correspondence
Address: |
PATTON BOGGS LLP
8484 WESTPARK DRIVE
SUITE 900
MCLEAN
VA
22102
US
|
Assignee: |
Flamel Technologies
Venissieux
FR
|
Family ID: |
9552460 |
Appl. No.: |
11/783801 |
Filed: |
April 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10130783 |
Aug 5, 2002 |
7226618 |
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PCT/FR00/02831 |
Oct 11, 2000 |
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11783801 |
Apr 12, 2007 |
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Current U.S.
Class: |
424/499 ;
424/184.1; 514/11.3; 514/14.1; 514/15.2; 514/34; 514/44R; 514/5.9;
514/54; 514/56; 514/7.7 |
Current CPC
Class: |
A61K 9/5138 20130101;
A61K 2800/413 20130101; A61Q 19/00 20130101; A61P 3/10 20180101;
B82Y 5/00 20130101; A61K 8/90 20130101; A61K 8/044 20130101; B01J
13/0021 20130101 |
Class at
Publication: |
424/499 ;
424/184.1; 514/002; 514/034; 514/044; 514/054; 514/056 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/7042 20060101 A61K031/7042; A61K 31/7088
20060101 A61K031/7088; A61K 31/715 20060101 A61K031/715; A61K
31/727 20060101 A61K031/727; A61K 39/00 20060101 A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 23, 1999 |
FR |
FR 99 14751 |
Claims
1-19. (canceled)
20. A pulverulent solid, obtained from a colloidal suspension of
submicronic particles which can be used, in particular for carrying
active principle(s) (PA(s)), these particles being individualized
supramolecular arrangements: based on linear, amphiphilic polyamino
acids (PAA), with a-peptide linkages and comprising at least two
different types of recurring amino acids: hydrophilic AAI and
hydrophobic neutral AAO, the amino acids of each type being
mutually identical or different, and capable of combining in
colloidal suspension, in the nondissolved state, at least PA and of
releasing it, in particular in vivo, in a prolonged and/or delayed
manner characterized: in that the AAI(s) is (are) chosen from amino
acids with an ionizable side chain, the natural amino acids Glu an
Asp in carboxylic form and/or in the form of salts being
particularly preferred, in that the AAO(s) is(are) chosen from the
group comprising natural neutral amino acids, preferably those
belonging to the subgroup comprising: Leu, Ile, Val, Ala, Gly, Phe;
in that it is stable at pH between 4 and 13 in the absence of
surfactant(s), by a load factor Ta with insulin, expressed as % of
combined insulin mass relative to the mass and measured according
to a procedure Ma, Ta being such that: 7.ltoreq.Ta, preferably,
8.ltoreq.Ta.ltoreq.50, and, still more preferably,
10.ltoreq.Ta.ltoreq.30, and by a mean hydrodynamic diameter Dh
expressed in nm and measured according to a procedure Md, Dh being
such that: 10 nm.ltoreq.Dh.ltoreq.150 nm, preferably, 20
nm.ltoreq.Dh.ltoreq.1100 nm.
21. A method for preparing the pulverulent solid of claim 20,
comprising: 1) copolymerization of monomers consisting of
anhydrides of N-CarboxyAmino acids (NCA) of at least two different
types, on the one hand, NCAs-pAAI ("pAAI" designated precursors of
AAI) and, on the other hand NCAs-AAO, is carried out in the
presence: of at least one nonaromatic polar solvent, preferably
chosen from the group comprising: N-MethylPyrrolidone (NMP),
DiMethylFormamide (DMF), DiMethyl SulfOxide (DMSO),
DiMethylAcetamide (DMAc), pyrrolidone; NMP being most particularly
preferred; and optionally of at least one cosolvent selected from
aprotic solvents (preferably 1,4-dioxane) and/or protic solvents
(preferably pyrrolidone) and/or water and/or alcohols, methanol
being particularly preferred; 2) the recurring pAAI motifs of the
copolymer obtained in step 1 are converted to recurring AAI motifs,
using hydrolysis, preferably acid hydrolysis, for which the
copolymer obtained in step 1 is brought into contact with an
aqueous phase for acid hydrolysis+water; 3) the reaction medium is
neutralized; 4) optionally, the reaction medium is dialyzed in
order to purify the aqueous suspension of structured particles; 5)
optionally, this suspension of step 4 is concentrated; 6) the
liquid medium is removed in order to collect the pulverulent solid
comprising the particles.
22. The method of claim 21, wherein, at the end of step 1, the
copolymer poly(AAO) (PAAI) obtained is precipitated--preferably in
water--and the precipitate is recovered.
23. A method for preparing the pulverulent solid of claim 20,
comprising 1) copolymerization of monomers consisting of anhydrides
of N-CarboxyAmino acids (NCA) of at least two different types, on
the one hand, NCAs-pAAI ("pAAI" designating precursors of AAI) and,
on the other hand, NCAs-AAO, is carried out in the presence: of at
least one nonaromatic polar solvent, preferably chosen from the
group comprising: N-MethylPyrrolidone (NMP), DiMethylFormamide
(DMF), DiMethyl SulfOxide (DMSO), DiMethylAcetamide (DMAc),
pyrrolidone; NMP being most particularly preferred; and optionally
of at least one cosolvent selected from aprotic solvents
(preferably 1,4-dioxane) and/or protic solvents (preferably
pyrrolidone) and/or water and/or alcohols, methanol being
particularly preferred; 2) the recurring pAAI motifs of the
copolymer obtained in step 1 are converted to recurring AAI motifs,
using hydrolysis, preferably acid hydrolysis, for which the
copolymer obtained in step 1 is brought into contact with an
aqueous phase for acid hydrolysis+water; 3) the reaction medium is
neutralized; 4) optionally, the reaction medium is dialyzed in
order to purify the aqueous suspension of structured particles.
24. A method for preparing the pulverulent solid of claim 20,
wherein the combination of PA with the particles is carried out by
bringing a liquid phase containing the PA into contact with the
colloidal suspension of particles.
25. A method for preparing the pulverulent solid of claim 20,
wherein the combination of the PA with the particles is carried out
by bringing a PA in the solid state into contact with the colloidal
suspension of particles.
26. A method for preparing a colloidal suspension of submicronic
particles, comprising contacting the pulverulent solid of claim 20
with a liquid phase containing the PA.
27. A method for preparing a colloidal suspension of submicronic
particles, comprising contacting the pulverulent solid of claim 20
with the PA in solid form to form a mixture, and dispersing this
mixture of solids in a liquid phase, preferably an aqueous
solution.
28. An intermediate product of the method of claim 21, comprising
PAA copolymers which are precursors of particles.
29. A pharmaceutical, nutritional, plant-protection or cosmetic
proprietary product, comprising the pulverulent solid of claim 20,
comprising at least one active principle selected from the group
consisting of: Vaccines, Proteins and/or peptides, among which
those most preferably selected are: hemoglobins, cytochromes,
albumins, interferons, antigens, antibodies, erythropoietin,
insulin, growth hormones, factors VIII and IX, interleukins or
mixtures thereof, hematopoiesis-stimulating factors,
polysaccharides, heparin being more particularly selected, nucleic
acids and, preferably, RNA and/or DNA oligonucleotides,
non-petido-protein molecules belonging to various anticancer
chemotherapy classes and, in particular, anthracyclines and
taxoids, and mixtures thereof.
30. A method for preparing a copolymer comprising precursors of
hydrophilic amino acids with ionizable side chains, comprising
copolymerizing N-carboxy anhydride (NCA) forms of i) monomers of
precursors of hydrophilic amino acids with ionizable side chains
and ii) monomers of hydrophobic neutral amino acids in the presence
of at least one non-aromatic polar solvent selected from the group
consisting of N-methylpyrrolidone (NMP)), dimethylformamide (DMF),
dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc) and
pyrrolidone, and optionally in the presence of at least one
cosolvent selected from the group consisting of aprotic solvents,
protic solvents, water, and alcohols, precipitating the copolymer
to thereby recover a copolymer comprising precursors of hydrophilic
amino acids with ionizable side chains.
31. A copolymer comprising precursors of hydrophilic amino acids
with ionizable side chains produced by the method of claim 30.
Description
TECHNICAL FIELD
[0001] The field of the present invention is that of Vector
Particles (PV), which are useful for the administration of active
principles (PA). The latter are preferably medicaments or nutrients
for administration to an animal or human organism by the oral,
nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular,
intradermal, intraperitoneal, intracerebral or parenteral route, or
the like. However, this may also involve cosmetic products or
plant-production products, such as herbicides, pesticides,
insecticides or fungicides, or the like. In terms of chemical
nature, the PAs most particularly, but without limitation, involved
in the invention are, for example, proteins, glycoproteins,
peptides, polysaccharides, lipopolysaccharides, oligonucleotides,
polynuclides and organic molecules.
[0002] The present invention relates, more precisely, to colloidal
suspensions of Vector Particles, advantageously of the submicronic
type, based on polyamino acids (PAA). The present invention relates
to both uncoated particles as such, and vector systems for PAs,
consisting of particles loaded with the PA(s) considered. The
present invention also relates to pulverulent solids comprising
these PVs. The invention also relates to methods for preparing said
colloidal suspensions of particles, with or without PAs.
PRIOR ART
[0003] The encapsulation of PA into the PVs is intended in
particular to modify their duration of action and/or to convey them
to the site of treatment and/or to increase the bioavailability of
said PAs. Numerous encapsulation techniques have already been
proposed. Such techniques are intended, on the one hand, to allow
the transport of the PA to its site of therapeutic action, while
protecting it against attacks by the body (hydrolysis, enzymatic
digestion and the like) and, on the other hand, to control the
release of the PA at its site of action, in order to maintain the
quantity available for the body at the desired level. The PAs
involved in these vicissitudes of transport and of existence in the
body are, for example, proteins, but may also be products which are
completely different, organic molecules of synthetic or natural
origin.
[0004] The review by M. J. HUMPHREY (Delivery system for peptide
Drugs, published by S. DAVIS and L. ILLUM, Plenum Press, N.Y. 1986)
reports the problem relating to the enhancement of the
bioavailability of the PAs and the advantage of vector and
controlled release systems.
[0005] Among all the materials which can be envisaged for forming
PVs, polymers are increasingly used because of their intrinsic
properties. As regards the specifications which it is desired to
obtain for the PVs, they are particularly demanding and comprise,
in particular, the following specifications. [0006] 1 The first
specification desired for the PVs would be that the polymer
constituting the PVs is biocompatible, capable of being eliminated
(by excretion) and/or biodegradable and, even better, that it is
metabolized into products which are not toxic for the body. In
addition, it would be appropriate for the biodegradation in the
body to be of a sufficiently short duration. [0007] 2 It would be
advantageous for the PVs to be able to form a stable aqueous
suspension without the aid of an organic solvent and/or a
surfactant. [0008] 3 It would also be desirable for the PVs to have
a sufficiently small size to be able to undergo, in suspension in a
liquid, a sterilizing filtration by a filter whose pore diameter is
less than or equal to 0.2 .mu.m. [0009] 4 It is desirable for the
PVs and the PV-PA systems to be obtained by a method which is
nondenaturing for the PA. [0010] 5 The PVs advantageously ought to
make it possible to control the rate of release of the PA. [0011] 6
Another important specification would be that the PV-PA systems can
constitute excellent injectable medicaments. This enhanced capacity
for administration by injection--e.g. intravenous or intramuscular
injection--"injectability" is characterized by: [0012] (i) a
reduced injected volume (for a given therapeutic dose) [0013] (ii)
a low viscosity. [0014] These two properties are satisfied when the
therapeutic dose of PA is associated with a minimal quantity of PA.
In other words, the PVS should have a high PA load factor. [0015] 7
The cost specific to the PVs in an injectable preparation should be
reduced and, here again, it is appropriate for the PVs to have a
high PA load factor. In the final analysis, the small size and a
high load factor are major specifications sought for the PVs.
[0016] 8 It is also advantageous for the polymer constituting the
PVs not to induce an immune response.
[0017] The earlier technical proposals, which are described below,
have tried to satisfy all these specifications.
[0018] By way of illustration, there may be mentioned the earlier
proposals (a) to (h): [0019] (a) U.S. Pat. No. 5,286,495 relates to
a method of encapsulation by vaporization of proteins in aqueous
phase, using materials having opposite charges, namely: alginate
(negatively charged). and polylysine (positively charged). This
method of manufacture makes it possible to produce particles having
a size greater than 35 .mu.m. [0020] (b) Moreover, emulsion
techniques are commonly used to prepare microparticles loaded with
PA. For example, patent applications WO 91/06286, WO 91/06287 and
WO 89/08449 disclose such emulsion techniques in which organic
solvents are used to solubilize polymers, for example of the
polylactic type. However, it was found that the solvents may be
denaturing, in particular for peptide or polypeptide PAs. [0021]
(c) Biocompatible PVs called proteinoids, which have been described
since 1970 by X. FOX and K. DOSE in "Molecular Evolution and the
Origin of Life", Ed. Marcel DEKKER Inc (1977), are also known.
Thus, patent application WO 88/01213 proposes a system based on a
mixture of synthetic polypeptides, whose solubility depends on the
pH. To obtain the matrix microparticles according to this
invention, they solubilize the mixture of polypeptides, and then
with a change of pH, they cause the precipitation of proteinoid
particles. When the precipitation is carried out in the presence of
a PA, the latter is encapsulated into the particle. [0022] (d)
There may also be mentioned, as a reminder, U.S. Pat. No. 4,351,337
which belongs to a field which is different from that of the
vectorization of PA which is specific to the invention. This patent
discloses mass implants which are attached and located at quite
precise sites in the body. These implants are hollow tubes or
capsules of microscopic size (160 .mu.m and having a length equal
to 2 000 .mu.m), consisting of copolymers of copoly(amino acids)
e.g. poly(glutamic acid-leucine) or poly(benzyl
glutamate-leucine)--which are obtained by copolymerization of
monomers of N-carboxyanhydrides of amino acids (NCA). The inclusion
of a PA occurs through a technique for evaporation of solvent for a
mixture of polymer and of PA. U.S. Pat. No. 4,450,150 belongs to
the same family as U.S. Pat. No. 4,351,337 studied above and
essentially has the same object. The constituent PAAs are
poly(glutamic acid-ethyl glutamate). [0023] (e) Patent application
PCT/FR WO 97/02810 discloses a composition for the controlled
release of active principles, comprising a plurality of lamellar
particles of a biodegradable polymer, which is at least partially
crystalline (lactic acid polymer) and of a PA absorbed onto said
particles. In this case, the release of the active principle occurs
by desorption. [0024] (f) The publication "CHEMISTRY LETTERS 1995,
707, AKIYOSHI ET AL" relates to the stabilization of insulin by
supramolecular complexing with polysaccharides hydrophobized by
grafting of cholesterol. [0025] (g) The article which appeared in
"MACROMOLECULES 1997, 30, 4013-4017" describes copolymers composed
of a polypeptide block based on L-phenylalanine,
(-benzyl-L-glutamate or
O-(tetra-O-acetyl-D-glucopyranosyl)-L-serine, and a synthetic
block, such as poly(2-methyl-2-oxazoline) or
poly(2-phenyl-2-oxazoline). Polymers aggregate in aqueous medium to
form particles of 400 nm, which are capable of combining with an
enzyme, lipase. The term combined means here that the protein
adsorbs onto the particle by a physical phenomenon (no covalent
bonding). [0026] (h) Patent application FR 2 746 035 describes in
particular, page 28, lines 3 to 16, a colloidal suspension of
composite gel microparticles, obtained from a polyamino acid of the
polypolyleucine/sodium glutamate type, fractionated coconut oil
(miglyol.RTM.) and deionized water or buffered saline solution
(phosphate buffer pH 7.4 at 25.degree. C.). The mean reference
diameter D[4,3] of these composite gel microparticles is 2 800 nm.
It is evident from all the examples of FR 2 746 035, that the
smallest mean reference diameter D[4,3] is equal to 1 900 nm.
[0027] Moreover, these composite gel microparticles cannot combine
with insulin in the nondissolved state in colloidal suspension,
according to a factor Ta.gtoreq.7%. Under these conditions, it is
obvious that the composite gel microparticles do not meet the
specifications, and in particular not the specifications relating
to injectability and to the capacity for combination and for
release in relation to insulin. [0028] In addition, the method
according to FR 2 746 035 does not involve a nonaromatic polar
solvent and the formation of microparticles does not occur
spontaneously in aqueous medium, but involves the use of vigorous
homogenization with the aid of a rotor/stator type device. [0029]
(i) The subject of PCT application WO 96/29991 is polyamino acid
particles useful for the vectorization of PA. These particles have
a size of between 10 and 500 nm, preferably between 30 and 400 nm.
In the examples of this PCT application, the size of the particles
is measured by the radius of gyration. The radius of gyration of
the particles obtained in these examples varies from 55 to 280 nm.
Other techniques exist for measuring the size of colloidal
particles. The determination of the mean hydrodynamic diameter (Dh)
of the particles by quasi-elastic light scattering (QELS) is an
example of a convenient method of measurement. In the whole of the
present disclosure, an Md procedure for measuring Dh is taken as
reference. Md is described later. Thus, the Dh of the particles
according to the examples of the PCT WO 96/29991 extends from 150
nm to 750 nm. It is to be noted that the PVs in question here
consist of a hydrophobic core surrounded by hydrophilic hair. The
hydrodynamic diameter of these objects is less than double their
radius of gyration, as will be explained, for example, in the books
"Dynamic Light Scattering", B. J. Berue and R. Pecaran (Wiley,
1976) and "Physicochemical Hydrodynamics", R. F. Probstein (Wiley
1994). The load factor Ta for the particles is conveniently
expressed by the ratio of the mass of insulin to the mass of dry
PV. According to the examples of WO 96/29991, with a PA consisting
of insulin, is at best 0.065 mg/mg, that is 6.5% by dry weight of
insulin relative to the mass of PAA. Ta is measured according to a
procedure Ma described later. The particles according to WO
96/29991 form spontaneously by bringing PAA into contact with an
aqueous solution. The PAAs comprise neutral and hydrophobic amino
acid monomers AAO and ionizable and hydrophilic monomers AAI. These
PAAs are prepared by copolymerization of NCA of AAI precursors
(e.g.: Glu-OMe) and of NCA of AAO (e.g. Leu) in solution in a
dioxane/toluene mixture. The copoly(Glu-OMe) (Leu) obtained in
solution is recovered by precipitation in water, filtration and
drying. This copolymer is then subjected to acid hydrolysis by
incorporating it into TriFluoroAcetic acid (TFA), in which it
dissolves. A copolymer (Glu-O--Na)(Leu) is recovered after
neutralizing, dialysis, filtration and freeze-drying. This coPAA is
dispersed in an aqueous solution of NaCl and a suspension of
nanoparticles spontaneously forms. As indicated above, the latter
have a Dh size greater than 150 nm and an insulin load factor Ta of
6.50%.
[0030] It is therefore evident from the above that the earlier
technical proposals described above, and in particular proposal
(i), incompletely satisfy the new specifications indicated above,
and in particular a capacity for sterilizing by filtration, a high
rate of degradation, adaptability to constraints for administering
medicaments by injection, low cost and high PA load factor.
[0031] As regards the sterilizing filtration capacity, it is
important that the PV particles are sufficiently small to pass, in
suspension in a liquid, across filters whose cut-off is less than
or equal to 0.2 .mu.m, without clogging. Such ease and efficiency
of filtration sterilization are particularly appreciated for
injectable medicaments.
[0032] As regards the capacity for injection of the PVs, it is
appropriate, for a given dose of PA, to be able to inject small
volumes of liquid suspension, and that this suspension is not very
viscous. This involves being able to reduce the quantities of
excipient (PV) compared with the targeted therapeutic dose of PA
and to provide PVs having a size which is as small as possible,
while increasing the loading capacity of PA.
[0033] As regards the specification relating to biodegradability of
the PVs, the smaller the size of the PVs, the better it is and it
allows their rapid elimination.
[0034] In addition, it is appreciable to be able to reduce the
quantities of excipient (PV) for economic reasons and so as to
enhance the tolerance of the injectable medicament.
BRIEF DISCLOSURE OF THE INVENTION
[0035] Under these circumstances, an essential objective is to be
able to provide novel PVs which spontaneously form, and without the
aid of surfactants or of organic solvents, stable aqueous
suspensions of PV. Another essential objective of the present
invention is to provide novel PVs in stable aqueous colloidal
suspension or in pulverulent form and based on poly(amino acids)
(PAA), these novel PVs having to satisfy as much as possible
specifications 1 to 8 of the abovementioned specifications.
[0036] Another essential objective of the invention is to improve
the particles disclosed in PCT application WO 96/29991.
[0037] Another essential objective of the invention is to provide a
novel suspension of PV whose characteristics are perfectly
controlled, in particular in terms of PA load factor and in terms
of control of kinetics of release of PA.
[0038] Another essential objective of the invention is to provide
injectable medicinal suspensions. The specifications, which are
required for such suspensions, are a low volume for injection and a
low viscosity. It is important for the mass of colloidal particles
per injection dose to be as low as possible, without limiting the
quantity of active principle PA transported by these particles, so
as not to damage the therapeutic efficacy.
[0039] Another essential objective of the invention is to provide
an aqueous colloidal suspension or a pulverulent solid comprising
particles for carrying active principles satisfying the
specifications targeted above and which constitutes an appropriate
and suitable galenic form for administration, for example oral
administration, to humans or animals.
[0040] Another essential objective of the invention is to provide a
colloidal suspension comprising particles for carrying active
principles which can be filtered on 0.2 .mu.m filters for
sterilization purposes.
[0041] Another essential objective of the invention is to propose a
method for preparing PAA particles (dry or in suspension in a
liquid) which are useful in particular as vectors for active
principles, it being necessary for said method to be simpler to
use, nondenaturing for the active principles and, in addition, to
always allow fine control of the mean particle size of the
particles obtained.
[0042] Another essential objective of the invention is the use of
the abovementioned particles in aqueous suspension or in solid form
for the preparation: [0043] of medicaments (e.g. vaccines), in
particular for administration, in particular oral, nasal, vaginal,
ocular, subcutaneous, intravenous, intramuscular, intradermal,
intraperitoneal, intracerebral or parenteral administration, it
being possible for the active principles of these medicaments to
be, in particular, proteins, glycoproteins, peptides,
polysaccharides, lipopolysaccharides, oligo-nucleotides and
polynucleotides, [0044] and/or of nutrients, [0045] and/or of
cosmetic or plant-protection products, [0046] and/or of organic
medicinal molecules.
[0047] Another essential objective of the present invention is to
provide submicronic PV suspensions based on PAA and capable of
serving as vector for a PA, in particular one which is medicinal,
for administration of said PA to a human or animal organism, or
alternatively for a nutritional, plant-protection or cosmetic
PA.
[0048] Another objective of the present invention is to provide a
medicament, such as the system for prolonged release of active
principles, which is easy and economical to produce and which is,
in addition, biocompatible and capable of providing a very high
level of bioavailability of the PA.
[0049] Another essential objective of the invention is to provide a
system for carrying a vaccine, which is intrinsically
nonimmunogenic and in combination with one or more antigens.
[0050] The objectives relating to the products (inter alia) are
achieved by the present invention which relates, first of all, to a
stable colloidal suspension of submicronic structured particles
which can be used, in particular for carrying active principle(s)
PA(s), these particles being individualized (discrete)
supramolecular arrangements: [0051] based on linear, amphiphilic
polyamino acids (PAA), with peptide linkages and comprising at
least two different types of recurring amino acids: hydrophilic AAI
and hydrophobic neutral AAO, the amino acids of each type being
mutually identical or different, [0052] and capable of combining in
colloidal suspension, in the nondissolved state, at least one PA
and of releasing it, in particular in vivo, in a prolonged and/or
delayed manner, characterized: [0053] in that the AAI(s) of the
polymer chains is(are) chosen from amino acids with an ionizable
side chain, the natural amino acids Glu and Asp in carboxylic form
and/or in the form of salts being particularly preferred, [0054] in
that the AAO(s) of the polymer chains is(are) chosen from the group
comprising natural neutral amino acids, preferably those belonging
to the subgroup comprising: Leu, Ile, Val, Ala, Gly, Phe; [0055] in
that the particles are stable in aqueous phase at pH between 4 and
13 in the absence of surfactant(s), [0056] by a load factor Ta for
the vector particles with insulin, expressed as % of combined
insulin mass relative to the mass and measured according to a
procedure Ma, Ta being such that: [0057] 7.ltoreq.Ta [0058]
preferably, 8.ltoreq.Ta.ltoreq.50 [0059] and, still more
preferably, 10.ltoreq.Ta.ltoreq.30 [0060] and by a mean
hydrodynamic diameter Dh expressed in nanometers (nm) and measured
according to a procedure Md, Dh being such that: [0061] 10
nm.ltoreq.Dh.ltoreq.150 nm [0062] preferably, 20
nm.ltoreq.Dh.ltoreq.100 nm.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The procedures Md and Ma for the Dh and Ta measurements are
detailed below.
Procedure Md:
[0064] The pulverulent PAA powder is suspended in a 0.15 M aqueous
sodium chloride solution at pH 7.4, 25.degree. C. and at a polymer
concentration of between 0.01 and 0.5 g/l and, preferably, equal to
0.1 g/l. This suspension is stirred for 4 hours, and then
introduced into the scattering cell of a light scattering
apparatus, of the Brookhaven type, functioning with a laser beam
having a wavelength of 488 nm and vertically polarized. The
hydrodynamic diameter is calculated from the electric field
autocorrelation function by the cumulant method, as described in
the manual "Surfactant Science Series" volume 22, Surfactant
Solutions, Ed. R. Zana, chap. 3, M. Dekker, 1984. Procedure Ma:
[0065] (a) Preparation of an aqueous insulin solution: freeze-dried
human recombinant insulin (Sigma No. 10259) is poured into a 0.1 N
HCl solution over 5 min at 25.degree. C. This solution is then
poured into a phosphate buffer solution which is finally
neutralized by adding 0.1 N NaOH. The solution is then allowed to
stand for 30 min at room temperature, and then filtered on
0.8-0.2.mu. acrodisc membrane. The mass of insulin is calculated
according to the desired volume of solution, in order to obtain a
concentration of 60 IU/ml. [0066] (b) Dispersion of the vector
particles in PAA to be combined in the insulin solution: the
freeze-dried PVs are added to the insulin solution, in an amount of
10 mg PV/ml of solution. This mixture is stirred on a vortex two or
three times, and then placed in a rocking shaker at room
temperature for 18 hours. The colloidal suspension is then stored
at 4.degree. C. [0067] (c) Separation of free insulin from combined
insulin and assay of free insulin: the solution containing the
insulin and the PVs is centrifuged for 1 hour at 60 000 g at
20.degree. C. The supernatant is placed in tubes provided with an
ultrafiltration membrane (cut-off 100 000 Da) and centrifuged at 3
000 g, 2 hours at 20.degree. C. The insulin in the filtrate is
assayed by HPLC.
[0068] One of the inventive bases of these novel vector particles
PV, in stable aqueous colloidal suspension or in the form of
pulverulent solid, is due to the innovative selection of a group of
polymers and of an innovative methodology which make it possible to
obtain particles of submicronic size, which form a stable aqueous
colloidal suspension in the absence of surfactants or solvents.
[0069] Another inventive basis of these novel vector particles PV,
in stable aqueous colloidal suspension or in the pulverulent solid
state, is due to the innovative selection of a group of particular
submicronic structured particles by their load factor Ta.gtoreq.7%
and their size Da.ltoreq.150 nm. This selection is the result of
major and long research studies on the method for producing the
particles. Indeed, the reduction in size and the increase in the
loading capacity of the particles of polyamino acids was a priori
not obvious. Thus, using the method for producing nanoparticles of
polyamino acid taught in PCT application WO 96/29991, persons
skilled in the art were not able to obtain, "to measure", particles
which correspond to the new specifications, as defined above.
[0070] Finally, it is by modifying the compositions of the polymers
and the operating conditions that the inventors were able to
isolate these structured particles of very small size which are
based on PAA and which have, quite surprisingly and unexpectedly, a
load capacity Ta for insulin which may be up to three times higher
than that characteristic of the particles according to WO
96/29991.
[0071] Advantageously, the suspension according to the invention is
characterized in that the submicronic particles do not acquire
their cohesion from the presence of the following three compounds:
[0072] I) oil [0073] II) aqueous phase [0074] III) and at least one
synthetic non-crosslinked linear copolyamino acid comprising at
least two different types of amino acid comonomer: hydrophilic AAI
and hydrophobic AAO, unlike the suspension of microparticles
according to patent application FR 2 746 035.
[0075] The structure of the PAA polymers and the nature of the
amino acids are chosen such that: [0076] the polymer chains are
spontaneously structured in the form of small-sized particles (PV),
[0077] the particles form a stable colloidal suspension in water
and in physiological medium, [0078] the PVs combine with proteins
or other PAs in aqueous medium, by a spontaneous mechanism which is
nondenaturing for the protein, [0079] the PVs release the PAs in
physiological medium and, more precisely, in vivo; the kinetics of
release depend on the nature of the PAA polymer which is the
precursor for the PVs.
[0080] Thus, by modifying the particular structure of the PAA, it
is possible to control the phenomena for combining and releasing
the PA from the kinetic and quantitative point of view.
[0081] It is to the applicant's credit to have chosen, as
constituent material of the PVs, a particular composition of
polyamino acids which are amphiphilic and which therefore possess
properties of the PVs in PAA, namely: [0082] possibility of
spontaneously forming colloidal suspensions of PV which are
compatible with the pH of the physiological media encountered in
the therapeutic applications targeted, [0083] spontaneous
combination of the PAs with PVs in the absence of another agent
apart from water which serves as solvent for them and which, in the
case of proteins, is not denaturing, [0084] possibility of
releasing the PA from the PA-PV combination complex, under
physiological conditions, with pharmacokinetic and pharmacodynamic
profiles, which make it possible to envisage advantageous uses in
the therapeutic field (PA vectorization), and which, moreover, have
novel properties which are: [0085] filterability with a cut-off of
less than or equal to 0.2 .mu.m for sterilization purposes, [0086]
improved biodegradability, [0087] optimized injection capacity.
[0088] It was possible to obtain these new properties by virtue of
the primary technical functions of the PVs which are the small
nanometric size and the high load factor.
[0089] To define these PAAs a little further, it is possible to
indicate that they can be of the alternating sequential, ordered
(block) type or of the random sequential, disordered type.
[0090] Thus, according to a first embodiment of the PVs according
to the invention, the constituent PAAs are of the "block" type and
are characterized by an AAO/(AAI+AAO) molar ratio such that: [0091]
10%.ltoreq.AAO/(AAO+AAI).ltoreq.70%, [0092] preferably, 20%
AAO/(AAI+AAO).ltoreq.60%, [0093] and still more preferably,
35%.ltoreq.AAO/(AAI+AAO).ltoreq.50%.
[0094] Advantageously, the absolute length of each AAO block,
expressed as number of AAO is such that: [0095] preferably,
AAO>10, [0096] and, still more preferably,
20.ltoreq.AAO.ltoreq.100.
[0097] According to a second embodiment of the PVs according to the
invention, the constituent PAAs are of the "random" type, that is
to say are prepared by simultaneous copolymerization of AAI and AAO
monomers, and the AAO/(AAO+AAI) molar ratio is such that: [0098]
AAO/(AAO+AAI)>10%, [0099] and, preferably,
AAO/(AAO+AAI).gtoreq.20%, [0100] and, still more preferably,
30%.ltoreq.AAO/(AAI+AAO).ltoreq.70%.
[0101] Advantageously, the molar mass Mw of these random PAAs is
such that: [0102] Mw.gtoreq.2 000 g/mol, [0103] preferably,
Mw.gtoreq.5 500 g/mol, [0104] and still more preferably, 5 500
g/mol.ltoreq.Mw.ltoreq.200 000 g/mol.
[0105] According to a preferred characteristic of the invention,
the constituent block or random PAAs of the particles have degrees
of polymerization DP of between 30 and 600, preferably between 50
and 200, and still more preferably between 60 and 150.
[0106] Advantageously, the constituent PAAs of the PV particles are
"diblock" PAAs.
[0107] The present invention relates, not only to suspensions of
uncoated particles, as defined above, but also to particles
comprising at least one active principle PA. Preferably, the
suspension according to the invention is aqueous and stable. These
particles, loaded or not with PA, are advantageously in dispersed
form in a liquid (suspension), preferably an aqueous liquid, but
may also be in a pulverulent solid state, obtained from the
suspension of PV as defined above.
[0108] Accordingly, the invention relates, apart from to a
colloidal suspension (preferably aqueous suspension) of PV, to a
pulverulent solid comprising PVs and obtained from the suspension
according to the invention.
[0109] Another essential subject of the invention relates to the
preparation of the selected particles (as described above), both in
the form of a colloidal suspension and in the form of a pulverulent
solid. The method of preparation considered essentially consists in
synthesizing precursor PAAs and in converting them to structured
particles.
[0110] More precisely, this includes, first of all, a method for
preparing submicronic structured particles capable of being used,
in particular for carrying active principle(s), these particles
being discrete supramolecular arrangements: [0111] based on linear
amphiphilic polyamino acids (PAA), with linkages (-AAI hydrophilic
and AAO hydrophobic, the amino acids of each type being mutually
identical or different; [0112] having a mean diameter Dh, expressed
in nm and measured according to a procedure Md, such that: [0113]
10.ltoreq.Dh.ltoreq.150 [0114] preferably, 20.ltoreq.Dh.ltoreq.100;
[0115] on the one hand capable of forming a stable colloidal
suspension by simple mixing in an aqueous medium, without it being
necessary to add a solvent or surfactants thereto; [0116] and on
the other hand, capable of combining in a liquid medium, with at
least one PA and, in particular, with insulin according to a load
factor Ta, expressed as %, and measured according to a procedure Ma
such that: 7.ltoreq.Ta, preferably 8.ltoreq.Ta.ltoreq.25, and, on
the other hand, of releasing it, in particular in vivo, in a
prolonged and controlled manner.
[0117] This method is characterized in that: [0118] 1. a
copolymerization of monomers N-Carboxy-Anhydrides of amino acids
(NCA) of at least two different types, on the one hand, NCAs-pAAI
("pAAI" designating a precursor of AAI) and, on the other hand,
NCAs-AAO, is carried out in the presence: [0119] of at least one
nonaromatic polar solvent, preferably chosen from the group
comprising: N-MethylPyrrolidone (NMP), DiMethylFormamide (DMF),
Dimethyl Sulfoxide (DMSO), DiMethylAcetamide (DMAc), pyrrolidone,
NMP being most particularly preferred, [0120] and, optionally of at
least one protic cosolvent preferably chosen from the group
comprising pyrrolidone, water, alcohols; methanol being
particularly preferred; [0121] 2. the recurring pAAI motifs of the
precursor PAA copolymer of the particles are converted to recurring
AAI motifs, using hydrolysis, preferably acid hydrolysis, for which
an aqueous acid phase is added to the organic medium described
above; [0122] 3. optionally, the reaction medium is neutralized;
[0123] 4. optionally, the reaction medium is purified by dialysis
in order to obtain an aqueous suspension of structured particles;
[0124] 5. optionally, this suspension is concentrated; [0125] 6.
optionally the liquid medium is removed in order to collect the
pulverulent solid comprising the particles.
[0126] The first step of the method is based on known techniques of
polymerization of anhydrides of N-carboxy-(-amino acids (NCA),
described, for example, in the article "Biopolymers, 15, 1869
(1976)" and in the book by H. R. KRICHELDORF "(-Aminoacid-N-carboxy
Anhydride and Related Heterocycles" Springer Verlag (1987). The use
of judiciously chosen polar nonaromatic aprotic copolymerization
solvents, while avoiding any precipitation and the use of acid
hydrolysis in the presence of water and of nonaromatic polar
organic solvent, constitute novel and inventive modalities which
lead to structured, discrete and submicronic particles with a high
PA load capacity, and which form a stable colloidal suspension in
aqueous medium. These particles are not at all comparable to a
macroscopic agglomerated precipitate of the type mentioned above in
relation to the earlier proposal (d).
[0127] According to one variant, at the end of step 1, the
copolymer poly(AOO) (pAAI) obtained is precipitated--preferably in
water--and this precipitate is recovered. This variant corresponds
to a batch mode for preparing particles, in which the copolymer
poly(AAO) (pAAI) is isolated in the form of a precipitate forming a
stable intermediate product. This precipitate may be, for example,
filtered, washed and dried.
[0128] Still more preferably, the NCAs-pAAI are NCAs of O-alkylated
glutamic or aspartic acid, for example NCA-Glu-O-Me, NCA-Glu-O-Et
or NCA-Glu-O-Bz (Me=methyl-Et=ethyl-Bz=benzyl).
[0129] In a known manner, the copolymerization takes place at a
temperature between 20 and 120.degree. C., at atmospheric pressure
and in the presence of an amine-containing initiator, e.g.:
NH.sub.3. Other experimental parameters, such as the concentration
of NCA and/or polymer in the nonaromatic polar solvent (preferably
NMP), and/or the concentration or the nature of the protic
cosolvent, during the synthesis, will be adjusted according to the
desired effects known to persons skilled in the art.
[0130] The acid hydrolysis (step 2) is carried out using water and
at least one inorganic acid such as phosphoric or hydrochloric
aid--the latter being preferred--and/or an organic acid, such as
TriFluoroAcetic acid (TFA), acetic acid, dichloroacetic acid or
organosulfonic acids.
[0131] The water/acid ratios--expressed in parts by weight--in an
acidic aqueous phase for hydrolysis are advantageously: [0132] from
60/1 to 2/1, [0133] preferably 40/1 to 2/1, [0134] and, still more
preferably, 20/1 to 2/1.
[0135] The acidic aqueous phase for hydrolysis/NMP
ratios--expressed in parts by weight--are advantageously: [0136]
from 5/100 to 200/100 [0137] preferably 10/100 to 100/100 [0138]
and still more preferably from 20/100 to 80/100.
[0139] Other parameters, such as the polymer concentration, the
temperature of the reaction mixture, the mode of adding the acidic
aqueous phase for hydrolysis, the use of reduced pressure, the
duration of the reaction, and the like, are adjusted according to
the desired effects and are well known to persons skilled in the
art.
[0140] The neutralization (step 3) is carried out in practice, for
example, using sodium hydroxide.
[0141] The salt formed after neutralization as well as the solvent
are then removed by any appropriate physical separation treatment,
for example by diafiltration (dialysis) (step 4), filtration, pH
modification, chromatography and the like.
[0142] This gives an aqueous suspension of structured particles
which may be concentrated, for example, by distillation or any
other suitable physical means: ultrafiltration, centrifugation.
[0143] To separate, in step 6, the particles from their liquid
suspension medium, the aqueous phase is optionally removed, for
example, by drying (e.g. in an oven) by freeze-drying or any other
suitable physical means: ultrafiltration, centrifugation. A white
pulverulent solid is recovered at the end of this step 6.
[0144] According to one variant, the concentration step may be
carried out by a chemical treatment, such as a reduction in the pH,
which converts to an acid the hydrophilic part of the glutamate
monomers, making them insoluble in water. These acidic PAA
intermediates may be filtered, washed and dried. Said acidic
intermediates may be neutralized with a chemical base in a
subsequent step in order to obtain a suspension of particles.
[0145] It should be noted that the use of steps 1, 2, 3, 4 and
optionally 5 of the above method corresponding to a preparation of
a colloidal suspension of submicronic particles and to a high load
factor with the PAs.
[0146] During this preparation of colloidal suspension, the
amphiphilic PAAs poly(AAO)(AAI) of step 2 are placed in an aqueous
medium in which at least part of the AAIs is soluble and at least
part of the AAOs is insoluble. The PAAs exist in the form of
nanoparticles in this aqueous medium.
[0147] An alternative for preparing the PV suspension according to
the invention consists in bringing the pulverulent solid, as
described above and as product and by its method of production,
into contact with a nonsolvent aqueous medium for the AAOs.
[0148] To carry out the combination of one or more PAs with the
particles, it is possible to use several methods in accordance with
the invention. Nonlimiting examples of these methods are listed
below.
[0149] According to a first method, the combination of PA with the
particles is carried out by bringing a liquid phase (aqueous or
otherwise) containing the PA into contact with the colloidal
suspension of particles.
[0150] According to a second method, the combination of the PA with
the particles is carried out by bringing a PA in the solid state
into contact with the colloidal suspension of particles. The solid
PA may be, for example, in freeze-dried, precipitate or powdered
form or the like.
[0151] According to a third method, the pulverulent solid (PAA), as
described above as product and by its production characteristics,
is brought into contact with a liquid phase (aqueous or otherwise)
containing the PA.
[0152] According to a fourth method, the pulverulent solid, as
described above as product and by its production characteristics,
is brought into contact with the PA in solid form. This mixture of
solids is then dispersed in a liquid phase, preferably an aqueous
solution.
[0153] In all these methods, the PA used may be in pure or
preformulated form.
[0154] Given the nanometric size of the particles, the suspension
may be filtered on sterilizing filters, which makes it possible to
obtain, easily and at a lower cost, sterile injectable medicinal
liquids. The fact that it is possible, by virtue of the invention,
to control the size of the particles and reach Dh values of between
25 and 100 nm, is a major advantage.
[0155] The present invention also relates to novel intermediate
products of the method described above, characterized in that they
consist of PAA copolymers which are precursors of particles.
INDUSTRIAL APPLICATION
[0156] According to another of its aspects, the invention relates
to a suspension and/or a pulverulent solid, as defined above and/or
as obtained by the method presented above, this suspension and this
solid comprising at least one active principle preferably chosen
from: [0157] vaccines, [0158] proteins and/or peptides, among which
those most preferably selected are: hemoglobins, cytochromes,
albumins, interferons, antigens, antibodies, erythropoietin,
insulin, growth hormones, factors VIII and IX, interleukins or
mixtures thereof, hematopoiesis-stimulating factors, [0159]
polysaccharides, heparin being more particularly selected, [0160]
nucleic acids and, preferably, RNA and/or DNA oligonucleotides,
[0161] non-petido-protein molecules belonging to various anticancer
chemotherapy classes and, in particular, anthracyclines and
taxoids, [0162] and mixtures thereof.
[0163] The invention also relates to a suspension and/or the
pulverulent solid loaded with nutritional, plant-protection or
cosmetic PA.
[0164] Finally, the invention relates to a pharmaceutical,
nutritional, plant-protection or cosmetic proprietary product,
characterized in that it comprises a suspension and/or the
pulverulent solid loaded with PA and as defined above.
[0165] According to another of its subjects, the invention also
relates to the use of these PVs (in suspension or in solid form)
loaded with PA, for the manufacture of medicaments such as systems
with controlled release of PA.
[0166] In the case of medicaments, they may be, for example, those
which can be administered, preferably by the oral, nasal, vaginal,
ocular, subcutaneous, intravenous, intramuscular, intradermal,
intraperitoneal, intracerebral or parenteral route.
[0167] The cosmetic applications which may be envisaged are, for
example, compositions comprising a PA combined with the PVs
according to the invention and which can be applied by the
transdermal route.
[0168] The relevant plant-protection products may be, for example,
herbicides, pesticides, insecticides, fungicides and the like.
[0169] The following examples will make it possible to better
understand the invention in its various product/method/application
aspects. These examples illustrate the preparation of particles of
polyamino acids loaded or otherwise with active principles, and
they likewise present the structural characteristics and the
properties of these particles.
LEGEND TO THE FIGURES
[0170] FIG. 1: Nanoparticles corresponding to a block copolymer Ia:
leucine 50/glutamate 50 obtained according to the teaching of
patent WO 96/29991.
[0171] FIG. 2: Nanoparticles obtained with the block copolymer
according to the present invention (example 2). It will be noted
that the bar now represents here only 50 nm.
[0172] FIG. 3: Variation in the glucose concentration (mean at %
basal on 4 dogs) after injection of a PV formulation loaded with
insulin in an amount of 2 IU/kg.
[0173] FIG. 4: Variation in the serum insulin concentration (mean
on 4 dogs) after injection of a PV formulation loaded with insulin
in an amount of 2 IU/kg.
EXAMPLES
Example 1
Production, in Aqueous Stable Colloidal Suspension and in
Pulverulent Solid Form, of Vector Particles, from a Block Polyamino
Acid, Poly(Leu/Glu) 40/80 Diblock
[0174] 112.4 g of NCA-GluOMe (0.60 mol) and 449 g of
N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into
a 1 liter reactor thermostated at 20.degree. C. After dissolution,
21.38 g of a 0.34 M solution of ammonia in 1,4-dioxane (1.25 mol
%/NCA) are added. The polymerization is monitored by measuring the
carbon dioxide emitted into a gas bell jar and verified by
disappearance of vibration bands characteristic of the NCAs at 1860
and 1790 cm.sup.-1. After 30 min, a solution of 47.17 g of NCA
Leucine (0.30 mol) in 631 g of NMP is introduced. After 10 min of
reaction, the temperature is increased to 60.degree. C. The
polymerization is monitored as above and is complete after 2 hours.
The temperature of the reaction mixture obtained is increased to
80.degree. C. 31.5 g of aqueous concentrated hydrochloric acid
(35%, 12 M) are added, with mechanical stirring over 30 min to 350
g of the reaction mixture obtained at the end of step 1. The
reactor is then placed under reduced pressure regulated at 600 mbar
for 6 hours. A mixture of 31.5 g of 35% hydrochloric acid and of
126 g of water is then added over 60 min, followed by a second
phase of vacuum at 250 mBar for 18 hours. In this example, the
overall water/pure hydrochloric acid ratio is 7.6/1 by mass and the
acidic aqueous phase/NMP ratio is 60/100 by mass.
[0175] The reaction mixture is then cooled to 50.degree. C. and
then neutralized with aqueous sodium hydroxide (35% by mass). The
NMP and the sodium chloride formed during the neutralization are
removed by diafiltration against 20 volumes of Milli Q water, on a
membrane with an MWCO of 1 000 Daltons (Pellicon II system,
Millipore). A stable aqueous colloidal suspension of vector
nanoparticles is thus obtained. The suspension of nanoparticles is
finally freeze-dried.
[0176] The contents of leucine motifs are determined by proton
nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for
4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic
diameter (Dh) is 70 nm (according to Md).
Example 2
Combination of Insulin with the Nanoparticles of Poly(Leu/Glu)
40/80
[0177] The procedure Ma is used. The concentration of free insulin,
assayed by HPLC chromatography is equal to 0.59 mg/ml and the
combined insulin concentration equal to 1.51 mg/ml is deduced
therefrom. The load capacity for a colloidal solution of 10 mg/ml
reaches 1.51 mg/ml of insulin. Thus, the ratio of the mass of
combined insulin to the bLE (Ta) mass is 15.1%.
Example 3
Production, in Stable Colloidal Aqueous Suspension and in
Pulverulent Solid Form, of Vector Particles from a Block PAA
Poly(Leu/Glu) 25/70 Biblock
[0178] 146.4 g of NCA GluOMe are dissolved in 586 g of NMP to which
18.43 g of a 0.48 M solution of ammonia in methanol are added. When
the polymerization of the NCA GluOMe is complete, a solution of
43.9 g of NCA Leu in 708 g of NMP is introduced and the
polymerization of the NCAs Leu is continued until disappearance of
the monomers is obtained. The medium is then heated to 80.degree.
C. and 129.4 g of 35% HCl are added dropwise thereto over 30 min to
1 hour. A 600 mBar vacuum is applied for 6 hours, and then an
additional 129.4 g of 35% HCl are added as a mixture with 517.5 g
of water. A 250 mBar vacuum is then applied for 18 hours. After
this step, the temperature is reduced to 50.degree. C., 1 liter of
water is introduced, followed by 280 ml of 35% NaOH in order to
bring the pH to 7.4. The suspension is then filtered (5 .mu.m),
dialyzed (cut-off 1 000 Da) in water, in order to remove the
solvent and the salts, and finally filtered (0.22 .mu.m). This
suspension may be directly used or may be subjected to subsequent
treatments, such as distillation of the water (step 5) or
freeze-drying (step 6).
[0179] The mean hydrodynamic diameter Dh (according to Md) is
14.8%. The insulin load factor Ta, determined according to the
procedure Ma, is 35 nm.
Example 4
Production, in Stable Aqueous Colloidal Suspension, of Vector
Nanoparticles, from a Block Polyamino Acid, Poly(Leu/Glu) 50/70
Diblock and Characteristics of the Nanoparticles
[0180] 38.9 g of NCA-GluOMe (0.208 mol) and 156 g of
N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into
a 0.5 liter reactor thermostated at 30.degree. C. After
dissolution, 5.79 g of a 0.407 M solution of ammonia in methanol
(1.25 mol %/NCA) are added. The polymerization is monitored by
measuring the carbon dioxide emitted into a gas bell jar and
verified by disappearance of vibration bands characteristic of the
NCAs at 1860 and 1790 cm.sup.-1. After 30 min, a solution of 23.3 g
of NCA Leucine (0.148 mol) in 263 g of NMP is introduced. After 10
min of reaction, the temperature is increased to 60.degree. C. The
polymerization is monitored as above and is complete after 1-2
hours. The temperature of the reaction mixture obtained previously
is increased to 80.degree. C. 41.9 g of aqueous hydrochloric acid
(35% of the mass) are added, with mechanical stirring over 30 min,
to the reaction mixture. The reactor is then placed under reduced
pressure regulated at 600 mbar for 6 hours. A mixture of 41.9 g of
35% hydrochloric acid and of 167.5 g of water is then added over 60
min, followed by a second phase of vacuum at 250 mbar for 18 hours.
The reaction mixture is then cooled to 50.degree. C. and then
neutralized with aqueous sodium hydroxide (35% by mass). The NMP
and the sodium chloride formed during the neutralization are
removed by diafiltration against 20 volumes of Milli Q water, on a
membrane with an MWCO of 1 000 Daltons (Pellicon II system,
Millipore). A stable aqueous colloidal suspension of vector
nanoparticles is thus obtained. The suspension of nanoparticles is
finally freeze-dried.
[0181] The mean hydrodynamic diameter Dh is measured according to
Md on aqueous suspensions of the freeze-dried products. The insulin
load factor Ta is determined according to the procedure Ma.
Example 5
Production, in Stable Aqueous Colloidal Suspension of Vector
Nanoparticles, from a Block Polyamino Acid, Poly(Leu/Glu) 25/35
Diblock and Characteristics of the Nanoparticles
[0182] 38.9 g of NCA-GluOMe (0.208 mol) and 156 g of
N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into
a 0.5 liter reactor thermostated at 30.degree. C. After
dissolution, 5.78 g of a 0.452 M solution of ammonia in methanol
(1.25 mol %/NCA) are added. The polymerization is monitored by
measuring the carbon dioxide emitted into a gas bell jar and
verified by disappearance of vibration bands characteristic of the
NCAs at 1860 and 1790 cm.sup.-1. After 30 min, a solution of 23.3 g
of NCA Leucine (0.149 mol) in 5 219 g of NMP is introduced. After
10 min of reaction, the temperature is increased to 60.degree. C.
The polymerization is monitored as above and is complete after 1-2
hours. The temperature of the reaction mixture obtained previously
is increased to 80.degree. C. 42.0 g of aqueous hydrochloric acid
(35% of the mass) are added, with mechanical stirring over 30 min,
to the reaction mixture. The reactor is then placed under reduced
pressure regulated at 600 mBar for 6 hours. A mixture of 42.0 g of
35% hydrochloric acid and of 167.9 g of water is then added over 60
min, followed by a second phase of vacuum at 250 mBar for 18 hours.
The reaction mixture is then cooled to 50.degree. C. and then
neutralized with aqueous sodium hydroxide (35% by mass). The NMP
and the sodium chloride formed during the neutralization are
removed by diafiltration against 20 volumes of Milli Q water, on a
membrane with an MWCO of 1 000 Daltons (Pellicon II system,
Millipore). A stable aqueous colloidal suspension of vector
nanoparticles is thus obtained. The suspension of nanoparticles is
finally freeze-dried.
[0183] The contents of leucine motifs are determined by proton
nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for
4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic
diameter Dh is measured according to Md on aqueous suspensions of
the freeze-dried products. The insulin load factor is determined
according to Ma.
Example 6
Production, in Stable Aqueous Colloidal Suspension of Vector
Nanoparticles, from a Block Polyamino Acid, Poly(Leu/Glu) 50/150
Diblock and Characteristics of the Nanophases
[0184] 46.4 g of NCA-GluOMe (0.248 mol) and 186 g of
N-methyl-2-pyrrolidinone (NMP) are introduced, with stirring, into
a 0.5 liter reactor thermostated at 30.degree. C. After
dissolution, 6.90 g of a 0.19 M solution of ammonia in methanol
(1.25 mol %/NCA) are added. The polymerization is monitored by
measuring the carbon dioxide emitted into a gas bell jar and
verified by disappearance of vibration bands characteristic of the
NCAs at 1860 and 1790 cm.sup.-1. After 30 min, a solution of 12.97
g of NCA Leucine (0.083 mol) in 218 g of NMP is introduced. After
10 min of reaction, the temperature is increased to 60.degree. C.
The polymerization is monitored as above and is complete after 1-2
hours. The temperature of the reaction mixture obtained previously
is increased to 80.degree. C. 40.3 g of aqueous hydrochloric acid
(35% of the mass) are added, with mechanical stirring over 30 min,
to the reaction mixture. The reactor is then placed under reduced
pressure regulated at 600 mBar for 6 hours. A mixture of 40.3 g of
35% hydrochloric acid and of 161.3 g of water is then added over 60
min, followed by a second phase of vacuum at 250 mBar for 18 hours.
The reaction mixture is then cooled to 50.degree. C. and then
neutralized with aqueous sodium hydroxide (35% by mass).
[0185] The NMP and the sodium chloride formed during the
neutralization are removed by diafiltration against 20 volumes of
Milli Q water, on a membrane with an NWCO of 1 000 Daltons
(Pellicon II system, Millipore). A stable aqueous colloidal
suspension of vector nanoparticles is thus obtained. The suspension
of nanophases is finally freeze-dried.
[0186] The contents of leucine motifs are determined by proton
nuclear magnetic resonance (signals at 2.10, 2.22 and 2.58 ppm for
4H of Glu and at 0.85 ppm for 6H of Leu). The mean hydrodynamic
diameter Dh is measured according to Md. The insulin load factor is
determined according to Ma.
Example 7
Comparative Example of the Nature of the Particles Formed with the
Teaching of PCT Patent WO 96/29991
[0187] The particles obtained by the teaching of patent WO 96/29991
are those which appear in FIG. 1. Advantageously, the particles
according to the invention are those which appear in the appended
FIG. 2 corresponding to a photograph taken under a transmission
electron microscope.
[0188] The differences in morphology and size appear blatantly on
comparing FIG. 1 which represents PVs according to the prior art,
on the one hand, and FIG. 2 showing PVs according to the invention,
on the other hand. A notable difference in morphology is observed
here. The PVs of FIG. 2 are such that the majority of the
larger-sized particles exhibit an oblong shape.
Example 8
Test of Stability of a Colloidal Suspension Prepared According to
Example 2 with the Polymer Poly(Leu/Glu) 40/80
[0189] The pulverulent powder of Example 2 is dissolved in an
amount of 60 mg/ml of powder in a phosphate buffer. The pH was
adjusted to 7.3 and the osmolality of the suspension was adjusted
to 300 mOsm/kg using a 5 M NaCl solution. The solution was filtered
(0.22 .mu.m) before being distributed at the rate of 5 ml into
sterile 10 ml bottles. The stability of the samples was evaluated
over a period of 4 months. Half of the samples were kept at
4.degree. C. (.+-.2.degree. C.) while the other samples were
maintained at laboratory temperature: 25.degree. C. (.+-.5.degree.
C.). At given times, the samples are collected from the site of
storage and equilibrated for 1 hour at room temperature before the
analysis. The analytical methods are detailed, the results being
presented in the form of two tables.
[0190] 1) Verification of the homogeneity of the colloidal
solution: Without stirring the suspension, 100 .mu.l samples are
collected three times in order to represent the state of the
solution at the top, in the middle and at the bottom of the bottle.
The refractive index of each sample is measured at 25.degree. C. on
an Abbe refractometer calibrated relative to pure water. Three
readings are made for each sample and the three mean values are
compared. Any variation in the concentration of the solution
results in a difference in refractive index.
2) Measurement of the hydrodynamic diameter: A 100 .mu.l sample of
the solution to be analyzed is diluted 120-fold with a 0.15 M NaCl
solution and the Dh of the colloidal particles is measured
according to the protocol Md.
[0191] 3) Measurement of the viscosity: The measurements are
carried out on 0.75 ml samples using an AR1000 rheometer (TA
instruments) equipped with a Cone/Plane geometry (cone 4
cm/2.degree. C.) at a temperature of 20.0.degree. C.+/-0.1.degree.
C. (regulation by Pelletier effect). The viscosity curve as a
function of the shear gradient is recorded for gradients varying
from 1 to 100 s.sup.-1. At these concentrations, the solutions are
slightly rheofluidizing and the viscosity value selected is taken
for a gradient of 10 s.sup.-1.
[0192] The results obtained after aging at 4.degree. C. and
25.degree. C. are assembled in Tables I and II. TABLE-US-00001
TABLE I Aging at 40.degree. C. To T1 T2 T3 T4 T5 number of days 0 9
28 59 92 127 of aging homogeneity sample 1 cm 1.3443 1.3442 1.3447
1.3438 1.3440 1.3443 (index) sample 1.5 cm 1.3442 1.3442 1.3446
1.3439 1.3439 1.3440 sample 2 cm 1.3443 1.3442 1.3448 1.3439 1.3440
1.3440 hydrodynamic diameter 45 45 44 43 44 44 (nm) Viscosity (mPa
s) 246 246 250 250 262 250
[0193] TABLE-US-00002 TABLE II Aging at 25.degree. C. To T1 T2 T3
T4 T5 number of days 0 9 28 59 92 127 of aging homogeneity sample 1
cm 1.3443 -- 1.3448 1.3441 1.3440 1.3442 (index) sample 1.5 cm
1.3442 -- 1.3448 1.3441 1.3440 1.3442 sample 2 cm 1.3443 -- 1.3447
1.3441 1.3440 1.3442 hydrodynamic diameter 45 -- 44 44 45 46 (nm)
viscosity (mPa s) 246 -- 246 250 284 240
Example 9
Test of Release Df Insulin in Animals after Administration of a
Suspension of Particles Containing Insulin
[0194] A formulation is prepared from PV (of Example 3) and
insulin, the quantity of each being determined according to
measurements of combination rate (Ma).
[0195] A group of 4 beagle dogs (males and females) weighing
between 10 and 12 kg are fasted for 18 hours. A preparation is
formulated and is composed of 80 IU of insulin t 56 mg of PV in 1
ml of PBS buffer. The dogs then receive a subcutaneous
administration of this insulin preparation at the rate of 2 IU/kg
of weight. Blood sample collected for glucose and insulin assay
before (-2 h, -1 h and 0 h) and after (1 h, 2 h, 4 h, 6 h, 12 h, 16
h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h) the injection.
The glucose concentrations are measured in the samples by the
glucose oxidase method and serum insulin is assayed using a
radioimmunological method. FIG. 3 gives the mean of the variation
in glucose for this formulation. FIG. 4 gives the mean of the
variation in serum insulin for this formulation.
[0196] This example shows, through the biological activity, the
nondenaturation of the protein as well as the possibility of
prolonging the release by >24 h, two advantageous aspects of the
present invention.
* * * * *