Note: Descriptions are shown in the official language in which they were submitted.
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PCT/EP99/00782
PHARMACEUTICAL COMPOSITIONS IN FORM OF NANOPARTICLES
COMPRISING LIPIDIC SUBSTANCES AND AMPHIPHILIC SUBSTANCES AND
' RELATED PREPARATION PROCESS
Prior art
' s In the research field of the new vehicles suitable to the administration
of active
principles, a great interest has been directed towards the polymeric systems
having size in the micrometer range and to the polymeric systems having size
in
the manometer range.
Among the mostly used polymers the polyalkylcyanoacrylates and the poly-lactic
~o acid (PLA) and poly-lactic glycolic acid (PLA-PLGA) derivatives are to
remember.
Such systems show however some disadvantages.
For example the polyalkylcyanoacrylates are metabolized by the organism in a
24
hours interval and release formaldehyde, a potentially toxic derivative; the
PLA
and PLA-PLGA polymers do not produce toxic metabolites but they have long
is degradation times ranging from some weeks to some months, and then they may
show dangerous accumulation phenomena.
Moreover, the preparation methods of these systems need the use of potentially
toxic organic solvents which may remain in traces in the final form.
In the end, the size of the majority of said systems exclude their use for
2o intravenous way because extraneous bodies having size higher than 5 pm
injected in vein may cause embolisms.
These negative aspects generated greater attention for administration systems
having greater biocompatibility and lower toxicity: the first among all these
are the
lipidic colloidal systems such as oil/water emulsions, liposomes, lipidic
micro- and
2s nanoparticles.
Oil/water emulsions, consisting of lipidic droplets having size in the range
of
manometers, dispersed in an external aqueous phase, have been used as a
vehicle for the parenteral feeding (JP Patent No. 55,476, 1979, Okamota, Tsuda
and Yokoama).
3o Oil/water emulsions containing active principles have been described in the
Patent
WO 91102517, 1991, Davis and Washington. Such systems have a high capacity
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to incorporate active principles in the internal lipidic phase, but the active
principles easily diffuse from such phase towards the external phase
originating
stability problems and limitations for the optional development of a
protracted
release form.
s The liposomes are colloidal structures having an aqueous internal phase
sorrounded by one or more layers of phospholipids. The use of liposomes as
vehicles for the administration of drugs is described for example in the U.S.
Patent
No. 3,993,754 (1976, Rahman and Cerny).
However typically, such systems show stability problems during the stocking, a
io poorly reproducible preparation method and a low potentiality to
incorporate and
retain active principles.
Fountain and others invented lipidic microparticles in globular form having
size
ranging from 0.5 pm to 100 ~m as vehicles for the administration of active
principles. Such invention is disclosed in the U.S. Patent No. 4,610,868
(1986).
is Domb and others (US Patent 435,546) invented the LiposheresT"", insoluble
particles having size about equal to 40 pm, suspended in an aqueous
environment, consisting of a lipophilic internal phase surrounded by external
layers of phospholipids, added to the composition and adsorbed on the surface
of
the particles themselves. These systems were developed for the controlled
2o release of anaesthetic drugs (Domb and others, US Patent 5227165) and of
active
principles having insecticide and pesticide activity (Domb and others US
Patent
5227535). However the technique for the preparation of such systems requires
the
help of solvents which remain in traces in the final form.
The per os administration turns out to be difficult for active principles
which are not
2s much soluble, not much absorbed in the gastroenteric tract or which are
sensible
to the pH or the action of the proteolytic enzymes (proteins and peptides).
The .
incorporation of such substances in lipidic nanoparticles allows to overcome
such
difficulties because these nanoparticle systems may be absorbed along the '
gastrointestinal tract. Their reduced size allow to exploit the mechanisms of
the
3o passive transmucosal absorption, or to pass through the intercellular
junctions or
the ionic channels or to use the endocytosis mechanism or to enter the
lymphatic
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flux.
Solid lipidic systems consisting of nanopellets were developed by Speiser and
' others (US Patent 4,880,634, 1989), and destined to the oral administration
of
poorly absorbed drugs. The fipidic pellets are prepared emulsifying lipidic
s substances in an aqueous environment with a high energy mixer, then cooling
the
emulsion at room temperature and obtaining the pellets by sonication.
Gasco (EP 0526666A1, 05/08/1991) invented a technique for the preparation of
lipidic nanoparticles. A microemulsion is prepared adding to an aqueous phase
a
lipid melted in the presence of surfactants and cosurfactants, which is then
io dispersed in an aqueous environment maintained at a temperature around 10
°C.
The solid nanoparticles are obtained in an aqueous suspension, but may be
subsequently deprived of the residual surfactants by ultrafiltration and
recovered
by filtration or freeze-drying.
Such technique turns out to be advantageous from the point of view of the
saving
is of energy with respect to the high energy homogenization, it allows to
obtain
smaller nanoparticles, having average diameters ranging from 90 nm to 900 nm,
with a more uniform size distribution and a low polydispersion index. However
the
preparation of a microemulsion needs the melting of the fipidic material which
is
for most used lipidic substances about 70 °C, which limits the use of
such
2o technique for the thermoiable substances.
Summary of the invention
The invention relates to pharmaceutical compositions in form of nanoparticles,
having a diameter lower than 1000 nm and preferably ranging from 50 to 500 nm,
comprising a composite material;,, consisting of at least one lipidic
substance and
2s at least one amphiphilic substance, and a pharmaceutically active
principle.
We have unexpectedly found that, operating according to the present invention,
said composite material and the relative particles have characteristics not
achievable by an usual mixing of a lipidic substance with an amphiphilic
substance
or by the adsorption of an amphiphilic substance on lipidic particles.
3o The amphiphilic substance may be preferentially distributed on the surface
of the
nanoparticles or it may be preferentially distributed inside the nanoparticles
or it
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may be homogeneously distributed on the surface of and inside the
nanoparticles.
The formation of the composite material allows to obtain nanoparticles:
1. with surface characteristics helping the oral administration absorption and
the '
half-life time in the circulatory system;
s 2. with mass characteristics, as the low melting temperature, allowing to -
incorporate thermolable drugs;
3. suitable, thanks to the presence of lipophilic zones and partially
hydrophilic
zones in the composite material, to the vehiculation both of hydrosoluble
drugs
and of liposoluble drugs;
~0 4. able to homogeneously incorporate the hydrophilic drugs (for example
peptides)
inside an essentially lipophilic matrix.
Detailed description of the invention
The invention relates to the preparation of compositions for pharmaceutical
use in
form of particles having size lower than one micrometer (nanoparticles),
is comprising a composite material consisting of lipidic and amphiphilic
substances,
the latter being of lipidic or polymeric kind.
Generally, the nanoparticles according to the invention are prepared starting
from
a composite material obtained by comelting or cosolubilization of the lipidic
material and the amphiphilic substances. The comelted mixture, at the
subsequent
2o cooling, results in a composite material having new characteristics with
respect to
the two starting materials, showing more hydrophilic zones and more lipophilic
~ \~,
,,
zones thanks to the reciprocal disposition of the components or to the
segregation
of the amphiphilic material towards the surface or inwards the mass of the
nanoparticles. These characteristics are substantially different from the
surface !~
2s adsorption of an amphiphilic substance on a lipophilic surface. Such
properties will
be described in detail in the Characterization Examples reported below. ,
The drug may be dissolved or suspended in said comelted mixture during the
preparation process and, thanks to the new properties of the composite
material, it
may divide, according to its characteristics, preferentially inside the more
3o hydrophilic areas or the more lipophilic areas. Moreover, the hydrophilic
drugs (for
example peptides) charged on the nanoparticles turn out to be unexpectedly
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distributed in an homogeneous way inside the nanoparticles themselves while
the
peptide fraction adsorbed on the surface turns out to be very low, and lower
with
respect to the Examples of the prior art (particles consisting of lipidic core
and
adsorbed amphiphilic substance).
s The nanoparticles obtained from the comelted mixture maintain the same
characteristics of the starting composite material.
The nanoparticles may be obtained with different preparation techniques:
- 1, a technique providing for the dispersion of a oil in water microemulsion
(consisting of, as oil phase, fipidic and amphiphilic materials kept at a
temperature
~o higher than the melting point of the composite mixture and one or more
surfactants and cosurfactants) in an aqueous medium, utilising the temperature
gradient.
- 2. A technique providing for the high pressure homogenization of a fine
emulsion
of a composite material, at a temperature higher than the melting temperature
of
is the materials forming the composite, or of a fine suspension of a composite
material, below the melting temperature of the composite, in presence of
surfactant agents.
The preparation of the invention according to the microemulsion-dispersion
process (technique 1 ), provides for the initial comelting or cosolubilization
of two
20 or more lipidic and amphiphilic components, taken to the melting
temperature of
the components themselves or at least to the melting of one of the two
components, when the latter is soluble in the former; an appropriate volume of
an
aqueous solution containing ore or more surfactants and cosurfactants, warmed
at
the same temperature of the composite material melted, under mild stirring is
2s added to such melted composite material.
It is also possible to form the microemulsion simultaneously taking to the
melting
temperature the lipidic and amphiphitic components in presence of the water
and
the surfactants and cosurfactants needed to formation of the microemulsion
itself.
The active pharmaceutical principle may be dissolved or dispersed in the
starting
3o melted composite material or added directly to the microemulsion during the
preparation of the microemulsion itself, depending on the properties of the
active
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principle itself. The distribution of the active principle occurs into the
composite
material, allowing an unexpected decrease of the drug amount adsorbed on the
surface and submitted to the degradating action of the enzymes and of the
external environment.
s The so formed oil/water microemulsion is subsequently dispersed in water or
in
aqueous medium, in controlled volume and stirring conditions, at a temperature
generally ranging from +1 ° to +10 °C, but that may also range
from -15 to -30 °C
using non aqueous solvents miscible with water, originating in this way the
composite nanoparticles in solid form in aqueous suspension. Said
nanoparticles
io have a diameter lower than 1000 nm. The nanoparticles turn out to be
different
with respect to the systems obtained by the techniques of amphiphilic
substances
adsorption on the surface of the lipidic particles (Domb) or by the use of
amphiphilic substances as surfactants for the formation of lipidic
nanoparticles
(fiasco).
is Subsequently, the nanoparticle suspensions may be washed with water or
aqueous solutions through an ultrafiltration system (or dialysis) which allows
to
remove the surfactant, cosurfactant and free drug excess. Therefore such
process
allows to remove the undesired possible effects due to the surfactants
presence in
the pharmaceutical form. Moreover, with such a procedure it is possible to
2o quantitatively determine the percentage of the active principle not
incorporated or
adsorbed on the nanoparticles.
The composition described above may be administered as an aqueous
suspension or it is recovered as a solid by freeze-drying, filtration,
evaporation of
the aqueous solvent or spray-drying techniques.
2s The nanoparticles according to the present invention have the following
quantitative composition by weight:
- lipidic substances from 0.5 to 99.5%, and preferably from 10% to 90%;
- amphiphilic substances from 0.5 to 99.5% and preferably from 10% to 90%;
- pharmacologically active principle from 0.001 to 99%, and preferably from
0.01
3o to 50% with respect to the sum of the lipidic substances and amphiphilic
substances.
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In the preparation process of the nanoparticles according to the technique (1
), the
component substances are used in the following proportions by weight:
' in the microemulsion:
- lipidic components, from 0.1 % to 50% by weight and preferably from 10 to
25%;
- s - amphiphilic components, from 0.1 % to 50% by weight and preferably from
0.5%
to 25%;
- surfactants, from 5% to 30% and preferably from 10% to 20%;
- cosurfactants, from 0% to 15% and preferably from 3% to 7%;
- water, or aqueous solutions, from 40% to 75% by weight and preferably from
l0 50% to 70%;
- pharmaceutical active principles, directly incorporated in the composite
material
or dissolved in the microemulsion, in concentrations variable on the base of
the
incorporation efficacy and of the desired dosages and ranging from 0.001 % to
99% and preferably from 0.001 % to 50% by weight with respect to the sum of
the
is lipidic components and amphiphilic components.
In the dispersion:
- the microemulsion prepared as described above is dispersed in aqueous
environment (water or aqueous solutions) with volumetric dilutions from 1:2 to
1:200, preferably from 1:5 to 1:50.
2o To the dispersion
- coadjuvants of the dispersion, from 0.05% to 5% by weight;
- viscosizing agents, of polymeric kind, from 0.05% to 5% by weight
may be added.
The preparation according to the high pressure homogenization technique
2s (technique 2) provides for the dispersion of the composite material, added
with
one or more adjuvant substances, in an aqueous environment. The composite
material is homogenized to form nanoparticles maintaining the system at the
melting temperature of the material itself or just below such temperature
("softening") or at temperatures maintaining the composite material at the
solid
3o state.
The composite material may be initially prepared by comelting or
cosolubilization,
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analogously to what is reported for the technique 1, proceeding to the
comeiting of
two or more lipidic and amphiphilic components taken to the melting
temperature
of the components themselves or at least to the melting of one of the two
components when the latter is soluble in the former.
s The composite material may be preliminarly dispersed in an aqueous solution
containing surfactant substances, stabilizing substances and/or viscosizing
substances by dispersion or low energy homogenization techniques (for example
using Silverson L2R or Ultra-Turrax kind equipments). After such treatment,
which
may be not necessary if the composite material shows surface characteristics
io such as to help its dispersion in water, the system is submitted to high
pressure
homogenizator (for example of APV Gaulin, APV Rannie Mini-Lab, Microfluidizer
kind) to repeated homogenization cycles which cause nanoparticle dispersions.
The high pressure homogenization treatment may occur at the composite material
melting temperature, at "softening" temperature or at temperatures at which
the
is material is present in a solid state in micronized form.
The active principle may be comelted, dissolved or dispersed in the composite
material or in each of its constituents during the comelting of the system, or
added
during the subsequent process phases, as it is or in presence of surfactants
which
helps its incorporation in the nanoparticles or the adsorption on their
surface.
2o Subsequently, the nanoparticle suspensions may be washed with water or
aqueous solutions, analogously to what is described for the technique (1 ),
through
a ultrafiltration system.
Analogously, the composition may be administered as aqueous suspension or
recovered as a solid by freeze-drying, filtration or aqueous solvent
evaporation or
2s spray-drying techniques.
In the preparation process of the nanospheres according to the technique (2),
the
substances composing the invention are used in the following proportions by
weight:
- lipidic components, from 0.1 % to 50% by weight, preferably from 0.5 to 15%;
30 - amphiphilic components, from 0.1 % to 50%, preferably from 0.5% to 15%;
- surfactants, from 0.05% to 10%, preferably from 0.5% to 5%;
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- water, or aqueous solutions of hydrosoluble components, from 45% to 99.5%,
preferably from 50% to 80%;
- dispersion coadjuvants from 0.05% to 5%;
viscosizing agents, of polymeric kind from 0.05% to 1 %;
- s - pharmaceutical active principles, directly incorporated in the composite
material
or dissolved in the microemulsion, in concentrations variable on the base of
the
incorporation efficacy and of the desired dosages, and ranging from 0.001 % to
99.9% and preferably from 0.001 % to 50% by weight with respect to the sum of
the lipidic components and the amphiphilic components.
io Among the lipidic materials usable according to the invention we can
mention both
natural products and synthetic or semi-synthetic kind products definable as
"fats"
in that they are not miscible or only partially miscible with water:
1 ) natural fats either saturated or unsaturated and partially or totally
hydrogenated
vegetal oils, for example hydrogenated cotton oil (LubritabT""), hydrogenated
palm
is oil (DynasanT"" P60) and hydrogenated soy-bean oil (SterotexT"" HM);
2) semi-synthetic and synthetic mono-, di- and triglycerides containing
saturated
andlor unsaturated fatty acids (having aliphatic chain length ranging from C~o
to
C22) and their polyhydroxyethylated derivatives, for example tristearine,
caprico-
caprylic triglycerides (MygIioITM, CaptexTM, LabrafacT"" Lipo), behenic
triglycerides
20 (CompritolT"") monoglycerides as glyceril monostearate (MyvapIexT"" 600) or
glyceril palmitostearate (PreciroITM) and saturated or unsaturated
polyhydroxylated
triglycerides (series of LabrafilT"", LabrafacT"" Hydro, GelucireT"");
3) "liquid waxes", for example isopropyl myristate, isopropyl-caprinate, -
caprylate, -
laurate, -palmitate, -stearate and esters of fatty acids, such as ethyl oleate
and
2s oleyl oleate;
4) "solid waxes", for example carnauba wax and bees-wax;
aliphatic alcohols, for example cetyl alcohol, stearyl alcohol, lauryl
alcohol,
cetylstearyl alcohol and their polyhydroxyethylated derivatives;
6) aliphatic carboxylic acids preferably having medium and long chain (C~o-
C22),
3o saturated (decanoic acid, lauric acid, palmitic, stearic, docosanoic acid,
etc.),
unsaturated (oleic, linoleic, etc.) and their polyhydroxyethylated
derivatives.
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Among the amphiphilic materials of lipidic kind one can use lipids having in
their
structure some hydrophilic components, such as for example:
1 ) phospholipids belonging to the series: phosphatidyl glycerol,
phosphatidylcholine and phosphatidic acid (e.g. dimiristoyl phosphatidyl
glycerol); .
s 2) mono- and di-glycerides such as glyceril monostearate (MyvapIexT"" 600)
or
glyceril palmitostearate (PrecirolT"");
3) triglycerides and saturated or unsaturated polyhydroxylated triglycerides
(e.g.
series of LabrafilT"", LabrafacT"" Hydro, GelucireT"");
4) esters of fatty acids, such as decylester of oleic acid: CetioIT"" V and
to isopropylmyristate;
5) medium chain fatty acids (such as capric, caproic and lauric acids).
Among the amphiphilic materials of polymeric kind, polymers may be used such
as:
1 ) Polyethylene glycols (PEG), both liquid (from PEG 200 to PEG 1000) and
solid
is (from PEG 1500 to PEG 20,000);
2) poly-(propyleneoxide) poly-(ethyleneoxide) copolymers, Poloxamer (LutroIT""
188, LutrolT"" 407);
3) Polyvinyl alcohol;
4) Polyacrylates (CarbopoiT"", PemulenT"", NoveonT"");
5) Poly-(methylvinyl ether) -malefic anhydride (GantrezT"") copolymers;
6) Polysaccharides of natural origin such as chitosan and derivatives,
ialuronic
acid and derivatives, xanthan, scleroglucan, gellan, guar gum, locust bean
gum,
alginate and dextran;
7) Polyesters such as for example poly-e-caprolactone.
2s As reported above the composite materials according to the invention may be
prepared by mixing, comelting or cosolubilization of the components selected
among the lipidic materials and among the amphiphilic materials of lipidic or
polymeric kind. For example, composite materials according to the invention,
may
be formed from mixtures of fatty acids (stearic acid-decanoic acid), of fatty
acids
3o and phospholipids (stearic acid-dimiristoyl phosphatidyl glycerol or
dimiristoyl
phosphatidylcholine), fatty acids and triglycerides or polyhydroxylated
triglycerides
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(stearic acid and LabrafilT"" 2130), mono- or di-glycerides with fatty acids
(glyceril
palmitostearate with stearic acid), fatty acids with polyethylene glycol
(stearic acid-
' PEG), mono and diglycerides with copolymers of polyethylene oxide-
polypropylene oxide (glycerilpalmitostearate with poloxamer).
- s As aqueous phases according to the invention (aqueous phase of the
microemulsion according to the technique 1 and/or dispersing phase according
to
the techniques 1 and 2) may be mentioned:
1 ) water as it is or buffered at different pH and ionic strength;
2) aqueous solutions of hydrophilic, hydrosoluble or hydrodispersable polymers
to such as polyethylene glycol, polyvinyl pyrrolidone, polyacrylic acids and
derivatives (e.g. Carbopol~, Pemulen~, etc.), polymethacrylic acids and
derivatives (e.g. Eudragit~), copolymers of polyoxyethylene-polyoxypropilene
(e.g.
Poloxamer, Lutrol~), polysaccharides of various nature such as for example
dextran, xanthan, scleroglucan, gum arabic, guar gum, chitosan, cellulose and
is starch derivatives;
3) aqueous solutions of saccharides (e.g. sorbitol, mannitol, xylitol);
3) mono or polyhydroxylic aliphatic alcohols, preferably having short chain
(C2-
C4);
4) polyethylene glycols (e.g. PEG 200, PEG 400, PEG 600, PEG 1000);
20 5) polyglycolic glycerides (e.g. LabrasolTM);
6) polyglycols, such as for example propylene glycol, tetraglycol,
ethoxydiglycol
(TranscutolT"" ).
Among the surfactants, to use in techniques 1 and 2, we may not exhaustively
mention all the non ionic surfactants with a HLB value generally but non
2s necessarily greater than 7, such as for example: sorbitan-esters of fatty
acids (e.g.
Span, Artacel~, Brij~), polyoxyethylen sorbitan esters of fatty acids (e.g.
Tween~, Capmul~, Liposorb~), copolymers of polypropileneoxide-
polyethyleneoxide (Poloxamer), esters of polyethylene glycol (PEG)-glycerol
(Labrasol~, Labrafil~ with HLB 6-7), esters of PEG and acids or long chain
3o aliphatic alcohols (e.g. Cremophor0), polyglycerid esters (Plurol~), esters
of
saccharides and fatty acids (sucro-esters). When needed, even anionic
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surfactants (e.g. sodium lauryl sulfate, sodium stearate, sodium oleate), bile
salts
(e.g. sodium glycocholate, taurodeoxycholate, taurocholate, ursodeoxycholate)
or
cationic (e.g. tricetol), as well as low HLB surfactants, lecithins as they
are (Lipoid
S75) and hydrogenated (e.g. Lipoid S75, S75-3), phospholipids and their
s semisynthetic or synthetic derivatives may be used.
Among the cosurfactants needed for the formation of the microemulsion we
remember short chain alcohols such as for example ethanol, 2-propanol, n-
butanol, isopropanol; short and medium aliphatic acids (e.g. butyric acid,
valeric
and capronic acids), aromatic alcohols (e.g. benzyl alcohol); medium chain
to alcohols and aliphatic acids (C8-C~2) such as decanoic acid, lauric acid,
caprynil
alcohol and lauryl alcohol. Moreover, as cosurfactants may be used also esters
or
ethers of acids or medium-tong chain aliphatic alcohols with mono- or
polyhydroxylated alcohols. Some of the components mentioned among the
cosurfactants may at the same time form the oil phase of the microemulsion.
is The pharmaceutical active principles usable in the invention may be both
~hydrosoluble (e.g. peptides or proteins) and liposoluble (e.g. steroidal
hormones),
as well as'poorly soluble in both vehicles (e.g. acyclovir). The surface and
mass
properties of the nanoparticles according to the invention allow important
advantages such as for example:
20 't ) the possibility of administering by oral or transmucosal way molecules
usually
not absorbable by such a way (e.g. polypeptides and proteins);
2) the possibility of administering by oral and/or parenteral way lipophilic
highly
insoluble and poorly absorbable molecules;
3) an improvement in the biopharmaceutical properties of the active principles
2s (e.g. controlled or prolonged release and increase of the plasmatic half-
life time);
4) the possibility of administering by topical way molecules active at the
mucosal
or dermal level (e.g. antiviral, antimicotic, antipsoriatic drugs);
5) the possibility of encapsulating active principles having unpleasant
flavour,
administrable in immediate release formulations.
3o The active principle groups which may be advantaged from the invention
include:
non steroidal (NSAID) and steroidal (SAID) anti-inflammatories, estrogenic or
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progestational hormones, cardiovasculars, antivirals, antimycotics,
antineoplastics, hypolipidemics, peptides and proteins having different
action.
Among said active principles we may mention, however as a not exhaustive
example:
- s ergot alkaloids and derivatives: dihydroergotamine, didhydroergotoxine and
bromocriptine.
Analgesics and non steroidal anti-inflammatories, and their salts: diclofenac
sodium, diclofenac hydroxyethil pyrrolidine, diclofenac diethylamine,
ibuprofen,
flurbiprofen, ketoprofen, indomethacin, mefenamic acid, naproxen, nimesulide
and
to piroxicam.
Antiarrhythmics: amiodarone, diisopyramide, propranolol and verapamil.
Antibactericals: amoxicillin, flucloxacillin, gentamicin, rifampicin,
erythromycin and
cephalosporins.
Antifungins and antipsoriatics: amphotericin, butoconazole nitrate,
ketoconazole,
is econazole, etretinate, tluconazole, flucytosine, griseofulvin,
itraconazole,
miconazole, nystatin, sulconazole and tioconazole.
Antivirals: Acyclovir, ganciclovir, AZT and protease inhibitors.
Antihypertensives: amlodipine, clonidine, diltiazem, felodipine, guanabenz
acetate,
isradipine, minoxidil, nicardipine hydrochloride, nimodipine, nifedipine,
prazosin
2o hydrochloride and papaverine.
Antidepressants: carbamazepine.
Antihistaminics: diphenhydramine, chlorpheniramine, pyrilamine,
chlorcyclizine,
promethazine, acrivastine, cinnarizine, loratadine and terfenadine.
Antineoplastics and immunosuppressants: cyclosporin, dacarbazine, etretinate,
2s etoposide, lomustine, melphaian, mitomycin, mitoxantrone, paclitaxel,
procarbazine, tamoxifen, taxol and derivatives and taxotere.
Anxiolytics, sedatives, hypnotics: alprazolam, bromazepam, diazepam,
lorazepam, oxazepam, temazepam, sulpiride and triazolam.
p-Blockers: alprenolol, atenolol, oxprenolol, pindolol and propranolol.
30 (3-Agonists: salbutamol, salmeterol.
Cardiac and cardiovascular inotropics: amrinone, digitoxin, digoxin,
lanatoside C,
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medigoxin and ubidecarenone.
Corticosteroids: beclomethasone, betamethasone, budesonide, cortisone acetate,
desoximethasone, dexamethasone, fludrocortisone acetate, flunisolide,
hydrocortisone, methylprednisolone, methylprednisone and triamcinolone.
s Gastrointestinals and anti H2-histaminics: cimetidine, cisapride,
domperidone,
famotidine, loperamide, mesalazine, omeprazole, ondansetron hydrochloride and
ranitidine.
Hypolipidemics: bezafibrate, clofibrate, gemfibrozil, probucol and lovastatin.
Anti-anginals: amyl nitrate, glyceryl trinitrate, isosorbide dinitrate and
mononitrate
io and pentaerythritol tetranitrate.
Central Action Drugs: for example nicotine.
Vitaminic and Nutritional Agents: betacarotene, vitamin A, Vitamin B2, Vitamin
D
and derivatives, vitamin E and derivatives and vitamin K.
Opioid Analgesics: codeine, dextropropoxyphene, diihydrocodeine, morphine,
is pentazocine and methadone.
Sexual Hormones: danazol, ethinyl estradiol, medroxyprogesterone acetate,
methyltestosterone, testosterone, norethistrone, norgestrel, estradiol,
estriol,
progesterone, stilbestrol and diethylstilbestrol.
Peptidic, proteic or polysaccharidic molecules having different activity:
20 leuprolide and t_H-RH analogues, calcitonin, glutathione, somatotropin
(GH),
somatostatin, desmopressin (DDAVP), interferon, molgramostin, epidermic growth
factor (EGF), nervous growth factor (NGF), insulin, glucagon, toxins or
toxoides
(for example tetanus toxin), antigenic factors of proteic or polysaccharidic
kind,
heparin, heparin having low molecular weight and heparinoids.
2s Molecules having specific topical activity: e.g. sun protectors (UV
Absorbers); skin
nutrients, ceramides and glycolic acid.
The characteristics of the compositions according to the invention may be
evaluated by several physico chemical methods, such as for example:
- thermal analysis (DSC, TGA and "hot stage" microscopy) in order to verify
the
3o change of the mass properties,
- surface analysis (angle of contact method) to determine the variation of the
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Is
surface of the surface properties,
- laser light scattering (LLS) techniques for the determination of the size
' distribution of the nanoparticles,
- Zeta potential measurement techniques for the determination of the
superficial
- s charge properties of the nanoparticles,
- active principles incorporation efficiency determination techniques (marking
techniques with fluorescent molecules, separation and analysis techniques),
- morphological observation techniques (electron transmission microscopy:
TEM).
Such techniques allow to demonstrate that the composite materials forming the
Io nanoparticles according to the invention and the nanoparticles themselves
show
innovative and advantageous properties. Using the suitable combinations of
lipidic
and amphiphilic material, it is possible to prepare materials and composite
nanoparticles which may:
- increase the incorporation efficiency of hydrophilic drugs (e.g. peptides)
in a
Is lipidic matrix, helping the dissolution/dispersion of the active principle
in the most
hydrophilic zones of the nanoparticles themselves,
- modify in an unexpected way the localization of the molecules incorporated
into
the nanoparticles: for example, allowing a homogeneous distribution, inside
the
lipophilic nanoparticles, of peptidic molecules usually adsorbed on the
surface,
20 - show a surface energy lower with respect to the starting components, thus
resulting more biocompatible,
- show surfaces more lipophilic than the single components, and thus result
more
absorbable by oral way,
- when useful to the application, show surfaces more hydrophilic than the
single
2s components and increase the plasmatic half life times of the particles
injected by
parenteral way,
- at certain proportions of amphiphilic and lipidic substance, result in two
phases,
with peculiar characteristics, wherein the amphiphilic compound:
1 ) may be preferentially located on the surface of the nanoparticles
("segregated
3o in surface");
2) may be preferentially located inside the nanoparticles ("segregated
inside");
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3) may be homogeneously located on the surface and inside the nanospheres
themselves,
- at certain proportions the nanoparticles, consisting of composite materials
having _
peculiar and different characteristics with respect to the single original
materials,
s may melt at physiological temperatures releasing the active principle in a
fast way
(e.g. for topical/percutaneous treatments and flavour masking),
- at other proportions, the nanoparticles may stay as they are at
physiological
temperatures ensuring the release of the active principle by diffusion and/or
degradation of the nanoparticles themselves,
to - at certain proportions of lipidic and amphiphilic material, in the
preparation
technique (1 ), based on the oil-water microemulsion of the composite mixture,
it is
possible to extend the thermal interval of existence of the microemulsion
itself to
lower temperature values, allowing the incorporation of thermolable molecules.
Fundamental and innovative characteristic of the invention, independently from
is the process of preparation of the nanoparticles, is thus the unexpected
formation
of a composite material having new and different surface and mass
characteristics
with respect to the single components. On the base of the qualitative
characteristics of the chosen starting materials (e.g. chemical structure,
melting
point, hydrophobicity and wettability), and at the relative percentages of the
2o materials in a determined mixture, it is possible to obtain a very high
number of
composite materials, and thus of nanoparticles having different
characteristics.
Advantage of the invention is the possibility to increase the incorporation of
the
hydrophilic active principle inside a lipophilic nanoparticle, and to be able
to modify
its distribution.
2s An important advantage of the invention thus consists in the possibility to
have
vector systems (composite nanoparticies) consisting of new materials
originated
by exclusively physical changes of the component substances and thus not
requesting the long toxicological experimental tests iter.
3o For illustrative aim examples of preparation of the compositions according
to the
invention are reported, with the technique 1 (Examples 1-36), and with the
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17
technique 2 (Examples 37-45), and Comparative Examples with preparations
according to the prior art {A-E). The characterization of the obtained
products and
the advantageous behaviour in vivo with respect to the products of the prior
art
are reported in the Examples {F-Z 1 ).
s EXAMPLE 1
4 grams of stearic acid and 19.9 mg of di-myristoil-phosphatidyl glycerol
(DMPG
percentage in the mixture: 0.5%} are mixed, and they are heated to about 72
°C
with the formation of the melted composite .mixture to which 4 ml of n-butanol
are
added. In 20 ml of water acidified to pH 3, 2 g of sodium taurodeoxycholate
and
l0 3.85 mg of salmon calcitonin are dissolved and the solution heated to 72
°C is
added to the melted composite mixture. To the so formed emulsion, kept under
stirring, 3 ml of Tween 20 are added, to form a transparent and anisotropic
microemulsion. The microemulsion, at about 60 °C, is dispersed in 5
volumes of
water at pH 3 cooled to 3-5 °C, under constant stirring (250 rpm), to
form
is composite solid lipidic nanopartictes consisting of stearic acid -
dimyristoil-
phosphatidyl glycerol containing calcitonin. The suspension is washed by
ultrafiltration in order to remove the surfactant excess. The calcitonin
incorporation
efficacy in the nanoparticles, determined by fluorimetric and chromatographic
techniques (see characterization Examples), is about 10.5 %. The average
2o diameter of the nanoparticles is 226 nm and the polydispersion index is
0.200.
EXAMPLE 2
The preparation of the Example 1 is repeated, using a DMPG percentage in the
composite mixture equal to 1.0%. The calcitonin incorporation efficacy is
10.2%,
the average diameter of the nanoparticles 220 nm and the polydispersion 0.268.
25 EXAMPLE 3
The preparation of the Example 1 is repeated, using a DMPG percentage in the
composite mixture equal to 4.5%. The calcitonin incorporation efficacy is
13.2%,
the average diameter of the nanoparticles 199 nm and the polydispersion 0.212.
EXAMPLE
3o The preparation of the Example, 1 is repeated, using a DMPG percentage in
the
composite mixture equal to 5.2%. The calcitonin incorporation efficacy is
13.4%,
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the average diameter of the nanoparticles 195 nm and the polydispersion 0.200.
EXAMPLE 5
The preparation of the Example 1 is repeated, using a DMPG percentage in the
composite mixture equal to 9.5%. The calcitonin incorporation efficacy is
9.19%,
s the average diameter of the nanoparticles 231 nm and the polydispersion
0.186.
EXAMPLE 6
The preparation of the Example 1 is repeated, using a DMPG percentage in the
composite mixture equal to 25%. The calcitonin incorporation efficacy is
9.51%,
the average diameter of the nanoparticles 205 nm and the polydispersion 0.275.
io EXAMPLE 7
The preparation of the Example 1 is repeated, using as an amphiphilic material
the distearoil phosphatidic acid (DSPA) phospholipid in a percentage in the
composite mixture equal to 1.2%. The calcitonin incorporation efficacy is
9.9%, the
average diameter of the nanoparticles 245 nm and the polydispersion 0.261.
is EXAMPLE 8
The preparation of the Example 7 is repeated, using a DSPA percentage in the
composite mixture equal to 5.2%. The ~alcitonin incorporation efficacy is
14.4%,
the average diameter of the nanoparticles 324 nm and the polydispersion 0.341.
EXAMPLE 9
2o The preparation of the Example 1 is repeated, using low molecular weight
heparin
as an active principle (average molecular weight 4000 Da) and, the dimyristoil
phosphatidylcholine (DMPC) phospholipid as amphiphilic material, in a
percentage
in the composite mixture equal to 4.0%. The heparin incorporation efficacy is
about 1.0%, the average diameter of the nanoparticles is 213 nm and the
2s polydispersion 0.186.
EXAMPLE 10
The preparation of the Example 9 is repeated, using the dimyristoil
phosphatidylcholine (DMPC) phospholipid, in a percentage in the composite
mixture equal to 8.0%. The average diameter of the nanoparticles is 179 nm and
3o the polydispersion 0.249.
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The preparation of the Example 9 is repeated, using the dimyristoil
phosphatidylcholine (DMPC) phospholipid, in a percentage in the composite
mixture equal to 10%. The average diameter of the nanoparticles is 290 nm and
the polydispersion 0.270.
- s EXAMPLE 12
The preparation of the Example 9 is repeated, using the dimyristoil
phosphatidylcholine (DMPC) phospholipid, in a percentage in the composite
mixture equal to 14%. The average diameter of the nanoparticles is 369 nm and
the polydispersion 0.360.
to EXAMPLE 13
The preparation of the Example 1 is repeated, using the Labrafac HydroT""
polyhydroxylated triglyceride as 'amphiphilic material, in a percentage in the
composite mixture equal to 1.0%. The average diameter of the nanoparticles is
307 nm and the polydispersion 0.234.
15 EXAMPLE 14
The preparation of the Example 13 is repeated, using the Labrafac HydroT""
polyhydroxylated triglyceride, in a percentage in the composite mixture equal
to
2.5%. The average diameter of the nanoparticles is 307 nm and the
polydispersion 0.234.
2o EXAMPLE 1_5
The preparation of the Example 13 is repeated, using the Labrafac HydroT""
polyhydroxylated triglyceride, in a percentage in the composite mixture equal
to
50%. The average diameter of the lipidic nanoparticles is 310 nm and the
polydispersion 0.332.
2s EXAMPLE 16
The preparation of the Example 13 is repeated, using the Labrafac HydroT""
polyhydroxylated triglyceride, in a percentage in the composite mixture equal
to
10.0%. The average diameter of the nanoparticles is 326 nm and the
polydispersion 0.325.
3o EXAMPLE 17
The preparation of the Example 13 is repeated, using the Labrafac HydroT""
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polyhydroxylated triglyceride, in a percentage in the composite mixture equal
to
10.0% and incorporating in the composite mixture 3.87 mg of calcitonin in 3.8
g of
the mixture itself. The average diameter of the nanoparticles is 326 nm and
the
polydispersion 0.325.
s EXAMPLE 18
2.18 g of stearic acid and 67 mg of LabrafilT"" M 2130CS polyhydroxylated
triglyceride (Labrafil percentage in the composite mixture: 3.1 %) are mixed,
and
they are heated to about 75 °C with the formation of the melted
composite
mixture. The mixture is solidified by cooling. In 10 ml of water, acidified to
pH 3,
io 1.34 g of sodium taurodeoxycholate and 3.85 mg of salmon calcitonin are
dissolved, the solution is heated to 72 °C and it is added to the
melted composite
mixture at about 72 °C, to which 1 ml of n-butanol had been added. To
the so
formed emulsion kept urider stirring, 4 ml of Tween 20 are added, to form a
transparent and anisotropic microemulsion. The microemulsion, heated to about
is 50 °C is dispersed in 50 volumes of water at pH 3 cooled to about 3-
5 °C, under
constant stirring (250 rpm), to form the solid nanoparticles containing
calcitonin.
The suspension is washed by ultrafiltration in order to remove the surfactant
excess. The calcitonin incorporation efficacy in the nanoparticles is about
11.5 %.
The average diameter of the nanoparticles is 185 nm and the polydispersion
index
2o is 0.300.
EXAMPLE 19
The preparation of the Example 18 is repeated without the incorporation of
calcitonin. The average diameter of the composite nanoparticies is 182 nm and
the polydispersion index 0.295.
2s EXAMPLE 20
The preparation of the Example 18 is repeated, using the Labrafil 2130CS
polyhydroxylated triglyceride at 8.9% in the composite mixture. The composite
mixture is melted at about 70 °C. The average diameter of the
nanoparticles is
173 nm and the polydispersion index 0.268.
3o EXAMPLE 21
The preparation of the Example 18 is repeated, using the Labrafil 2130CS
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polyhydroxylated triglyceride at 10% in the composite mixture. The composite
mixture is melted at 66 °C. The average diameter of the nanoparticles
is 216 nm
' and the polydispersion index 0.287.
EXAMPLE 22
s The preparation of the Example 18 is repeated, using the Labrafil 2130CS
polyhydroxylated triglyceride at 15% in the composite mixture. The average
diameter of the nanoparticles is 188 nm and the polydispersion index 0.247.
EXAMPLE 23
The preparation of the Example 18 is repeated, using the Labrafil 2130CS
to polyhydroxylated triglyceride at 50% in the composite mixture and the
composite
mixture is melted at 60 °C. The average diameter of the nanoparticles
is 312 nm
and the polydispersion index 0.424.
EXAMPLE 24
The preparation of the Example 18 is repeated, using the Labrafil 2130CS
is polyhydroxylated triglyceride at 75% in the composite mixture and the
composite
mixture is melted at 54 °C. The average diameter of the nanoparticles
is 162 nm
and the polydispersion index 0.315.
EXAMPLE 25
The preparation of the Example 18 is repeated, using the Labrafil 2130CS
2o pofyhydroxylated triglyceride at 95% in the composite mixture and the
composite
mixture is melted at 35 °C. The average diameter of the nanoparticles
is 205 nm
and the polydispersion index 0.281.
EXAMPLE 26
1.5 g of stearic acid and 0.5 g of decanoic acid (decanoic acid percentage in
the
2s mixture: 25%) are mixed, and the mixture is heated to about 75 °C
with the
formation of the composite mixture. The mixture is solidified by cooling. In
10 ml of
water acidified to pH 3 1.30 g of sodium taurodeoxycholate are dissolved and
the
solution heated to 55 °C is added to the melted composite mixture at
about 55 °C,
to which 1 ml of n-butanol had been added. To the so formed emulsion, kept
3o under stirring, 2.6 ml of Tween 20 are added to form a transparent and
anisotropic
microemulsion. The microemulsion maintained at about 45 °C is dispersed
in 50
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22
volumes of water at pH 3 cooled to about 3-5 °C, under constant
stirring (250
rpm), to form low melting solid nanoparticles. The suspension is washed by
ultrafiltration in order to remove the surfactant excess. The average diameter
of
the nanoparticles is 280 nm.
s EXAMPLE 27
The preparation of the Example 26 is repeated, using the decanoic acid at 50%
in
the composite mixture. The composite mixture is melted at 50 °C. The
average
diameter of the nanoparticles is 310 nm and the polydispersion index 0.280.
EXAMPLE~8
io The preparation of the Example 26 is repeated, using the decanoic acid at
75% in
the composite mixture. The composite mixture is melted at 35 °C. The
average
diameter of the nanoparticles is 300 nm and the polydispersion index 0.250.
EXAMPLE 29
1.8 g of stearic acid and 0.2 g of polyethylene glycol PEG 4000 (PEG 4000
is percentage in the mixture: 10.0%) are mixed, and they are heated to about
75 °C
with the formation of the composite mixture. In 10 ml of water, acidified to
pH 3
1.30 g of sodium taurodeoxycholate are dissolved and the solution heated to 50
°C is added to the melted composite mixture at about 50 °C, to
which 0.5 ml of n-
butanol had been added. To the so formed emulsion, kept under stirring, 2.4 ml
of
2o Tween 20 are added to form a transparent and anisotropic microemulsion. The
microemulsion maintained at about 45 °C is dispersed in 50 volumes of
water at
pH 3, at about 3-5 °C, under constant stirring (250 rpm), to form the
composite
lipidic solid nanoparticles. The suspension is washed by ultrafiltration in
order to
remove the surfactant excess. The average diameter of the nanoparticles is 184
2s nm and the polydispersion 0.302.
EXAMPLE 30
The preparation of the Example 29 is repeated using the polyethyleneglycol PEG
4000 at 20% in the composite mixture. The composite mixture is melted at 50
°C.
The average diameter of the nanoparticles is 263 nm and the polydispersion
index
30 0.334.
EXAMPLE 31
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The preparation of the Example 29 is repeated, using the poly-(propyleneoxide)
poly-{ethyleneoxide) copolymer as amphiphilic material, LutroIT"" 188 at 10%
in the
composite mixture. The composite mixture is melted at 50 °C. The
average
diameter of the nanoparticles is 353 nm and the polydispersion index 0.314.
,EXAMPLE 32
The preparation of the Example 29 is repeated, using LutroIT"" 188 at 20% in
the
composite mixture. The composite mixture is melted at 50 °C. The
average
diameter of the nanoparticles is 375 nm and the polydispersion index 0.300.
EXAMPLE 33
io 1.4 g of stearic acid and 607 mg of LabrafilT"" M2130CS polyhydroxylated
triglyceride (Labrafil percentage in the mixture: 30.1 %) are mixed, and they
are
heated to about 70 °C with the formation of the composite mixture. To
such a
mixture 1.06 g of partially hydrogenated soybean lecithin (Lipoid S75-35) are
added. 10 ml of an aqueous solution at pH 3 are added to the composite mixture
is melted at about 70 °C, to which 1.0 ml of n-butanol has been added.
To the so
formed emulsion, kept under stirring, 6 ml of Tween 20 are added to form a
transparent and anisotropic microemulsion. In the microemulsion about 200 mg
of
ciclosporin are added. The microemulsion, heated to about 50 °C is
dispersed in
50 volumes of water at pH 3, cooled to about 3-5 °C, under constant
stirring (250
2o rpm), to form the solid nanoparticles containing ciclosporin. The
suspension is
washed by ultrafiltration in order to remove the surfactant excess. The
average
diameter of the nanoparticles containing ciclosporin is 304 nm and the
polydispersion index 0.365.
EXAMPLE 34
2s The preparation of the Example 33 is repeated, incorporating in the
microemulsion
100 mg of etoposide. The composite mixture is melted at 50 °C. The
average
diameter of the composite nanoparticles is 288 nm and the polydispersion
0.211.
The preparation of the Example 29 is repeated, using LutroIT"" 188 at 20% in
the
3o composite mixture. The composite mixture is melted at 50 °C and
added with
acyclovir in an amount equal to 100 mg per gram of composite mixture. The
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average diameter of the nanoparticles containing acyclovir is 360 nm and the
polydispersion index 0.302.
EXAMPLE ~6
980 mg of stearic acid and 510 mg of LabrafilT"" M2130CS polyhydroxylated
s triglyceride (Labrafil percentage in the mixture: 30%) are mixed, and they
are
heated to about 70 °C with the formation of the composite mixture. To
such a
mixture 300 mg of Q10 coenzyme (Ubidecarenone) and 1.06 g of partially
hydrogenated soybean lecithin (Lipoid S75-35) are added. 10 ml of an aqueous
solution at pH 3 are added to the composite mixture melted at about 70
°C, to
io which 1 ml of n-butanol has been added. To the so formed emulsion, kept
under
stirring, 6 ml of Tween 20 are added to form a transparent and anisotropic
microemulsion. The microemulsion, heated to about 50 °C is dispersed in
50
volumes of water at pH 3, at about 3-5 °C, under constant stirring (250
rpm), to
form the solid nanoparticles containing ubidecarenone. The suspension is
washed
is by ultrafiltration in order to remove the surfactant excess. The
ubidecarenone
incorporation percentage in the nanoparticles is 99%, the average diameter of
the
nanoparticles is 195 nm and the polydispersion index 0.214.
EXAMPLE 37
6 g of stearic acid and 0.9 g of LabrafilT"" M2130CS polyhydroxylated
trigtyceride
20 (15% of the mixture) are mixed, which are melted at a temperature about
equal to
70 °C. To the melted composite mixture 1.5 g of soybean lecithin
(Lipoid S75-35)
are added, and 300 ml of an aqueous solution at pH 5.5 containing 3 g of Tween
20. The so formed emulsion is passed in a high pressure Rannie-MiniLab 8.30
homogenizer, at a temperature equal to 70 °C and a pressure equal to
750 bar for
2s times ranging from 0 to 15 min. The dispersions are recovered and
instantaneously cooled to 4 °C, by constant stirring at 250 rpm in a
thermostated
bath, giving origin to solid nanoparticles. The average diameters are:
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2s
Process Time (min)Diameter (nm) Polydispersion
257 0.36
264 0.392
- 15 282 ~ x.400 -
EXAMPLE 38
The preparation of the Example 37 is repeated, maintaining the temperature of
the
s system, once prepared the composite material by comelting, below the melting
temperature of the composite itself (T < 50 °C). The obtained
dispersion is pre-
homogenized for 5 minutes in a low energy (Silverson mod. L2R) homogenizer-
mixer, and subsequently homogenized at high pressure (750 bar) at constant
T°
(45 °C).
1o EXAMPLE 39
0.39 g of stearic acid and 5.5 g of LabrafilT"" M2130CS polyhydroxylated
triglyceride (95% of the mixture) are mixed, which are melted to a temperature
equal to 75 °C. To the melted composite mixture 3 g of soybean lecithin
(Lipoid
S75-35) are added and 300 ml of an aqueous solution at pH 5.5 containing 12 g
of
is Tween 20. The so formed emulsion is passed in a high pressure Rannie-
MiniLab
8.30 homogenizer at a temperature equal to 70 °C and a pressure equal
to 750
bar for times ranging from 0 to 15 min. The dispersions are instantaneously
cooled to 4 °C by constant stirring at 250 rpm in a thermostated bath,
giving origin
to composite lipidic solid nanoparticles. The average diameters are:
Process Time (min)Diameter (nm) Polydispersion
3 106 0.205
5 72 0.161
10 91 0.176
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The preparation of the Example 39 is repeated, maintaining the temperature of
the
system, once prepared the composite material by comelting, under the melting
temperature of the composite itself (T < 35 °C). The obtained
dispersion is pre-
homogenized in a low energy (Silverson mod. L2R) homogenizer-mixer, and
s subsequently homogenized at high pressure (750 bar) at constant T (30
°C).
EXAMPLE 41
3.75 g of stearic acid and 3.75 g of polyethylene glycol 20000 (PEG 20000)
(50%
in the mixture) are mixed, which are melted to a temperature equal to 75
°C. To
the melted composite mixture 3 g of soybean lecithin (Lipoid S75-35) are added
io and 300 ml of an aqueous solution at pH 5.5 containing 6 g of Tween 20. The
so
formed emulsion is passed in a high pressure Rannie-MiniLab 8.30 homogenizer,
at a temperature equal to 70 °C and a pressure equal to 750 bar for
times ranging
from 1 to 10 min. The dispersions are instantaneously cooled to 4 °C by
constant
stirring at 250 rpm in a thermostated bath, giving origin to solid
nanoparticles. The
is average diameters are:
Process Time Diameter (nm) Polydispersion
(min)
156 ~ 0.274
188 0.308
EXAMPLE 42
The preparation of the Example 41 is repeated, maintaining the temperature of
the
2o system, once prepared the composite material by comelting, under the
melting
temperature of the composite itself (T < 45 °C). The obtained
dispersion is pre-
homogenized in a low energy (Silverson mod. L2R) homogenizer-mixer, and
subsequently homogenized at high pressure (750 bar) at constant T (45
°C).
EXAMPLE 43
2s The preparation of the Example 41 is repeated, with a PEG percentage equal
to
15% in the composite mixture and incorporating in the composite mixture itself
300 mg of salmon calcitonin. The efficiency of the calcitonin incorporation in
the
PEG-stearic acid mixture is equal to 35%.
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EXAMPLE 44
The preparation of the Example 42 is repeated maintaining the temperature of
the
system, once prepared the composite material by comelting below the melting
temperature of the composite itself (T < 45 °C). The obtained
dispersion is pre-
s homogenized in Silverson and subsequently homogenized at high pressure (750
bar) at constant T (45 °C).
EXAMPLE 45
A composite mixture containing 5% stearic acid and 95% LabrafilT"" M2130CS
polyhydroxylated triglyceride is prepared by comelting and cooling. To the
melted
io composite mixture (75 °C) 1 g of ibuprofen and 240 mg per gram of
mixture of
soybean lecithin (Lipoid S75-35) and an aqueous solution containing 1.2% of
Tween 20 in an amount of 40 ml of aqueous solution at pH 5.5 per gram of
composite mixture + drug are added. The so formed emulsion is treated in a
high
pressure Rannie-MiniLab 8.30 homogenizer, at a temperature equal to 70
°C and
is a pressure equal to 750 bar, for times ranging from 0 to 10 min. The
dispersions
are instantaneously cooled to 4 °C by constant stirring at 250 rpm in a
thermostated bath, giving origin to solid nanoparticles. The average diameters
are:
Process Time (min)Diameter (nm) Polydispersion
2 224 0.304
264 0.364
253 0.352
2o EXAMPLE A,~ACCORDING TO THE PRIOR ART EP 0526666A1~
4.2 grams of stearic acid are melt heated to about 72 °C. In 20 ml of
water
acidified at pH 3, 2.6 g of sodium taurodeoxycholate are dissolved and the
solution, warmed to 72 °C is added to the melted stearic acid, to which
2 ml of n-
butanol have been added. To the so formed emulsion, maintained under stirring
in
2s a mixer, 5 ml of Tween 20 are added obtaining a transparent and anisotropic
microemulsion. The microemulsion, taken to about 60 °C, is dispersed in
5
volumes of water at pH 3, at about 3-5 °C, under constant stirring (250
rpm), to
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form the nanoparticles according to the technique used in EP 0526666A1. The
suspension is washed by ultrafiltration to remove the surfactant excess. The
average diameter of the nanoparticles is 209 nm and the polydispersion index
is -
0.155.
s EXAMPLE B
The procedure of the Example A is repeated, adding 3.85 mg of salmon
calcitonin.
The incorporation efficacy of the calcitonin in the nanoparticles is 1.82%.
The
average diameter of the nanoparticles is 193 nm and the polydispersion index
is
0.235.
EXAMPLE C
The procedure of the Example B is repeated. To the so prepared nanoparticles
an
amount of amphiphilic material (dimyristoil phosphatydil glycerol, DMPG) is
added,
under stirring in a mixer, in a ratio equal to 1:10 with respect to the
lipidic mass of
the stearic acid, which is adsorbed on the surface of the nanoparticles
is themselves. The incorporation efficacy of the calcitonin on the
nanoparticles is
equal to 1.75%. Such composition according to the prior art is directly
comparable
with the Example 5 of the invention, deferring only for having the adsorbed
amphiphilic component on the surface and not as a component of the composite
material forming the nanoparticles. The suspension is washed by
ultrafiltration in
20 order to remove the surfactant excess. The average diameter of the
nanoparticles
is 215 nm and the polydispersion index is 0.175.
EXAMPLE D
1.7 g of stearic acid and 300 mg of ubidecarenone are mixed and they are melt
heated to about 70 °C. 0.5 ml of n-butanol, 1.30 g of sodium
taurodeoxycholate in
2s 10 ml of aqueous solution at pH 3, heated to 70 °C are added to the
comeited. To
the so formed emulsion, maintained under stirring, 3.25 g of Tween 20 are
added
to form a microemulsion. The microemulsion heated to about 70 °C is
dispersed in
50 volumes of water at pH 3 at about 3-5 °C under constant stirring
(250 rpm), to
form the lipidic nanoparticles according to the EP 0526666A1 technique. The
3o suspension is washed by ultrafiltration to remove the surfactant excess.
The
incorporation percentage of ubidecarenone in the nanoparticles is about 80%,
the
CA 02319565 2000-08-02
Wa'99/39700 PCT/EP99/00782
29
average diameter of the nanoparticles is 205 nm and the polydispersion index
is
0.244.
WCAMPLE E ACCORDING TO THE PRIOR ART WO 93/057681
7.5 g of stearic acid are melted at a temperature equal to 75 °C. 3 g
of soybean
s lecithin (Lipoid S75-35) are added and 300 ml of aqueous solution at pH 5.5
containing 6 g of Tween 20. The so obtained emulsion is treated in a high
pressure Rannie-MiniLab 8.30 homogenizes at a temperature equal to 70
°C and
a pressure equal to 750 bar, for times ranging from 0 to 10 min. The
dispersions
are instantaneously cooled to 4 °C giving origin to solid nanoparticles
according
io to the technique described in WO 93/05768.
The average diameters are:
Process Time (min)Diameter (nm) Polydispersion
190 0.254
178 0.308
CHARACTERIZATION EXAMPLE
is - Improvement of the incorporation characteristics of hydrophilic drugs
(peptides}
One of the innovative characteristics of the invention is the possibility to
improve
the efficacy of the incorporation of hydrophilic drugs (peptides) and to
improve the
distribution inside the composite nanoparticles. Such aspect has been shown by
2o the fluorescence techniques.
The calcitonin peptide has been marked by a fluorophor (7-nitrobenz-2oxa-
1,3diazol, NBD), according to a known technique (Biochem. J., 272, 713-719,
[1990]), and subsequently incorporated in the nanoparticles as described in
the
Examples 1-8 of the invention and the Comparative Examples B-C. The samples
2s have been washed according to the ultrafiltration procedures described in
the
Examples.
~ The percentage of peptide incorporated in the nanoparticles
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WO 99/39700 PCTlEP99/00782
~ The percentage of peptide superficially adsorbed, with respect to the
incorporated total, measuring the fluorescence before and after the treatment
of
the suspensions with the proteolytic enzyme tripsine, able to dissolve and _
degradate only the peptide fraction adsorbed on the surface of the particles
s themselves
have been calculated, by the measure of the emission values in fluorescence
with
respect to a standard curve, and with respect to 100% of fluorescence emitted
before the ultrafiltration.
I
The results, reported in the Example F, Table 1, show how the composite
io nanoparticles increase the incorporation efficacy of the peptide,
decreasing its
supeficially located fraction (adsorbed and attackable by the proteolytic
enzymes), and maintaining the majority inside the composite matrix.
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31
EXAMPLE F
Table 1. The calcitonin incorporation efficiency in composite lipidic
nanoparticles,
and percentage of peptide adsorbed on the surface of the particles themselves.
Example Materials CompositionIncorporationAdsorbed
(%) Efficiency Peptide
(%)
(%)
~1
______-TStearic
AcidT99.5
-___--,-10.5
-____-,-n.d-______~
I i
i
i
DMPG ; 0.5 i i i
2 : Stearic Acid ; 99.0~ 10.2 ~ 0.11
I I i i i
! ; DMPG ; 1.0 i i i
i
3 ; Stearic Acid ; 95.5;13.2 ~ 0.05
I I I I I
DMPG ; 4.5 ; i i
4 ; Stearic Acid ; 94.8;13.4 ~ 0.04
i I I ! i i
i ; DMPG i 5.2 ; ; i
_____--t.Stearic
Acid~91.5
-____
fi9.19
-____1-0.49
-____
I I
i
i
i
i DMPG ; 9.5 i i i
6 ; Stearic Acid ; 75 ; 9.51 ~ 3.98
I I I i
I ; DMPG ; 25 i i i
i
7 ; Stearic Acid ; 98.8; 9.9 ~ n.d.
I I i i
! I
i ; DSPA ;1.2 i i i
8 ; Stearic Acid ; 94.8;14.4 ~ n.d.
I I i i
I I ; ; i
DSPA ; 5.2
r43 t35 tn.d _
tStearic I i i
Acid~85
I I
I
PEG ; 15 i i i
B ; Stearic Acid ;100 ;1.82 ~ 0.93
! i
i
I _______ _
I ____
_ T0.95
_
_
I
_
____
_
_ ___
75
0 -
T1
~C
TStearic
AcidT90
1 . . I I
I I I
DMPG ; 10.0 i i i
- Modification of the mass characteristics
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r WC~ 99/39700 PCT/EP99/00782
32
Another innovative characteristic of the invention product is the modification
of the
mass properties of the composite materials forming the nanoparticles. The
modifications of the mass properties are quantitatively evaluated by thermal
'.
analysis techniques by a differential scanning calorimeter, DSC, Perkin Elmer
s mod. DSC 7. The nanoparticles according to the invention may be formed
either
by an homogenous composite material, or by a composite material presenting
separated phases with different properties. In the former case it is possible
for
example to observe that the homogeneous composite material shows the
,.:;
modification of ~a thermal event (melting temperature) as a function of the
io composition (Example G) (Table 2). In the.latter case more thermal events,
related
to the thermal transitions or to the melting of separated phases, occurring at
temperatures and with specific transition enthalpies for each per cent
composition
(Examples H, I) (Tables 3 and 4) are distinguishable. In both cases, the
characteristics of the composite material are unexpected and unforeseeable on
is the base of the single materials.
The modification of the thermodynamical behaviour ~ of the composite
nanoparticles as a function of the component percentage is observable also on
the nanoparticles in suspension, for example by techniques of laser light
scattering (Nlaser light scattering"). The phase transitions of the composite
2o nanoparticles are noticeable from the intensity variation of the scattering
as a
function of the temperature, measured by a Brookhaven mod. BI-90 Particle
Sizer.
By this technique, it is possible to point out how the composite nanoparticles
show
phase transitions and melting at temperatures different according to the
composition of the forming material; see: Example L, Figure 1.
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WO99/39700 PCT/EP99/00782
33
EXAMPLE G
Table 2. Melting point (°C) of binary composite mixtures of stearic
acid and
average chain fatty acids (amphiphilic) corresponding in part to the Examples
26-
28
Amphiphilic % 0 10 20 30 50 65 70 75 85 90 100
T-'T
Decanoic T
Acid T
T
T--T
T
'~'
T
;
71
:
;
60
;
56
;
50
;
43
;
;
35
;
28
;
26
;
32
c
i
i
i
i
~
~
i
Lauric Acid ; ; 69 ; ; 56 ; ; 44 ; ; ; ; 45
71 ; 62 43 42 ~ i
i i i i ~ i i i i ~
Myristic ~ ~ 68 ~ ~ 56 ~ ~ 50 ~ ~ ~ ~ 54
Acid 71 ~ 64 51 53 i i
i i i i i i i i i i
Palmitic ; : 68 ; : 60 : ; 56 ; ; ; ; 63
Acid 71 : 66 60 61 ~ i
i i > > ~ i i i i i
s
EXAMPLE H
Table 3. Phase transitions and enthalpies of the composite material stearic
acid-
dimyristoil-phosphatidyl glycerol (DMPG), and some significative compositions,
corresponding in part to the Examples 1-6.
OH (Jlg) Tf (°C) DH (J/g)
Tai (°C)
DMPG
0 _______r_ _______-T73.3 -___-.r210.5 '____~
i
_ _ i _ _ i _ _ i _ _ _
__
'____T
'____r
--___r
6 185.95
72.2
0.55
55.3
i i t i
12 _
55.0 ; 5.811 ; 71.8 160.02
i ~ i i
18 _
55.2 ; 6.71 ; 71.5 ~ 146.09
30 56.2 rnd r70.2 rnd ~ '
i
i _ i _ _ i__
-______~
-____r
--_____r
-____T
40 nd
68.8
nd
56.4
i i t i
50 57.2 ; 35.15 ; 69.7 ; 56.9
i i i i
100 ' ~ 125.21 ; nd
______! _________! _________! _________!
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34
EXAMPLE I
aI- ble 4. Phase transitions and enthalpies of the composite material stearic
acid-
Labrafil 2130 CS polyhydroxylated triglyceride, corresponding to the Examples
18-
T~ (°C) 0H (J/g) Tf (°C) DH (J/g)
s
Labrafil
0 _________~._________~73.9
-___-r210.5--___I
i I I
i
2 24. : 0.01 ; 74.1 ; 210.2
I I I i
5 25.1 ;0.4 ;73.3 ;204.4
I i
10 ___ I I _ ___r189.$-____~
25.2 ___~1.5 ___r71.6 i
-_ -__ -_ I
15 _ I _ I _ _ _
25.1-__ ___~2.5 ___r72.0 ___180.5-____~
--_ -_ 1 I
I I
50 25.3 ; 6.3 ; 65.4 ; 99.3
1 i I I
78 25.0 ; 8.4 : 56.0 ; 25.0
i
I
85 __ _ I _
I ___r55.1--____r15.2 -___
26.0 i
-____~8.5 I
--_
90 __ I I _ _ _
_ ___r52.0 ___~5.2~______~
27.1-_____,-10.2 -_ I i
'_ I
I
100 ~ 29.1 ~ 13.8 ~ 38.6 ; 0.77
i i I i
EXAMPLE L
Fi_ ucLre 1. Phase transitions of the composite nanoparticles (stearic acid-
DMPG),
wdetemiined by the scattering techniques, corresponding to the described
products
in the Examples 3-5_ and --the Comparative Example _ A;The phase transition
r--
~o (melting) is pointed out by the decrease of the intensity of the laser
light scattering
("scattering"), as a function of the temperature. The beginning of such a
variation
(withdrawal from the curve plateau) corresponds to the beginning of the
transition.
It is clear how, in the compositions according to the invention, such a
transition
may be changed and controlled varying the composition of the material forming
is the nanoparticles. The melting temperature of the particles (63 °C
in the Example
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WO 99/39700 PCT/EP99/00782
A), in fact decreases to 55 °C, 53 °C and 49 °C in
the Examples 3, 4, 5
respectively.
- Modification of the preparation characteristics (technique 1): interval of
microemulsion existence
- s The properties of the composite materials according to the invention allow
both to
obtain compositions having low melting point and to extend the existence
thermal
interval of the microemulsion, used as intermediate in the preparation of the
composite nanoparticles according to the technique 1 (Example M) (Figure 2),
at
temperatures very much lower than the melting temperature of the single
io component materials. Such interval has been determined by measuring of the
scattering frequency, which is minimum (< 10 KHz) in presence of a
microemulsion. Evident and principal advantage of the invention is to be able
to
formulate thermolable active principles in the composite nanoparticles; a
further
advantage is to be able to use, for the preparation of the nanoparticles, some
is materials having temperatures lower than their melting temperature.
EXAMPLE M
i r Thermal interval of existence of the microemulsion consisting, in oil
phase, of stearic acid and decanoic acid (preparative technique 1. See
Examples
26-28 and Example A).
20 - Modification of the surface characteristics
One of the innovative characteristics of the product of the invention is the
possibility to control the surface properties of the composite materials
forming the
nanoparticles and, consequently, of the nanoparticles themselves. The surface
characteristics of the nanoparticles obtained according to the techniques and
the
2s materials of the invention differ in substantial way with respect to the
surface
adsorption of amphiphilic components described in the state of art, because
they
are dependent from the formation of the composite material. Thus an advantage
of the invention is the possibility to obtain composite nanoparticles having
favourable characteristics of biocompatibility, hydrophobicity, hydropilicity
and
3o polarity according to the therapeutical aim to achieve.
The surface properties of the composite materials forming the nanoparticles
may
CA 02319565 2000-08-02
WO '99/39700 PCT/EP99/00782
36
be measured by the angle of contact method, using a Lorentzen & Wettre
apparatus. Such a method allows to calculate the surface energy of the
materials,
correlable to the wettability, through the equation (1 ),
Ysv = Ysl + Ylv (~os 8) (1 )
s wherein the indexes sv, sl, Iv, refer respectively to the surface energy (Y)
of the
solid-vapour, solid-liquid and liquid-vapour surfaces, and 8 is the angle of
contact
of the liquid with the solid surface. As the superficial free energy of each
material
may be divided into a polar component and a dispersion component, according to
(2):
ao Yt = YP + yd (2)
it is possible to calculate, from experimental measures elaborated by a series
of
equations, the values of such components and of the total free energy, as well
as
the polarity of the surfaces, expressed in percentage as:
%P = (YP ~ Yt ) * 100 (3)
is From the surface energy measures of the composite materials according to
the
invention it has been possible to determine that the amphiphilic component
may:
1 ) be preferentially arranged on the surface of the nanoparticles formed by
the
composite material ("segregated on the surface°);
2) be preferentially arranged inside the nanoparticles formed by the composite
2o material (°segregated inside");
3) be arranged homogeneously on the surface and inside the nanospheres
themselves.
The diagrams of the surface energies of the composite mixtures: stearic acid
DMPG (Example N), forming the nanoparticles described in the Examples 1-6,
2s stearic acid-Labrafac HydroT"" polyhydroxylated triglyceride (Example O),
forming
the nanoparticles described in the Examples 13-17, stearic acid-Labrafac Lipo
caprilic-capric triglyceride (Example P) are reported for exemplificative aim
in the
Figures 3, 4 and 5. In the Figures 3, 4 and 5 the curves A, B and C refer
respectively to the total surface free energy, to the dispersion component and
to
3o the polar component.
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WO 99/39700 PCT/EP99/00782
37
EXAMPLE N
Fi ur . Trend of the surface energy of the stearic acid-DMPG composition,
- referable to the Examples 1-6. The trend, characterized by a decrease of the
total
energy, shows a "preferential surface segregation" of the amphiphilic
component
_ s DMPG in the composite mixture.
EXAMPLE O
Figure 4. Trend of the surface energy of the stearic acid-Labrafac Hydro
composition, referable to the Examples 13-17. The trend shows a "preferential
inside segregation" of the amphiphilic component Labrafac Hydro in the
composite
io mixture (and of the stearic acid component inside).
EXAMPLE P
i r Trend of the surface energy of the stearic acid-Labrafac Lipo
composition. The invariant trend shows a homogeneous distribution of the
amphiphilic component Labrafac Lipo in the composite mixture.
is The variation of the surface properties of the composite nanoparticles as a
function of the used composite materials composition, is quantifiable also by
Zeta
potential measures carried out by a laser light scattering electrophoretic
analyzer,
ZetaMaster (Malvern, UK), on the aqueous suspensions of the composite
nanoparticles. The Zeta potential (shear plane potential) is a measure of the
2o surface charge of the nanoparticles in suspension. It is clear from the
Examples
Q-T that the Zeta potential of the nanoparticles, and consequently the surface
properties, range as a function of the amphiphilic material percentage in the
composite, with a trend proper to each composition.
The possibility to modify the surface properties and the Zeta potential of the
2s nanoparticles of the invention has moreover the important advantage to
decrease
or remove the sedimentation or the aggregation trend of the suspensions,
increasing then the stability. Such advantage may be then obtained modifying
the
composition of the material forming the nanoparticles themselves.
TALE 5. (EXAMPLES Q-T)
3o EXAMPLE Q
Zeta potential (mV) measured on the stearic acid-DMPG nanoparticles, prepared
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WO 99/39700 PCT/EP99/00782
38
analogously as the Examples 2-5 and A.
EXAMPLE R
Zeta potential (mV) measured on the stearic
acid-DMPG nanoparticles, prepared
analogously as the Examples 9-11 and .
A.
s EXAMPLE S
Zeta potential (mV) measured on the stearic acid-Labrafac Hydro
polyhydroxylated triglyceride nanoparticles,prepared analogously as the
Examples 13-16 and A.
EXAMPLE T
~o Zeta - potential (mV) measured on stearic acid-Labrafil 2130CS
the
polyhydroxylated triglyceride nanoparticles,prepared analogously as the
Examples 19-24 and A.
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WCs 99139700 PCT/EP99/00782
39
Table 5
Component DMPG DMPC Labrafac Labrafil
Hydro 2130CS
Q R S T
in Zeta Zeta Zeta Zeta
Composite PotentialPotential Potential Potential
(mV) (mV) (mV) (mV)
i i i i _ i
0 ~ -30.3(Ex. ; -30.3 ; -30.3
i (Ex. A) (Ex.
i A) ;
-30.3
(Ex.
A
i i i
i A) i i i i
1 -30.0 (Ex. ; -30.1 ~ -31.4 (Ex. ;
i i ~ i
i 2) i i 13) i i
i
1.5 ; ' ~ -33.7
i i i i i
2 ' ' ' -34.1
i i i i
2.5 ~ -30.3~ ~ -34.0 (Ex.
i i i i i
i i i 14) i i
3 ' -31.7' ' ' -21.2 (Ex.
i i i i i
i i i i 19) i
4 ' ' -32.2 (Ex '
i 9) i i i
i
4.5 ' -34
(Ex. i i i
3)
i i
~ -35.4(Ex. ; ; -34.2 (Ex. ;
i i -13.6 i
i
i 4) i i 15) i i
i
7 ~ -38.2~ -31.9 (Ex.
i i i i i
i i 10) i i i
~-40.4(Ex.' -33.7 . ; -33.6(Ex. ; (Ex.
(Ex i -11.3 i
r
i i ;16) i21)
;5) ;11)
12 t__~ ____t_34.3 '____...t._______~..t__~ _____.~
i i i i i
-; ~ ' -14.1 (Ex.
i i i i
i i ; ; 22)
Z________~_________..J-____-. ____.1_________J
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WO '99/39700 PCT/EP99/00782
Component ~ ~ DMPG ~ DMPC ~ Labrafac ~ Labrafil
Hydro ~ 2130CS
-19.8 (Ex.
i i i i
i i i i 23) i
_-____~._-___-__~t_-___-__-.t_ _- ...1
75 -18.9 (Ex. ,
i i i i i
24)
100 , , . . -25.4
- Composite nanoparticles morphology according to the invention and
according to the prior art.
In confirmation of the substantial differences between the nanoparticles
according
to the invention and the prior art, the morphology of some significant samples
has
s been observed (corresponding to the Example 6 and the Example A), by the
transmission electronic microscopy technique (TEM), using the "negative
coloration" (uracil citrate) process for the preparation of the nanoparticle
samples.
In Figure 6 the different morphology of the composite nanoparticles is evident
(Example 1, Fig. 6a), with respect to those ones prepared according to the
prior
~o art (Example A Fig. 6b): morphologically different, clear and dark areas
are in fact
noticed on the particles in 6a, corresponding to the phases of the composite
material ("preferential segregation"), while the system 6b appears
homogeneous.
Further ingredients of the sample corresponding to the Example 1 (Figure 7a
and
7b) suggest that the material present on the surface is different from a layer
of
is amphiphilic material adsorbed on the surface of the particle, but it is
integral part
of the particle itself.
- Improvement of the oral absorption of peptides (pharmacokinetics and
therapeutical efficacy)
In order to show an important advantage of the invention, composite lipidic
2o nanoparticles containing salmon calcitonin as active principle have been
administered °in vivo" (Examples 2, 4, 5), and nanoparticles containing
calcitonin
prepared according to the prior art (Examples A, B, C).
DOSAGE PROTOCOL AND SAMPLE ANALYSIS
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WO 99/39700 PCT/EP99/00782
41
Composite nanoparticles suspensions according to the invention (20 ml)
.'n
containing calcitonin (specific activity: 4000 Ullmg; nominal dose: 600 Ul/kg,
- incorporated effective dose in the nanoparticles: 60-80 Ul/kg), have been
administered per os to 4 Rhesus macaques. Samples of blood (1.5 ml) were taken
- s at determined times and immediately analyzed after the taking.
Pharmacokinetics: the peptide concentration in the plasma was determined by a
specific radioimmunoassay (RIA), expressed as milliunits/ml (mUl/ml).
Therapeutical Efficacy: the levels of the total calcium in the plasma were
determined by a colorimetric kit (Cobas Mira, Roche, CH). The levels of
ionized
to calcium were determined by injection in a device equipped with ion-
selective
membranes (IG Radiometer, Copenhagen, DK). The results were expressed as
per cent variation of the calcium levels with respect to the base line.
The results are reported in the Figures 8-11 related to the Examples U-Z1
reported below.
is EXAMPLE U
i r . Kinetics of absorption of calcitonin formulated in compositions
according
to the invention (Examples 2, 4, 5) and in compositions according to the prior
art
(Examples A, B, C), after oral administration.
EXAMPLE V
2o Figure 9. Kinetics of total calcium variation in blood after per os
administration of
calcitonin formulated in compositions according to the invention (Examples 2,
4, 5)
and in compositions according to the prior art (Examples A, B, C).
EXAMPLE Z
Figure 10. Kinetics of ionized calcium variation in blood after per os
administration
2s of calcitonin formulated in compositions according to the invention
(Examples 2, 4,
5) and in compositions according to the prior art (Examples A, B).
EXAMPLE Z1
Figure 11. Bioavailability, expressed as AUC (0-8 hours) (area subtended to
the
plasmatic kinetics curve) related to the oral administration of calcitonin
formulated
3o in compositions according to the invention (Examples 2, 4, 5, 7) and in
compositions according to the prior art (Examples A, B and C).
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WO 99/39700 PCT/EP99/00782
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It turns out to be evident from the pharmacokinetical reported tests that the
bioavailability of the calcitonin formulated in composite nanoparticles
according to
the invention and administered per os, is significatively increased (to 13.5
times)
with respect to the formulations of the prior art. Moreover, it is clear that
the
s nanoparticles according to the invention allow, beside an absorption
increment,
also a control of the peptide absorption which is present in active form in
plasma
to 8 hours from the administration. The therapeutical efficacy tests,
moreover,
point out how the effect on the haematic calcium is greater for the
formulations of
the invention (Examples 2, 4, 5) with respect to the formulations according to
the
io prior art (Examples A, B, C), and it does not depend on the simple mixture
of the
components (Example C), but on the composite material presence.
