(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual P roperty O rganization International Bureau (43) International Publication D ate 11 J a n u a r y 2001 (1 1 .0 1 .2 0 0 1 ) (51) International Patent Classification7: A61K 9/51, C08J 3/07 (21) International Application Num ber: (22) InternationaIFilingD ate:
WO 01/02087 A1 (10) International P ub lication N um ber
PCT B O lJ 13/04,
PCT/EP99/04677
6 July 1999 (06.07.1999)
(25) Filing Language:
English
(26) Publication Language:
English
(71) Applicant (for all designated States except US): UNIVERSITE DE GENEVE LABORATOIRE DE PHARMACIE GALENIQUE [CH/CH]; Section de Pharmacie, 30, quai Emest-Ansermet, CH-1211 Geneve 4 (CH).
WO 01/02087 Al
(72) Inventors; and (75) Inventors/Applicants (for US only): QUINTANAR-GUERRERO, David [MX/MX]; Rio Panuco 38, Colinas de Lago., Cuautitlan Izcalli, Mexico, 54744 (MX). WO 01/02087 A1ALLEMANN, Eric [CH/CH]; 113, route des Hospitaliers, CH-1257 Croix-de-Rozon (CH). GURNY, Robert [CH/CH]; 7, rue Calvin, CH-1204 Geneve (CH). FESSI, H atem [FR/FR]; 40, rue d’Aubigny, F-69003 Lyon (FR). DOELKER, Eric [CH/CH]; 56, Chemin de Conches, CH-1231 Conches (CH).
W O 01/02087 Al
(74) Agents: KLIGELE, B ernhard et al.; Novapat Interna tional SA, 9, rue du Valais, CH-1202 Geneve (CH). (81) Designated States (national): AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW. (84) Designated States (regional): ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, Cl, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG). Published: — With international search report. For two-letter codes and other abbreviations, refer to the "Guid ance Notes on Codes and A bbreviations " appearing at the begin ning o f each regular issue o f the PCT Gazette.
(54) Title: METHOD FOR PRODUCING AQUEOUS COLLOIDAL DISPERSIONS OF NANOPARTICLES (57) Abstract: The method for producing aqueous colloidal dispersions of nanoparticles comprises a first step which consists to emulsify a partially water-soluble organic solvent, containing a water-insoluble polymer in a weight/volume percentage at which nanoparticles are formed in a second step, in an aqueous solution containing optionally a stabilizing agent, using a low energy source, and a second step which consists to distillate the organic solvent from the oil-in-water emulsion formed in the first step, to cause the formation of nanoparticles, in suspension in the aqueous phase.
WO 01/02087
PCT/EP99/04677
I METHOD FOR PRODUCING AQUEOUS COLLOIDAL DISPERSIONS OF NANOPARTICLES The invention pertains to the area of dispersions in a 5
liquid phase of water-insoluble materials,
and more
particularly the invention relates to a method for producing aqueous colloidal dispersions of nanoparticles, using an emulsification-diffusion type technique. 10
Aqueous colloidal dispersions of nanoparticles,
and in
particular pseudolatexes which are aqueous colloidal, dispersions containing nanoparticles of water insoluble preformed polymers,
are currently offered for use as
aqueous coating means or as pharmaceutical vectors. 15 At present,
various techniques are known for preparing
aqueous colloidal dispersions containing nanoparticles, in particular pseudolatexes. emulsification-evaporation, 20
and emulsification-diffusion,
These techniques include nanoprecipitation,
phase during preparation,
salting-out
which have in common that
they involve the use of an organic solution, nanoparticle components,
and
containing the
which functions as an internal and of an aqueous solution,
containing stabilizers which will constitute the dispersion 25
medium for the nanoparticles. The emulsification-evaporation technique is a wellestablished technique based on the classical procedure disclosed in U S - A - 4 .1 7 7 .177
30
technique,
(Vanderhoff). In this
a polymer solution in a water-immiscible organic
solvent such as chloroform or methylene chloride is emulsified in an aqueous phase containing emulsifiers. This crude emulsion is then submitted to a high energy mixing step using a high energy source such as ultrasounds, 35' homogenizers,
high pressure dispersers,
micro fluidizers,
colloid mills or
in order to reduce the droplet size. The
CONFIRMATION COPY
WO 01/02087
PCT/EP99/04677
2
polymer emulsion resulting from such a treatment is very fine and contains very small droplets (below 0.5 Οπϊ in diameter). This emulsification is followed by the removal of the solvent, by vacuum distillation, producing a fine 5
aqueous dispersion of nanospheres. In this emulsificationevaporation method, each emulsion droplet will form one polymer particle when the solvent is removed. Consequently, the homogenization step is the determining factor in obtaining submicronic particles.
10
In order to avoid the problem of the homogenization step, which requires high energy, other techniques have been developed.
15
The nanoprecipitation technique was disclosed in EP-A-O.274.961, EP-A-0 .275.796 and EP-A-0 .349.428. In this method, polymer, drug and, optionally, a lipophilic stabilizer (e.g., phospholipids) are dissolved in a it
semipolar water-miscible solvent, such as acetone or 20
ethanol. This solution is poured or injected into an aqueous solution containing a stabilizer (e.g., poly(vinyl alcohol)
(PVAL) or poloxamer 188) under magnetic stirring.
Nanoparticles are formed instantaneously by the rapid diffusion of the solvent, which is then eliminated from the 25
suspension under reduced pressure. The usefulness of this simple technique is limited to water-miscible solvents in which the diffusion rate is sufficient to produce spontaneous emulsification. Also,
30
this technique can be used only for drugs soluble in this type of solvents. A major drawback with this technique is the difficulty to choose a drug/polymer/solvent/non-solvent system in which non-aggregated nanoparticles would be formed and the drug efficiently entrapped.
WO 01/02087
PCT/EP99/04677
3 The salting-out technique was first disclosed in the International Patent Application WO 88/08011. This technique is based on the separation of a totally water-miscible solvent, in particular acetone, from aqueous 5
solutions via a salting-out effect. Typically, the polymer and the drug are dissolved in acetone and this solution is emulsified under vigorous mechanical stirring in an aqueous gel containing the salting-out agent and a colloidal stabilizer. This oil-in-water emulsion is diluted with a
10
sufficient volume of water or of aqueous solution, in order to enhance the diffusion of acetone into the aqueous phase, thus inducing the formation of nanospheres. The utility of this technique is however generally limited
15
to drugs soluble in water-miscible solvents, in particular acetone-soluble drugs, to salting-out agents that enable phase separation without precipitation and to soluble stabilizers which are compatible with saturated aqueous solutions and which do not coacervate or precipitate in the
20
presence of the solvent. A major drawback is the use of a high quantity of salt which gives to the aqueous phase a fixed pH and which must be eliminated in a subsequent purification step. Another drawback is that it is necessary to remove the solvent and a considerable amount of water to
25
obtain a high polymer concentration in the final dispersion. Moreover, the acetone is mixed in the water which renders recycling of acetone problematic. The more recent technique for preparing nanoparticles is
30
the emulsification-diffusion technique proposed in Eur. J. Pharm. Biopharm, 41 14-18 (1995). The method involves the emulsification of a partially water-soluble (partially water-miscible) solvent, previously saturated with water and containing a polymer, in an aqueous phase
35
previously saturated with the solvent and containing a
WO 01/02087
PCT/EP99/04677
4 stabilizer. The subsequent addition of water to the system causes the solvent to diffuse into the external phase, resulting in the aggregation of polymer in nanoparticles. This method is of interest from a technological standpoint, 5
since it does not need comminuting forces as in the emulsification-evaporation technique does, it is highly efficient, reproducible and easy to scale-up. However, as in the salting-out technique, it is necessary
10
to remove the solvent and a considerable amount of water to obtain a high polymer concentration in the final dispersion. It is an object of the present invention to provide a
15
method for producing an aqueous colloidal dispersion
containing a high concentration of nanoparticles which is not affected by one or more of the above drawbacks of the known methods, and, in particular, which does not require +.
high shear forces and which does not require the removing 20
of toxic solvent, of stabilizer, of salting-out agent and/or of a considerable amount of water. The object of the present invention is achieved by a method for producing an aqueous colloidal dispersion of
25
nanoparticles, characterized in that it comprises : a) the emulsification of a partially water-soluble organic solvent, containing a water-insoluble material in a weight/volume percentage at which nanoparticles are formed in step b ) , in an aqueous solution containing optionally a
30
stabilizing agent, using a low energy source; b)
the distillation of the organic solvent from the
oil-in-water emulsion formed in step a) to cause the formation of nanoparticles in suspension in the aqueous phase .
WO 01/02087
PCT/EP99/04677
5
Thanks to the present invention, there is provided a method effective for producing aqueous colloidal dispersions containing a high concentration of nanoparticles, which is based on the emulsification-diffusion technique, which does 5
not require the use of high energy source for homogenization, which does not require the removing of a considerable amount of water, in which pharmaceutically acceptable organic solvents may be used with a possibility of solvent reuse, in which pharmaceutically acceptable
10
stabilizers may be used or not, and which is simple fco., implement, easy to scale up, of a low cost and reproducible . An advantage of the aqueous colloidal dispersions having a
15
high concentration of nanoparticles obtained by the method of the present invention is that they can be used directly for coatings or as pharmaceutical vectors without additional purification step.,*
20
The method of the present invention can be advantageously applied for producing aqueous colloidal dispersions of nanoparticles with ingredients entrapped therein, when the partially water-soluble organic solvent further contains additional ingredients.
25
It is to be noted that in the present description and claims, the terms "a partially water-soluble organic solvent" or "a partially water-miscible organic solvent" mean an organic solvent at least sparingly soluble in water 30
in the sense of the European Pharmacopoeia or a mixture of organic solvents containing at least one organic solvent at least sparingly soluble in water in the sense of the European Pharmacopoeia.
WO 01/02087
PCT/EP99/04677
6 Also, it is to be noted that in the present description and claims, the term "nanoparticles" means particles having a mean particle size not greater than I ΟπΚ. 5
We will now describe the present invention in a more detailed manner. To obtain an aqueous colloidal dispersion of nanoparticles according to the present invention, the first steps are to
10
prepare the organic phase, also named internal phase, and the aqueous phase, also named external phase. The organic phase is prepared by dissolving the selected water-insoluble material, and optionally additional
15
ingredients, in a partially water-soluble organic solvent. The aqueous phase is prepared separately, by providing water and optionally dissolving the selected stabilizing agent in water.
20 In a preferred embodiment of the present invention, the partially water-soluble organic solvent is previously added with a certain amount of water extending up to saturation and in a particularly preferred embodiment of the 2-5
invention, the partially water-soluble organic solvent is previously saturated with water in order to ensure initial thermodynamic equilibrium in the subsequent step. Preferably, the water is previously added with a certain
30
amount, extending up to saturation, of the same or another partially water-soluble organic solvent, and more preferably the water is previously saturated with the same or the other partially water-soluble organic solvent in order to ensure initial thermodynamic equilibrium in the
35
subsequent step. This means that the partially water-soluble organic solvent added to water may be identical or different to the
WO 01/02087
PCT/EP99/04677
7 partially water-soluble organic solvent in which waterinsoluble material is to be dissolved. In an other preferred embodiment of the present invention, 5
the water is previously added with a certain amount of a water-soluble organic solvent
(for example et ha nol ).
The water-insoluble material may be, for example, a polymer, a lipid, a wax and the like, or a mixture of two 10
or more polymers and/or lipids and/or waxes In a preferred embodiment of the invention,
and the like. the water-"
insoluble material is a polymer or a mixture of polymers, and in a particularly preferred embodiment, 15
the water-
insoluble material is a polymer selected from biodegradable polymer such as poly(D,L-Iactic acid) caprolactone)
(PLA) and poly(s-
(PCL) and non-biodegradable polymers such as
Eudragit速E, Eudragit速RS,
Eudragit速RL, cellulose acetate
phthalate (CAP), cellulose acetate trimellitate (CAT), and 20
ethylene vinyl acetate copolymer (EVAC). The partially water-soluble organic solvents should be selected on the basis of their volatility and low toxicity, in particular when a pharmaceutical application for the
25
resulting aqueous colloidal dispersion is considered. Examples of partially water-soluble organic solvents particularly preferred are ethyl acetate (AcEt) , methyl acetate (MeAc), isopropyl acetate and 2-butanone, because
30
of their widely recognized low toxicity, good solubilizing
properties and low boiling points. The additional ingredients may be any ingredients which can be entrapped in the nanoparticles or adsorbed at the 35
surface of the nanoparticles, such as for example drugs, cosmetics, products for veterinary and agricultural use, food products or additives, colorants or any other
WO 01/02087
PCT/EP99/04677
8 ingredients which can be useful when they are entrapped in nanoparticles or adsorbed at the surface of nanoparticles. When used, stabilizing agents particularly preferred for 5
the emulsification and for stabilizing the final dispersion are poly(vinyl alcohol)
(PVAL) and poloxamer 4 07 because of
their good water solubility, suitability for ingestion and compatibility with the system. 10
When used, the stabilizing agent is contained in the aqueous phase preferably in an amount of not more than 5 % w/v, and more preferably in an amount of not more than 1.25 % w/v.
15
The above-mentioned aqueous phase is then mixed with the organic solution of the material, using a low energy source such as, for example, a propeller, a magnetic stirrer, a shaker and the like, to produce, when the addition is finished, an emulsion of the oil-in-water type.
20 When using a propeller, a particularly advantageous stirring rate for the propeller is from 1500 to 2000 rpm. However, a stirring rate up to 5000 rpm may be acceptable. 25
The emulsification step is advantageously carried out at room temperature, but other temperatures may be used. In accordance with the invention, distillation of the 30
organic solvent from the oil-in-water emulsion is then carried out, to cause the displacement of the partially water-soluble organic solvent of the internal phase into the external phase and, consequently, to cause the formation of the particles in suspension in the aqueous
35
phase. In a particularly preferred embodiment of the present invention, the distillation is a vacuum distillation.
WO 01/02087
PCT/EP99/04677
9
In the method of the present invention, the formation of nanoparticles is highly dependent on the water-insoluble material concentration in the internal phase and a transition from nano- to microparticles is observed at high 5
water-insoluble material concentration. For this reason, the amount of water-insoluble material in the organic phase is critical for achieving the mean particle size desired for the final particles.
10 Thus, in the method of the present invention, the weight/volume percentage of the water-insoluble material in the organic solvent should be not greater than the critical weight/volume percentage at which the particles formed 15
during distillation have a mean particle size of I μιη. This critical percentage varies depending on the material/solvent/stabilizer system or material/solvent system and on the stirring rat‘e of the propeller,
20
in view
of the known effect of an increased stirring rate on decreasing the particle size. The critical percentage at which the particles formed during distillation have a mean particle size of I μπι is
25
easily obtained by an optimization for each material/solvent/stabilizer system or material/solvent system at a specific stirring rate. To illustrate the effect of the concentration of the water-
30
insoluble material in the organic solvent on particle size,
in the present invention, we will now explain the present invention, referring to Figures related to two examples of a non-biodegradable polymer particularly preferred of the present invention, named Eudragit®E-100 (obtained from 35' Rohm GmbH, Darmstadt, Germany), which
commercially available as a colloidal
however is not yet dispersion, and to an
WO 01/02087
PCT/EP99/04677
10 example of another non-biodegradable polymer, named cellulose acetate phthalate (CAP). Figure I represents the influence of the percentage of 5
Eudragit
E in the internal phase (ethyl acetate) on the
mean particle size, when the external phase contains 5 % w/v of PVAL as stabilizer and the rate of stirring is 150 0 rpm. 10
Figure 2 represents scanning electron micrographs (at x 4500) of Eudragit速E particles, prepared at different concentrations of Eudragit速E in the internal phase, namely : a) 10 % w/v in chloroform (Comparative Example hereafter)
15
b)
40 % w/v in ethyl acetate,
c)
3 0% w/v in ethyl acetate;
d)
20 % w/v in ethyl acetate (Example
3 hereafter);
e)
10 % w/v in ethyl acetate ;(Example
I hereafter)
when the external phase contains 5 % w/v of PVAL as 20
stabil izer and the stirring rate is 1500 rpm. Figure 3 represents the influence of the percentage of cellulose acetate phthalate in the internal phase (2-butanone) on mean particle size when the external phase
25
contains 5 % w/v of poloxamer 407 and the stirring rate is 1500 rpm. Figure 4 represents the influence of the percentage of Eudragit E in the internal phase (methyl acetate) on mean
30
particle
size when the external phase contains no
stabilizing agent and the stirring rate is 2000 rpm. The particle sizes were determined using a Coulter速 Nanosizer (Coulter Electronics, Harpenden (UK)).
35. For the scanning electron microscopy (SE), a concentrated dispersion of nanoparticles was finely spread over a slab
WO 01/02087
PCT/EP99/04677
11
and dried under vacuum. The sample was shadowed in a cathodic evaporator with a gold layer (~20 nm thick). The surface morphology of the nanoparticles was observed by SEM using a JSM-6400 scanning electron microscope (JEOL, Tokyo, 5
Japan). It is shown in Figure I that, for an Eudragit®E/ethyl acetate/PVAL system, when the PVAL content of the external phase is 5.00 % and the stirring rate of the propeller is
10
1500 rpm, the critical weight/volume percentage of Eudragit®E to ethyl acetate to obtain a mean particle size of 1000 nm is about 28 % w/v. However, in slightly different experimental conditions, this percentage varies.
15
The decrease of the size of the particles in relation with the diminution of the concentration of Eudragit®E in ethyl acetate is clearly apparent from Fig. 2 b) to Fig. 2 e) . Also, it is clear from Fig. 2/e) that only nanoparticles are obtained when Eudragit®E is contained in a partially
20
water-soluble organic solvent such as ethyl acetate at 10 % w/v or less. Conversely, in the Comparative Example when Eudragit®E is contained in a non water-miscible solvent such as
25
chloroform at a 10 % w/v, only microparticles are obtained, as shown in Fig. 2 a). Another non-biodegradable polymer preferred for the present invention is cellulose acetate phthalate (CAP).
30
It is shown in Figure 3 an example of a CAP/2-butanone/poloxamer system when the poloxamer 407 content of the external phase is 5.00 % w/v and the stirring rate of the propeller is about 1500 rpm. In this 35
case, the critical weight/volume percentage of CAP to 2-butanone to obtain a mean particle size of 1000 nm is
WO 01/02087
PCT/EP99/04677
12 about 38 % w/v. However, in slightly different experimental conditions, this percentage varies. It is shown is Fig. 4 an example of a Eudragit®E/methyl 5
acetate system when the internal phase contains no stabilizing agent and the stirring rate is about 2000 rpm. In this case, the critical weight/volume percentage of Eudragit®E to methyl acetate to obtain a mean particle size of 1000 nm is about 21 % w/v. However, in slightly
10
different experimental conditions, this percentage varies. As indicated above, it is pointed out that in the present invention, the water-insoluble materials are not limited to water-insoluble polymers.
15
The Examples below will illustrate the method of the present invention without limiting its scope in any way. EXAMPLES 20
Materials Water insoluble polymers used in the Examples were Eudragit®E, Eudragit®RS and Eudragit®RL (obtained from Rohm (GmbH Darmstadt, Germany); poly(ε-caprolactone)
(PCL)
((Tone®767) obtained from Union Carbide (Danbury, USA)); 25
ethylene vinyl acetate copolymer (EVAC, vinyl acetate content 40 %) (obtained from Fluka (Buchs, Switzerland)); poly(D,L-Iactic acid)
(PLA)
((Medisorb® obtained by
Medisorb (Cincinatti, OH, USA)); cellulose acetate phthalate (CAP) 30
(obtained from Fluka (Buchs, Switzerland));
and cellulose acetate trimellitate (CAT)
(obtained from
Eastman (Kingsport, USA)); Stabilizing agents used in the present Examples were poly(vinyl alcohol) 35
(PVAL)
(Mw 26 000)
(Mowiol®4-88,
Hoechst, Frankfurt-am-Main, Germany) and poloxamer 407 (Pluronic®F-127, BASF, Ludwigshafen, Germany).
WO 01/02087
PCT/EP99/04677
13
The partially water-soluble solvents used in the present Examples were ethyl acetate (EtAc)
(η20 = 1.372; water
solubility = 10 mg/ml), methyl acetate (η20 = 1.3614; water solubility = 243 mg/ml) and 2-butanone (η20 = 1.378 ; water 5
solubility = 275 mg/ml), of HPLC and of analytical grade, respectively (Fluka). Ethanol used in Example 12 is ethanol 96 % v/v (η20 = 1.361) (Fluka).
10
Distilled water used in the present examples was purified using a Milli-Q system (millipore, USA-Bedford, MO). A U other chemicals were of analytical grade and used 15
without further purification. Particle size analysis The average particle size and polydispersity index (scale from 0 to 9) were determined iising a Coulter® Nanosizer
20
(Coulter Electronics, Harpenden (UK). The measurements were made in triplicate for all the batches prepared. Example I Ethyl acetate and water were mutually saturated for I min
25
before use, in order to ensure initial thermodynamic equilibrium of both liquids. Typically, 4 g of Eudragit E were dissolved in 40 ml of water-saturated ethyl acetate and this organic solution (internal phase) was emulsified at room temperature with 80 ml of a 5 % w/v PVAL ethyl
30
acetate saturated aqueous solution (external phase), using a propeller stirrer (Heidolph-Elektro, KG type E-60, propeller : IKA 1381, Germany) at 1500 rpm for ten minutes. The oil-in-water emulsion formed was subjected to vacuum distillation at 35°C and 60 mmHg until complete solvent
35
evaporation. Generally, the solvent was recovered and used to prepare new batches. The mean particle size of the
WO 01/02087
PCT/EP99/04677
14
particles obtained by this method as well as the polydispersity are indicated in Table I below. Example 2 5
The method was repeated in the same manner as in Example I, except that 1.25 % w/v of PVAL was used in the external phase, instead of 5.0 % w/v of PVAL. The mean particle size of the particles obtained by this method and the polydispersity are indicated in Table I below.
10 Example 3 The method was repeated in the same manner as in Example I, except that 8 g of Eudragit E was used instead of 4 g of Eudragit E. The mean particle size of the particles 15
obtained by this method and the polydispersity are indicated in Table I below. Example 4 The method was repeated in the same manner as in Example I,
20
except that PCL was used, instead of Eudragit E. The mean particle size of the particles obtained by this method and the polydispersity are indicated in Table I below. Example 5
25
The method was repeated in the same manner as in Example I, except that EVAC was used, instead of Eudragit E. The mean particle size of the particles obtained by this method and the polydispersity are indicated in Table I below.
30
Example 5 The method was repeated in the same manner as in Example I, except that PLA was used, instead of Eudragit E .The mean particle size of the particles obtained by this method and the polydispersity are indicated in Table I below.
35
WO 01/02087
PCT/EP99/04677
15
Example 7 2-butanone and water were mutually saturated for I min before use, in order to ensure initial thermodynamic equilibrium of both liquids. Typically, 4 g of CAP were 5
dissolved in 40 ml of water-saturated 2-butanone and this organic solution (internal phase) was emulsified at room temperature with 80 ml of a 5 % w/v poloxamer 407 2butanone saturated aqueous solution (external phase), using a propeller (Heidolph-Elektro, KG type E-60, propeller :
10
IKA 1381, Germany) at 1500 rpm for ten minutes. The Šil-inwater emulsion formed was subjected to vacuum distillation at 35°C and 60 mmHg until complete solvent evaporation. Generally, the solvent was recovered and used to prepare new batches. The mean particle size of the particles
15
obtained by this method as well as the polydispersity are indicated in Table I below. Example 8 The method was repeated in the same manner as in Example 7,
20
except that 1.25 % w/v of poloxamer 407 was used, instead of 5.00 % w/v of poloxamer 407. The mean particle size of the particles obtained by this method as well as the polydispersity are indicated in Table I below.
25
Example 9 The method was repeated in the same manner as in Example 7, except that 8 g of CAP was used, instead of 4 g of CAP. The mean particle size of the particles obtained by this method as well as the polydispersity are indicated in Table I
30
below. Example 10 The method was repeated in the same manner as in Example 7, except that 12 g of CAP was used, instead of 4 g of CAP.
35
The mean particle size of the particles obtained by this
WO 01/02087
PCT/EP99/04677
16
method as well as the polydispersity are indicated in Table I below. Example 11 5
The method was repeated in the same manner as in Example 7, except that CAT was used, instead of CAP. The mean size of the particles obtained by this method as well as the polydispersity are indicated in Table I below.
10
Example 12 Ethyl acetate and water were mutually saturated for I min before use, in order to ensure initial thermodynamic equilibrium of both liquids. Typically, 4 g of Eudragit E were dissolved in 40 ml of water-saturated ethyl acetate
15
and this organic solution (internal phase) was emulsified at room temperature with 100 ml of a 80 : 20 v/v waterethanol mixture containing 0.21 % w/v of poloxamer 407 (external phase), using a propeller stirrer (HeidolphElektro, KG type E-60, propeller : IKA 1381, Germany) at
20
1500 rpm for ten minutes. The oil-in-water emulsion formed was subjected to vacuum distillation at 35째C and 60 mmHg until complete solvent evaporation. Generally, the solvent was recovered and used to prepare new batches. The mean particle size of the particles obtained by this method as
25
well as the polydispersity are indicated in Table I below. Example 13 2-butanone and water were mutually saturated for I min before use, in order to ensure initial thermodynamic
30
equilibrium of both liquids. Typically, 4 g of Eudragit
E
were dissolved in 40 ml of water-saturated ethyl acetate and this organic solution (internal phase) was emulsified at room temperature with 80 ml of a 2-butanone-saturated aqueous/solution (external phase), using a propeller 35
stirrer (Heidolph-Elektro, KG type E-60, propeller : IKA 1381, Germany) at 1500 rpm for ten minutes.
WO 01/02087
PCT/EP99/04677
17
The oil-in-water emulsion formed was subjected to vacuum distillation at 35째C and 60 mmHg until complete solvent evaporation. Generally, the solvent was recovered and used to prepare new batches. The mean particle size of the 5
particles obtained by this method as well as the polydispersity are indicated in Table I below. Example 14 Water was saturated with 2-butanone before use and methyl
10
acetate was saturated with water for I minute. Typically, 4 g of Eudragit E were dissolved in 40 ml of water-saturated methyl acetate and this organic solution (internal phase)
15
was emulsified at room temperature with 80 ml of
2-
butanone-saturated water (external phase),using
a
propeller stirrer (IKA, Eurostar; propeller : IKA 1381, Germany) at 2000 rpm for 15 minutes. The oil-in-water emulsion formed was subjected to vacuum distillation at 50째C and 150 mmHg until complete solvent evaporation. The mean particle size of the particles obtained by this method
20
as well as the polydispersity are indicated in Table I below. Example 15 The method was repeated in the same manner as in Example
25
14, except that propeller stirrer was used at 1500 rpm instead of 2000 rpm. The mean particle size of the particles obtained by this method as well as the polydispersity are indicated in Table I below.
30
Example 16 The method was repeated in the same manner as in Example 14, except that 6 g of Eudragit E was used, instead of 4 g of Eudragit. The mean particle size of the particles obtained by this method as well as the polydispersity are
35
indicated in Table I below.
WO 01/02087
PCT/EP99/04677
18
Example 17 The method was repeated in the same manner as in Example 14, except that 8 g of Eudragit E was used, instead of 4 g of Eudragit. The mean particle size of the particles 5
obtained by this method as well as the polydispersity
are
indicated in Table I below. Example IS
2-butanone and water were mutually saturated for I min 10
before use, in order to ensure initial thermodynamic equilibrium of both liquids. Typically, 4 g of Eudragit RS were dissolved in 40 ml of water-saturated 2-butanone and this organic solution (internal phase) was emulsified at room temperature with 80 ml of 2-butanone-saturated water
15
(external phase), using a propeller stirrer (IKA, Eurostar; propeller : IKA 1381, Germany) at 2000 rpm for 15 minutes. The oil-in-water emulsion formed was subjected to vacuum distillation at 50째C and 150 mmHg until complete solvent evaporation. The mean particle size of the particles
20
obtained by this method as well as the polydispersity
are
indicated in Table I below. Example 19 The method was repeated in the same manner as in Example 25
18, except that Eudragit RL was used, instead of Eudragit RS. The mean particle size of the particles obtained by this method as well as the polydispersity are indicated in Table I below.
30
Comparative Example The method was repeated in the same manner as in Example I, except that chloroform was used instead of ethyl acetate. The mean particle size of the particles obtained was 5006 nm.-and the polydispersity was 5.
WO 01/02087
PCT/EP99/04677
19
TABLE I Examples of nanoparticles obtained by the method of the invention. 5
Internal phase : 4 0 ml External phase : 80 ml (except for Example 12 wherein external phase is 100 ml) Stirring rate : 1500 rpm (except for Examples 14, 16, 17, 18 and 19 wherein the stirring
10
rate is 2000 rpm) Ex.
Polymer
Stabilizer
Mean Size
No.
(%w/v)
(% w/v)
(nm)
I
E u d r .E
(10)
EtAc
PVAL
(5.00)
573
4
2
E u d r .E
(10)
EtAc
PVAL
(1.25)
590
4
3
E u d r .E
(20)
EtAc
PVAL
(5.00)
817
4
4
PCL
EtAc
PVAL
(5.00)
543
3
5
EVAC
EtAc
PVAL
(5.00)
435
5
6
PLA
(10)
EtAc
P.VAL (5.00)
472
2
7
CAP
(10)
2-butanone
Poloxamer 407
(5.00)
260
2
8
CAP
(10)
2-butanone
Poloxamer 407
(1.25)
308
4
9
CAP
(20)
2-butanone
Poloxamer 4 07
(5.00)
614
5
10
CAP
(30)
2-butanone
Poloxamer 407
(5.00)
770
5
11
CAT
(10)
2-butanone
Poloxamer 407
(5.00)
811
7
>2
E u d r .E
(10)
EtAc
Poloxamer 407
(0.21)
248
5
13
E u d r .E
(10)
2-butanone
none
446
4
14
E u d r .E
(10)
MeAc
none
210
4
15
E u d r .E
(10)
MeAc
none
328
2
16
E u d r .E
(15)
MeAc
none
390
2
17
E u d r .E
(20)
MeAc
none
518
4
18
E u d r . RS
(10)
2 -butanone
none
114
3
19
E u d r . RL
(10)
2-butanone
none
63
3
CHCl3
PVAL
5006
5
Solvent
(10) (10)
Pd*
Comparative Example Eudr. E
*Pd
15,
(10)
(5.00)
: Polydispersity (index expressed from 0 to 9)
WO 01/02087
PCT/EP99/04677
20 As shown in Table I, nanoparticles were obtained with the polymers tested, indicating that the method of the present invention also can be advantageously applied to nonbiodegradable polymers commonly used in pharmaceutical 5
coating methods and to biodegradable polymers. The method of the present invention thus makes it possible to prepare aqueous colloidal dispersions containing high concentration of nanoparticles of water-insoluble material,
10
for example pseudolatexes, from a conventional oil-in-water emulsion with an ordinary mechanical stirring without requiring homogenization, without requiring dilution with water, with or without stabilizing agent, by direct displacement of a partially water-soluble solvent during
15
distillation. Further, the dispersion obtained by the method of the present invention may be used directly for coatings without additional treatments.
PCT/EP99/04677
WO 01/02087
21
CLAIMS 1. A method for producing an aqueous colloidal dispersion of nanoparticles, characterized in that it 5
comprises : a) the emulsification of a partially water-soluble organic solvent, containing a water-insoluble material in a weight/volume percentage at which nanoparticles are formed in step b), in an aqueous solution containing optionally a
10
stabilizing agent, using a low energy source; b) the distillation of the organic solvent from the oil-in-water emulsion formed in step a) to cause the formation of nanoparticles, in suspension in the aqueous phase .
15
2. The method for producing an aqueous colloidal dispersion according to claim I, characterized in that the aqueous solution contains no stabilizing agent. 20
3 . The method for producing an aqueous
colloidal
dispersion of nanoparticles according to claim I, characterized in that the aqueous solution contains no more than 5.00 % w/v of the stabilizing agent. 25
4 . The method for producing an aqueous
colloidal
dispersion of nanoparticles according to claim I, characterized in that the aqueous solution contains no more than 1.25 % w/v of the stabilizing agent. 30
5 . A method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims I, 3 and 4, characterized in that the stabilizing agent is selected from poly(vinyl alcohol) and poloxamer 407.
35
6. The method for producing an aqueous
colloidal
dispersion of nanoparticles according to any one of claims I to 5, characterized in that the partially water-soluble
WO 01/02087
PCT/EP99/04677
22 organic solvent is previously added with a certain amount of water extending up to the saturation. 7 . The method for producing an aqueous colloidal 5
dispersion of nanoparticles according to claim 6, characterized in that the partially water-soluble organic solvent is previously saturated with water. 8 . The method for producing an aqueous colloidal
10
dispersion of nanoparticles according to any one of claims I to 7, characterized in that the aqueous solution comprises water previously added with a certain amount, extending up to the saturation, of the same or another partially water-soluble organic solvent.
15
9 . The method for producing an aqueous colloidal dispersion of nanoparticles according to claim 8, characterized in that the aqueous solution comprises water previously saturated with the ,‘same or the other partially 20
water-soluble organic solvent. 10. The method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims I to 7, characterized in that the aqueous solution
25
comprises water previously added with a certain amount of a water-soluble solvent.
11. The method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims 30
I to 10, characterized in that the water-insoluble material is a polymer or a mixture of polymers. 12. The method for producing an aqueous colloidal dispersion of nanoparticles according to claim 11,
35
characterized in that the water-insoluble material is
a
polymer'' selected from biodegradable polymers such as poly(D,L-Iactic acid)
(PLA) and poly (Îľ-caprolactone)
(PCL)
WO 01/02087
PCT/EP99/04677
23
and non-biodegradable polymers such as Eudragit速E, Eudragit速RS,
Eudragit速RL, cellulose acetate phthalate
(CAP), cellulose acetate trimellitate (CAT), and ethylene vinyl acetate copolymer (EVAC). 5
13. The method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims I to 12, characterized in that the partially water-soluble organic solvent is selected from ethyl acetate (EtAc), 10
methyl acetate (MeAc) , isopropyl acetate or 2-butanone..14. The method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims I to 13, characterized in that the emulsification is made
15
by using a propeller stirrer rotating at a stirring rate from 1500 rpm to 2000 rpm.
15. The method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims 20
I to 14, characterized in that the distillation of the solvent is a vacuum distillation. 16. A method for producing an aqueous colloidal dispersion of nanoparticles according to any one of claims
25
I to 15, characterized in that the partially water-soluble organic solvent containing the water-insoluble material further contains one or more additional ingredients.
PCT/EP99/04677
WO 01/02087 1/6
2000 τ-
Ι 800 1600 --
Mean size (nm)
1400 -1200
- -
1000
- -
800 -600 -400 -200
- -
0
10
20
40
Eudragit E in the internal phase (% w/v)
F i gure
I
W O 01/0208?
PCT/EP99/04677
2 /6
I M ΓΓι
4.-5Β0
SUBSTITUTE SHEET (RULE 26)
3 9 m rn
W O 01/02087
3 /6
SUBSTITUTE SHEET (RULE 26)
PCT/EP99/04677
WO 01/02087
PC T/E P99/04677
4 /6
-
—
-I
l
I
fc
"/
'f t
-y rtfis >
I #
»
«
!
SUBSTITUTE SHEET (RULE 26)
PCT/EP99/04677
WO 01/02087
5/6
3500 -r
3000 --
Mean size (nm)
2 5 0 0 --
2000
--
1500 --
1000--
5 0 0 --
0
5
10
15
20
25
30
35
CAP in th e internal p h a se (% w/v)
Figure 3
40
45
50
WO 01/02087
PCT/EP99/04677
6/6
4000 3500
M ean size ( n m )
3000 2500
2000 1500 1000 500 0 10
12
14
16
20
22
24
26
E u d r a g i t E in t h e i n t e r n a l p h a s e ( % w / v )
Figure 4
28
30
INTERNATIONAL SEARCH REPORT Si A pplication No
PCT/EP 99/04677 ATlON OF SUBJECT HATTER A. CLASSIFICATION
B01J13/04
IPC 7
,
C08J3/07
A61K9/51
Acoordllng to Intemallonal Patent Claaelflcallon (IPC) or to both national daasffloaflon and IPC B. FIELDS SEARCHED_______________________________________________________________
Mlnfeniindocunenlatlonaeeiched (claaeltlcadon system folowed by ctassttlcadon symbols)
IPC 7
BOlJ A61K C08J
DocuTMntallon searched other than mlnknun docunentadon to the extent that such documents are Inchided Inthelleldeaearclied
Qectronlc data base consrited during the feitematlonal eeaich (name o( data beee and, where practical, eearch terma uaed)
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Relevanttoclakn N a
CltadonafdocunenLwlthfeKlcadon, where appropriate, of the relevant paseages
X
US 5 766 635 A (SPENLEUHAUER GILLES AL) 16 June 1998 (1998-06-16) claims 1,9 column 3, Hne 44 - line 59 example 7
E
MO 99 33558 A (ALLEMANN ERIC ;UNIV GENEVE (CH); DOELKER ERIC (CH); GURNY ROBERT () 8 July 1999 (1999-07-08) complete document
1,3-16
A
EP O 275 796 A (CENTRE NAT RECH SCIENT) 27 July 1988 (1988-07-27) cited In the application claim I column 5, line 27 - line 33 example 4
1-3
~
□
~
Furtherdoctinents are Ieted In the contkuatlon U box C.
• Special categories of cited docunente: “A" docunent defining the general elate of the art which Ie not considered to be of particular relevance "E" earlier docunent but publahed on or after the Irrtetrrallonal fling date "L" docunent which may throw doiiXe on priority dakn(e)or which Ia cited to establish the ptbilcaaon date of another citation or other special reason (as specified) "0“ document referring to an oral dadoaure, use, exhtrltlon or other means “P* docunent p tttsh e d prior to the krtematlonal tlferg date but later than the priority date claimed Date of the actual completion of the fertetnadonal search
20 March 2000 Name and mating address of the ISA European Patent Office, P.B. 5818 Patendaan 2 NL-2280 HV RIJawI^ Tel. (+81-70) 340-2040, Tx. 31 861 epo nl, Fax: (+81-70) 340-3018
ET
1,2,11, 12,16
- / -
El
Patent family members are Ieted fei annex.
lrP IaterdocumentpUiIIshedafterthe Intemadonai Illng date or priority date and not In conllct with the applcatlon but cited to uiderstarid the principle or theory urderiylng the Invention “X* document ofpardcUar relevance; the claimed kivendon cannot be considered novel or cannot be considered to feivotve an Invendve step when the document Is taken alone *Y* docunent of paidcUar relevance; the claimed Invention cannot be considered to Involve an feiverrtlve step when the docunent Iscombfeted with one or more other such docu ments, such combkradon being obvious to a person sidled InthearL docunent member of the same patent famly Date of mallng of the Intemadonal search report
30/03/2000 Authorized officer
Hallemeesch, A
Form PCTrtSAato (seoond sheet) (Jriy 1M2)
page I o f 2
INTERNATIONAL SEARCH REPORT IntMiM
il Application N o
PCT/EP 99/04677 O (C on lintiation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category 掳
Citation of docunent, wltii Indcadon1Where appropriate, of Itw relevant passage路
Relevant to dafen No.
LEROUW J -C ET AL: "NEW APPROACH FOR THE PREPARATION OF NANOPARTICLES BY AN EMULSIFICATION-DIFFUSION METHOD" EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS,NL,ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, vol. 41, no. I, I January 1995 (1995-01-01), pages 14-18, XP000482886 ISSN: 0939-6411 cited in the application
1,7-10
Rxm PCT/1SM210 (conttnuotlan of Mcond *w ct) (Jtiy 1*82)
page 2 o f 2
INTERNATIONAL SEARCH REPORT
IlAppIioalionNo
Imo n Mrtion o n p at»nt fam ily roem bw
PCT/EP 99/04677 Patent ckxximent cted In search report
PatentfamKy members)
PtMcaUon date
US 5766635
A
16-06-1998
NONE
UO 9933558
A
08-07-1999
AU
EP 0275796
A
27-07-1988
FR AT CA DE ES FR GR GR JP JP US US
Fam PCT/I8A/210 (peonttam ly annex) (Uiy 1M2)
PtMcaUon date
5860798 A 2608988 74024 1292168 3777796 2031151 2634397 3004152 3018122 2739896 63240936 5133908 5118528
A T A A T A T T B A A A
19-07-1999 01-07-1988 15-04-1992 19-11-1991 30-04-1992 01-12-1992 26-01-1990 31-03-1993 29-02-1996 15-04-1998 06-10-1988 28-07-1992 02-06-1992