Particularly, the invention relates to a process for the production of biodegradable nanoparticles made of lactic acid-glycolic acid copolymers, containing one or more active substances by the use of a double emulsion water-in-oil-in-water (W/O/W) process.

Prior Art As a result of extensive advances in the field of biotechnology and genetic engineering numerous novel protein drugs have been prepared, which are assumed to exhibit a high potential in the treatment of different diseases. Thus, for the treatment of cancer numerous proteins capable of impairing tumour growth, invasion and metastasis have been suggested as potential anti-tumour agents. These proteins also include protease inhibitors such as TIMP's (tissue inhibitors of metalloproteases), plasminogen activator inhibitors and cystatins.

One of the disadvantages of protein drugs is their instability in physiological environment, their tendency towards enzymatic degradation and thus a shorter half- life in vivo. Further, the proteins are large molecules that hardly pass biological membranes, which considerably reduces their availability.

In order to overcome some of the above problems, drug carrier systems for active substances have been developed which improve the half-life and the bioavailability of such substances. Among such systems nanoparticles represent a very promising approach since their properties of protecting active substances and of controlled release are considered to be very advantageous. In particular it has been found that nanoparticles allow the transport of active substances through biological membranes <BR> <BR> and specific targeting by a potential surface modification (Lamprecht A. , Ubrich N.,<BR> Perez M. H. , Lehr C. M., Hoffman M. , Maincent P. , Biodegradable monodispersed nanoparticles prepared by pressure homogenization-emulsification, Int. J. Pharm. 184 (1999) 97-105).

The main advantage of nanoparticles is the possibility of their various applications.

They allow parenteral and nonparenteral routes of administration. They can be injected intravenously and, since the protein is incorporated within a polymer matrix and thus protected from circulating enzymes, the plasma half-life of the incorporated protein is prolonged as compared with the injected protein solution. The release of the protein from the nanoparticles is also sustained, which results in a prolonged activity of the protein. Furthermore, by a selection of an appropriate polymer, nanoparticles may facilitate the absorption through biological membranes. An oral intake is possible as well. To this end, the size of nanoparticles is the crucial parameter determining the absorption from the gastrointestinal tract. Smaller nanoparticles are generally absorbed to a larger extent.

So far several encapuslation techniques for the preparation of nanoparticles using two main principles, i. e. single (W/O) and double (W/O/W) emulsion methods have been developed.

Thus, US 4,177, 177 discloses a process for a direct emulsification of a solution of water-insoluble polymers in appropriate organic solvents into an aqueous solution containing at least one emulsifier of nonionic, anionic or cationic type. Ultrasound (sonication) was used to reduce the size of emulsion droplets, whereby a particle size of less than 0. 5, um was achieved.

In EP 0 052 510 B2 a phase separation technique using coacervating agents or nonsolvents such as mineral oils or vegetable oils is described. The active substance, e. g. polypeptide, was first dissolved in the aqueous phase of a water-in-oil emulsion.

The polymer was precipitated around the aqueous droplets by the addition of a nonsolvent for the polymer such as silicon oil. Then a hardening agent was added to extract the organic solvent from the micro-or nanoparticles. However, one of the disadvantages of the said process is large consumption of organic solvents needed for the extraction and washing.

In US 5,019, 400 another process using spray drying and/or spray coating technique is described. For spray drying the polymer and the active aubstance are dispersed in a solvent for the polymer. Then the solvent is evaporated by spraying the solution, which results in the formation of polymer droplets containing the active substance.

However, sensitive substances such as proteins may become inactive during the process due to the elevated temperatures used and to the exposure to organic solvent/air interfaces.

In US 5,407, 609 a process utilizing solvent evaporation is used. Solvent evaporation technique (oil-in-water (O/W) process) involves a dissolution of the polymer and of the active substance in an organic solvent, whereupon the solution is added to a stirred aqueous outer phase, which is immiscible with the polymer. The aqueous outer phase usually contains surfactants stabilizing the oil-in-water emulsion and preventing agglomeration. The emulsifier used is typically polyvinyl alcohol. The organic solvent is then evaporated over a period of several hours or more, whereby a polymer is precipitated and a polymer matrix is formed.

Also a so-called double emulsion water-in-oil-in-water (W/O/W) method has been developed, which is particularly useful when water-soluble peptides are used that cannot be easily encapsulated by the common oil-in-water (O/W) process (EP 0 190 833 B1). The main advantage of this process is that during the process of formation of the nanoparticles, the protein remains in the inner aqueous phase, whereby a direct contact between the protein and the organic solvent is reduced. This process involves dissolving the protein in water, adding the aqueous protein solution to an organic polymer solution under stirring and forming a first W/O emulsion. This W/O emulsion is then poured into an aqueous phase containing an emulsifier and thus a double W/OW emulsion is formed under stirring. The organic solvent is subsequently removed by evaporation or extraction.

However, during the preparation of nanoparticles of such kind, characteristic destructive interactions between the solvent and the protein may occur, which are partly due to organic solvents utilized, the shear forces during stirring and sonication, which all decrease the biological activity of the encapsulated active substance.

The decisive step in the preparation of micro-and nanoparticles is the emulsification that may, in general, be carried out by sonication, high-pressure homogenization or high-shear homogenization. Stirring combined with a subsequent sonication during one hour usually results in nanoparticles of a diameter larger than 1400 nm (Ferdous A. J. , Stembridge N. Y. , Singh M. , Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro, J. Control. Rel. 50, 1998: 71-78). For obtaining particles in the nanometer range, a higher energy is required, which often results in a loss of the biological activity of the protein. When the process of evaporation of the solvent oil-in-water (O/W) was used and all the three processes were combined, i. e. the sample was homogenized (20000 rpm, 20 min), followed by simultaneous stirring (500 rpm) and sonication for 1 hour, particles of a size of less than 200 nm were obtained.

Solution of the Technical Problem With Examples Therefore, the task of the present invention is to overcome the disadvantages of the prior art and to provide a novel process for the production of nanoparticles, which does not have a substantial detrimental effect on the active substances that are to be incorporated.

During extensive studies leading to the present invention the inventors have found that a combination of different measures, namely stirring and sonication, both at a level at which each single measure as such does not result in a sufficiently intensive emulsification, yields nanoparticles of a small size, wherein the encapsulated active substance exhibits improved biological activity.

Thus, one aspect the present invention relates to a process for the production of nanoparticles with encapsulated one or more active substances, wherein an emulsification method is used, whereat stirring and sonication are carried out simultaneously, either at an energy level that alone is not sufficient for the formation of nanoparticles and which preserves the biological activity of the encapsulated active substance.

According to the invention stirring and sonication are carried out simultaneously and at moderate conditions. In the preferred embodiment the stirring speed is in the range from 4000 to 15000 rpm, preferably from 5000 to 10000 rpm, and more preferably from 5000 to 7000 rpm.

Sonication is preferably carried out at a frequency from 20 kHz to 70 kHz.

Process according to the invention particularly comprises: I) dissolving a biodegradable polymer in a suitable organic solvent therefor (preferably in a water-immiscible organic solvent), II) emulsifying, whereat the stirring and the sonication are carried out simultaneously, either at an energy level that alone is not sufficient for the formation of nanoparticles, comprising: a) emulsifying the active substance (preferably a polypeptide or peptide) dissolved in water or in a suitable aqueous solvent therefor (e. g. buffer), into the organic solution obtained in step I) to provide formation of a primary emulsion with the active substance in the inner aqueous phase, and b) emulsifying the primary emulsion obtained in step IIa) into an aqueous solution of an emulsifier as the continuous phase (preferably with an aqueous polyvinyl alcohol solution) so as to obtain nanoparticles having the active substance encapsulated therein, and III) isolating and drying the nanoparticles in a known manner (drying is preferably carried out by lyophilisation, whereat the active substance is in step IIa) dissolved in a suitable aqueous solvent therefor containing cryoprotectants).

In the process according to the invention a novel conveniently modified technique of emulsification is applied, wherein a high shear homogenization at low rpm and sonication are used simultaneously. Then the obtained primary emulsion is emulsified into the solution of an emulsifier to obtain a double emulsion (W/O/W). The double emulsion is diluted with excessive water to facilitate the removal of the organic solvent and the mixture is stirred to allow the solvent to evaporate, whereby the precipitation of the polymer and thus also the formation of solid nanoparticles with the encapsulated active substance are induced. The particles are isolated by centrifugation or filtration and washed several times with distilled water or suitable aqueous buffers to remove the excessive emulsifier from the surfaces. Then the particles are dried by conventional means, e. g. in vacuum, by streaming nitrogen gas or air, by lyophilisation or spray drying.

The organic solvent used in the step of dissolving the biodegradable polymer may be any solvent capable of forming an emulsion with a determined quantity of an aqueous emulsifier solution and which may be removed from the emulsion droplets by the addition of the excessive amount of aqueous emulsifier solution and which is further capable of dissolving biodegradable polymers. In other words, the solvent should be immiscible or essentially immiscible with water, but partly soluble in the cited aqueous emulsifier solution. Examples for organic solvents that may be used for dissolving the polymer are chloroform, benzene, dichloromethane, chloroethane, dichloroethane, trichloroethane, carbon tetrachloride, ethyl ether, cyclohexane, n-hexane, toluene, more preferably ethyl acetate, methylene chloride or a mixture of methylene chloride and acetone, most preferably ethyl acetate. It was surprisingly found that the use of ethyl acetate limits the loss of biological activity of proteins such as cystatin, and provides smaller particles as compared to other solvents. Without wishing to be bound to any theory, this may be due to a greater solubility of ethyl acetate in water, which leads to a faster diffusion of ethyl acetate into the outer aqueous phase, leaving behind insoluble polymer particles with incorporated protein.

Since emulsions are thermodynamically unstable systems, the use of an emulsifier is necessary. The emulsifier serves for several purposes: it assists in obtaining the correct droplet size distribution of the emulsion, stabilizes the W/O/W emulsion to avoid coalescence of droplets and prevents the precipitated nanoparticles from sticking to each other. The preferred examples of emulsifiers are anionic surfactants (e. g. sodium oleate, sodium stearate, sodium lauryl sulfate etc. ), nonionic surfactants (e. g. polyoxyethylene sorbitan fatty acid esters (Tween 80, Tween 60 etc.)), polyoxyethylene castor oil derivatives, polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin etc. , more preferably polyvinyl alcohol. Such emulsifiers may be used either alone or in a combination.

For encapsulating the active substance various different polymer materials may be used such as lactic acid-glycolic acid polyesters, polylactic acid, poly-P-hydroxybutyric acid, polyhydroxyvaleric acid, polycaprolactone, polyesteramides, polycyanoacrylates, poly (amino acids), polycarbonates, polyanhydrides, biodegradable polymers, whereat lactid acid-glycolic acid polyester is specifically preferred. The weight ratio of (poly) lactic acid/ (poly) glycolic acid is preferably from about 99/1 to 36/65, more preferably 95/5 to 50/50, with the exact composition of the polymer being selected by a person skilled in the art based upon his general knowledge and depending on the desired release kinetics. Lactic acid- glycolic acid copolymer (i. e. poly (lactic-co-glycolic acid), PLGA) microspheres and nanoparticles are biocompatible and degrade at forming non-toxic monomers, the polymer matrix perfectly protects the proteins and peptides against destructive environmental conditions, especially when administered orally. The release kinetics of the encapsulated active substances can be controlled as well by varying the molecular weight and the monomer ratio of PLGA.

As the active substance to be encapsulated according to the present invention, any medicinal active ingredient may be used, in particular substances having a short half- life in the body such as proteins and/or peptides. There can be used biologically active macromolecules such as interferons, interleukins, colony stimulating factors, tumour necrosis factors, other immunomodulators, growth factors, transforming growth factors, erythropoietin, albumin, blood proteins, hormones, vaccines, viruses, toxins, antibodies, antibody fragments, enzymes, enzyme inhibitors including cystatin.

In addition to the active substance also other substances may be incorporated, such as agents for controlling the stability and, if desired, agents for controlling the solubility of the biologically active substance. Such agents may be pH controlling agents, preservatives and stabilizers, and cryoprotectants that may include glycols, albumin, gelatin, amino acids, ethylenediamine tetraacetic acid, dimethyl sulfoxide, citric acid, dextrin, saccharose, fructose, mannose, trehalose, other sugars and all combinations thereof.

Another object of the present invention are nanoparticles obtained according to the process of the present invention and having a preferable particle size from about 100 to about 800 nm. These nanoparticles surprisingly convey an increased biological activity and stability to the active substances encapsulated therein.

The present invention is essentially based on the finding that the rotation of the homogenizer with high speed (15000 rpm or more), which is normally required for the production of nanoparticles, can be substituted by a lower speed if the system is simultaneously subjected to the action of ultrasound with low energy, whereat the biological activity and bioavailability of the encapsulated active substance is improved as compared to nanoparticles produced by common techniques of stirring or sonication alone or of a successive combination thereof.

It was also found that a shorter time of homogenization (2 minutes in comparison with 7 minutes) preserves the activity of the protein to a greater extent. This may, at least in part, be due to the reduction of time available for the contact of the protein with the organic solvent. A short production time may also lead to a higher efficacy of the protein encapsulated within the nanoparticles since it hinders the diffusion of the protein from the inner aqueous phase towards the outer large volume phase.

Nanoparticles according to the invention are suitable for the production of a drug for parenteral, nasal, pulmonal, peroral, oral, transdermal or rectal administration of the active substance.

In the drawings: Fig. 1 shows the size of nanoparticles obtained by the process according to the invention as compared to prior art nanoparticles, Fig. 2 shows cystatin activity under different experimental conditions, Fig. 3 shows cystatin activity during storage when it is encapsulated in nanoparticles (3a) obtained according to the present invention, as compared to the cystatin solution (3b).

The following examples illustrate the invention without limiting it thereto.

Example la The following process was used for the preparation of blank lactic acid-glycolic acid copolymer (i. e. poly (lactic-co-glycolic acid), PLGA) nanoparticles (nanoparticles without incorporated protein). A study was performed to evaluate how the experimental constrains affect the size of nanoparticles.

First, a polymer solution was prepared in a test tube by dissolving 50 mg of PLGA (Resomer RG 503H, Boehringer Ingelheim) in 1 ml of ethyl acetate. Then 200 p, l of water were added to the polymer solution and the mixture was homogeneously emulsified by a rotor-stator homogenizer (Omni Labtek, Omni International, USA) for two minutes. On two different samples two different rotation speeds, 15000 and 12500 rpm, respectively, were applied. After one minute of homogenization, 4 ml of a 5 % aqueous PVA (polyvinyl alcohol) solution were added to the homogenized mixture to form a stable double emulsion (W/O/W). When homogenization was completed, the resulting double emulsion was slowly poured into 100 ml of a 0.1 % aqueous PVA solution. The obtained mixture was then homogenized for 5 minutes at 5000 rpm.

Then the dispersion was ultracentrifuged at 15000 rpm for 15 minutes using Ultracentrifuge Sorvall RC 5C plus, Rotor SS 34, USA. After separation, the nanoparticles were washed three times with distilled water (20 ml) and collected by centrifugation at conditions mentioned before.

The obtained particles had a size ranging from 600 nm to 700 nm with the minimum particle size in the formulation prepared with the highest rotation speed.

Example lb The next study was performed to assess the loss of cystatin activity during homogenizing under various conditions and to identify the maximum rotation speed still possible for retaining the cystatin activity. Cystatin, a cystine proteinase inhibitor, was isolated from chicken egg white as described (Kos, J. , Dolinar, M., Turk, V.: Isolation and characterization of chicken L-and H-kininogens and their interaction with chicken cysteine proteinases and papain, Agents and Actions 38, 331- 339,1992). The biological activity of cystatin was determined on the plant proteinase papain using BANA (a-N-benzoyl-DL-arginine-ß-naphtylamide) as a substrate. The influence of different homogenization conditions on cystatin activity was observed on an aqueous cystatin solution. The same sequence of individual process steps as in the Example 1 a was performed. However, in the present Example no PLGA polymer was used and 1 ml of ethyl acetate, 4 ml of 5 % aqueous PVA solution and 100 ml of 0.1 % aqueous PVA solution were replaced with distilled water. Instead of 200 1ll of water a protein solution (2 mg of cystatin were dissolved in 200 p1 of 0.1 M phosphate buffer, pH = 6.0) was used. The same conditions as in Example la were applied and the effect of rotation speed on cystatin inhibitory activity was determined as described above.

It was found that the homogenization at 15000,12500 and 10000 rpm caused the respective losses of biological activity of cystatin for 82 %, 66 % and 30 %.

It was found that the rotation speed should be reduced to 5000 rpm to preserve the activity of cystatin at a level of about 98 %. However, such a reduction of the energy input does not enable the obtaining of nanoparticles.

Example 2a In order to enable the production of nanoparticles also at lower rotation speeds, the same procedure as in Example la was performed with the exception that, simultaneously with homogenization (10000,7500 and 5000 rpm), ultrasound was used as well. The formed nanoparticles had a particle size of 320,350 and 360 nm, respectively.

This shows that the energy input is utilized more efficiently if different types (processes) of emulsification are performed simultaneously, e. g. homogenization and ultrasonication. Thus, the synergistic effect of mechanical and ultrasonic energy enables the production of nanoparticles at lower rpm (5000 rpm) and mild ultrasonic waves.

Example 2b The following test was performed to determine a synergistic effect of emulsification (Example 2a) upon cystatin activity.

The same procedure as in the Example lb was performed, with exception that, simultaneously with homogenization at 5000 rpm, ultrasound was used. The loss of biological activity of cystatin was only 20 %. This indicates that this process is a good compromise between retaining a high level of protein activity and the formation of nanoparticles with the desired particle size.

Example 3a The next study was performed in order to determine the stability of protein within nanoparticles under simulated physiological conditions.

Nanoparticles were produced by the process according to the present invention. The same process as in Example la was carried out with the exception that instead of 200 jj. l of water there were used 200 1 of a solution of cystatin in water and that instead of homogenization at a high speed there was used homogenization at 5000 rpm with a simultaneous application of ultrasound.

The resulting nanoparticles were washed, centrifuged and collected as in Example 1 a and finally they were dispersed in 5 ml of 0.12 M phosphate buffer (PBS) with pH 7.2-7. 4 and incubated for 19 days at 37 °C under stirring on a magnetic stirrer (2 rpm).

Samples of 300 ul were taken at different time intervals (2 hours, 1,2, 5,7, 12,14, 16 and 19 days), then the samples were centrifuged (15 min at 16000 rpm) and the activity of cystatin in the supernatant was determined by enzymatic spectrophotometric assay (BANA test). The remaining nanoparticles were placed back into the incubation dispersion.

Example 3b Further, the nanoparticles with incorporated cystatin were prepared according to the present invention with the exception that cystatin was dissolved in a solution of 2 % albumin, 300 mM trehalose, 300 mM mannose, 300 mM fructose and 100 mM saccharose (cryoprotectant). After the preparation of nanoparticles as in Example la, the particles were centrifuged for 15 min at 7000 rpm using ultracentrifuge Sorvall RC 5C plus, Rotor SS 34, USA. The sediment was resuspended in water by means of ultrasound and dried by lyophilisation. The activity of cystatin in the dispersion of nanoparticles after lyophilisation was 90 % of that before lyophilisation. If the nanoparticles with cystatin were prepared as in 1b and lyophilized, the activity of cystatin was only 17 % of that before lyophilisation.

Example 3c In order to compare the stability of the protein released from nanoparticles with an ordinary protein solution, a control sample was prepared and treated under the same conditions as the dispersion of nanoparticles in Example 3a.

The control sample was prepared by dissolving an appropriate amount of cystatin in 5 ml of PBS (approximately the same concentration with regard to the protein in the released medium of the previous Example 3a).

The same procedure as in Example 3a was performed and the stability of incubated protein for different time intervals was obtained.