FR2776516A1 - Prolonged release microcapsules and implants - Google Patents

Abstract

Compositions in the form of microcapsules or implants comprising a) an excipient or mixture of excipients which are biodegradable polymers or copolymers such that when the composition is in the form of microcapsules either the (co)polymers have a viscosity in the range 0.7 - 1.6dl/g in chloroform, and the process for preparing the microcapsules does not involve the step of fusing the microcapsules, or the (co)polymers have a viscosity of 0.5 - 1.6dl/g in chloroform and the (co)polymers have a hydrophilic character, and when the composition is in the form of implants, the (co)polymers have a viscosity between 0.5 and 1.6dl/g in chloroform, and b) at least one active material such that the microcapsules or implants liberate the active material(s) over a period, of 3 months or more with a monophasic profile, are new. A water-soluble material in a form having a high specific surface area is also claimed as is its process for preparation in which the material, preferably a protein or peptide, is dissolved in water preferably at about half its saturation concentration, it is lyophilised rapidly at a temperature below -70 deg C, and the solid formed may be crushed, preferably ultrasonically.

Description

Prolonged release compositions and process for their preparation

  preparation The invention relates firstly to a composition in the form of microcapsules or implants comprising an excipient or a mixture of polymeric excipients or biodegradable copolymers with intrinsic viscosity of between 0.5 dl / g and 1.6 dl / g in CHCl3 and at least one active substance. The invention also relates to a composition in the form of microcapsules or implants comprising at least one biodegradable polymer or copolymer of high molecular weight and at least one water-soluble active substance having a high specific surface. Such compositions will be used to obtain regular release of the active substance over an extended period of up to

more than three months.

  These compositions and in particular the microcapsules find their main use in pharmacy, but can also be used in other fields, in particular in

  agrochemicals, ie in the phytosanitary field.

  The interest of the administration of active principles in the form of compositions with progressive release has been known for a long time, whether they are conventional pharmaceutical products, for example steroids, peptides or proteins (see for example the US patent 3, 773,919 of Boswell), or of products for phytosanitary use. The formulations adopted may take the form of microparticles in which the active principle is incorporated in a biodegradable polymer or copolymer such as the polylactide-co-glycolide copolymer.

(PLGA).

  It appeared that in particular when a relatively constant release mode, in any case without interruption, is sought, mode described for example as "monophasic" in European patent EP 58 481, polymers of PLGA type of relatively low molecular weight, therefore of low viscosity, are necessary. On this subject, mention may be made of European patents EP 21 234 (see Example 8.B.2. Describing a copolymer with an intrinsic viscosity of 0.5 dl / g), EP 52 510 o a copolymer having a viscosity of 0, 38 dl / g in hexaisofluoropropanol (HFIP) is tested in vivo, EP 26 599 which describes as an example polymers having viscosities of 0.12 to 0.20 dl / g and claims polymers having a viscosity of 0.08 at 0.30 dl / g. The polymers described in these patents are presented as leading to compositions with constant release. The compositions of patent EP 26 599 can for example contain

fertility control agents.

  It is also important to note in this regard that during the opposition procedure relating to European patent EP 58 481, procedure still in progress on the day of filing of this -2 - application, the applicant limited his main claim polymers of low viscosity (less than 0.3 or 0.5 dl / g), which alone are capable, according to the applicant, of allowing salting out of

monophasic type.

  Furthermore, when a longer release period is sought, for example more than a month, more complex problems appear and a solution proposed for example by patent EP 0 302 582 consists in mixing several types of microcapsules constituted

  by polymers of different viscosities.

  However, the Applicant has just found that certain polymers of high viscosity may be suitable for the preparation of compositions with gradual release over a long period of time. It has also been observed that the use of certain polymers leads to compositions having a monophasic release profile for a very long period and without an initial period comprising no release ("dead period"). This is particularly so of polymers having an intrinsic viscosity preferably at least equal to 0.5 dl / g in CHCl 3, and more preferably at least equal to 0.6 or 0.7 dl / g. However, the intrinsic viscosity of these polymers will in principle not exceed 1.6 dl / g in CHCl3, and may be less than 1.4 or 1.2 dl / g. Said polymers will preferably be PLGAs with a lactide / glycolide ratio

about 75/25.

  The polymers according to the invention can be prepared by the usual methods, in particular by opening the lactide or glycolide rings. Such a process is described for example in the

U.S. Patent 3,773,919.

  In the present invention, it is also possible to use a mixture of polymers of different high viscosities, but preference is given to compositions comprising only one polymer or copolymer. The invention therefore relates firstly to a composition in the form of microcapsules or implants comprising an excipient or a mixture of polymeric excipients or biodegradable copolymers with intrinsic viscosity of between 0.5 dl / g and 1.6 dl / g in CHC13 and an active substance or a mixture of active substances, these microcapsules or implants being able to release the active substance or the mixture of active substances over an extended period of at least 1 month, preferably at least 2 months and, more preferably, at least

3 months.

  By microcapsule is also meant to include microspheres, microparticles, nanocapsules, nanospheres or nanoparticles. By polymer is meant a polymer, a copolymer, or any mixture of these entities. Finally, by active substance is meant an active substance, one of its salts or one of its precursors, or any mixture of

these compounds.

  The polymers or copolymers which can be used in this aspect of the invention may in particular be polymers such as those of lactic acid, glycolic acid, citric acid or malic acid, or else other biocompatible polymers such as poly-B-hydroxybutyric acid, polyorthoesters, polyorthocarbonates, polyesters of c-cyanoacrylic acid, alkylene polyoxalates such as trimethylene or tetramethylene polyoxalate, polyamino acids, etc. They can also be copolymers such as PLGA, polystyrene, polymethacrylic acid, copolymers of methacrylic acid and acrylic acid, polyamino acids, polymers of maleic anhydride, ethylcellulose, nitrocellulose, acetylcellulose, etc. All these polymers or copolymers can be used alone or in any mixture. Preferably, D, L-PLGA will be used and, more preferably, a D, L-PLGA produced from 70 to 80% of D, L-lactide and from 20 to 30% of glycolide. A PLGA synthesized from 75% D, L-lactide and 25% glycolide

  will be particularly suitable for the invention.

  Another particularly preferred polymer for the invention is LPLGA, obtained from 15 L-lactide and glycolide. Compared to D, L-PLGA with the same viscosity, L-PLGA provides

  slower release and represents an alternative to D, L-PLGA of higher viscosity.

  Furthermore, preference will generally be given to PLGAs obtained by ring opening with hydrophobic initiators, such as those of lauric alcohol type, those obtained by ring opening, with hydrophilic initiators, such as those of lactic acid or glycolic acid type. The acid number, corresponding to the number of milliequivalents of KOH necessary per gram of polymer to neutralize the free acidity, seems to be the parameter best correlated with the hydrophilic or hydrophobic character of the polymer. In fact, it can be seen that as soon as it is less than 1 (hydrophobic polymer), a less homogeneous release profile is obtained, which can produce an interruption (or "gap") in the release after the initial release peak, whereas when it is greater than 1.5 and preferably greater than 2 (hydrophilic polymer), the release profile corresponds to a more rapid and homogeneous release. Hydrophilic polymers are therefore generally preferred, because they make it possible to avoid a "gap", but if the release is too rapid, then a polymer of the same viscosity but hydrophobic will not exhibit this "gap" and should be preferred. The charge of active substance ("core loading") of the microcapsules according to the invention, that is to say the ratio between the mass of the pure encapsulated peptide and the total mass of the microcapsule, will generally be between 0 and 20%, preferably between 2 and 15%. For triptorelin acetate, the charge will preferably be less than or equal to 10% and, more preferably, between 4 and 8% for forms allowing release over a period of approximately 3 months. For lanreotide acetate, the charge will preferably be understood

between 10 and 20%.

  4 - For implants, the load of active substance will generally be between 0 and 30% and, from

preferably between 15 and 25%.

  The encapsulation step can be a so-called coacervation step as described for example in

  American patents US 3,773,919 or European EP 52,510.

  One can also use a so-called fusion-extrusion process as described in European patent EP 58 481 or in American patent US 5,225,205, the products obtained being

  then optionally ground according to the usual methods, to result in microparticles.

  Furthermore, it is possible to use a water-soluble active principle such as a water-soluble salt of a peptide, for example acetate. It is also possible to use an insoluble salt of a soluble molecule such as a fatty acid salt of a peptide, for example, a peptide pamoate such as

  described in British patent GB 2,209,937.

  The compositions obtained by fusion-extrusion using the polymers according to the invention

  may also be in the form of implants and be used as such.

  These implants are preferably small implants (mini-implants or microimplants) with a diameter of the order of 1 mm, for example between 0.8 and 1.2 mm. The length of these implants can be for example between 10 and 35 mm, for example of the order of 25 mmn. These implants give very interesting results with low doses of active principle, for example of the order of 3 mg of triptorelin acetate per implant. Such implants can

  release the active ingredient for up to 3 months.

  In addition, it appeared that the configuration of the active principle could also have an influence on the diffusion of this product. In particular, when an active ingredient can be obtained

  in a crystallized or amorphous form, the choice of one or the other form is not indifferent.

  Patent application EP 709 085 describes microcapsules comprising a polymer and a water-soluble and amorphous active substance. In particular, it discusses the importance of obtaining particles of active substance of small size, and preferably of size less than lrm. This application does not however include any process for the preparation of said particles and no mention is made of the effect of the specific surface of the particles of active principle on the release profile of the compositions containing these particles. However, the Applicant has already used since 1986 microcapsules comprising an amorphous active substance, triptorelin acetate sold under the name Decapeptyl 3.75 mg, the particle size of which is only about 8 gm. However, it has found that the size of the particles is not the only determining parameter for promoting a release over an extended period which can range from

up to more than three months.

  -5- The question of the amorphous nature does not arise in principle for products such as peptides or proteins whose mode of production, especially lyophilization, leads to

  most cases to an amorphous product, which is the case for Decapeptyl 3.75 mg.

  The literature abundantly illustrates this phenomenon, and the following articles may in particular be cited: Hsu, C.C. et al., Pharmaceutical Research, 12 (1), 69-77 (1995) or Towns, J.K.,

  Journal of Chromatography, A, 705 (1), 115-27 (1995).

  The invention therefore also relates to a composition in the form of microcapsules or implants comprising at least one biodegradable polymer or copolymer of high molecular weight and at least one water-soluble active substance having a surface.

specific high.

  These microcapsules or implants allow a monophasic release profile in which the initial peak (or "burst" in English) is reduced compared to certain other preparations using a polymer of lower molecular weight, so that they make it possible to release the

  active substance over an extended period of up to more than three months.

  Among the active substances which can be used for the invention, mention may in particular be made of proteins, peptides chosen for example from the group consisting of the following substances: triptorelin or one of its salts, in particular triptorelin acetate, lanreotide or a of its salts, in particular lanreotide acetate, octreotide or one of its salts (as described for example in European patent EP 29 579), in particular octreotide acetate or pamoate, a compound having an LH-RH activity such as triptorelin, goserelin, leuprorelin, buserelin or their salts, an LH-RH antagonist, a GPIIb / Ila antagonist, a compound having an activity similar to a GPIIb / IIIa antagonist, erythropoietin (EPO) or one of its analogs, the various interferons cx, interferon 3 or y, somatostatin, a somatostatin derivative as described in European patent EP 215 171, an analogue of

  somatostatin as described in US patent US 5,552,520 (this patent itself

  even a list of other patents describing somatostatin analogs which are incorporated by reference to the present application), insulin, a growth hormone, a growth hormone releasing factor (GRF), a peptide releasing growth hormone (GHRP), epidermal growth factor (EGF), melanocyte-stimulating hormone (MSH), thyrotropin-releasing hormone (TRH) or one of its salts or derivatives, a thyroid stimulating hormone (TSH), a luteinizing hormone (LH), a follicle stimulating hormone (FSH), a parathyroid hormone (PTH) or a derivative thereof, lysozyme hydrochloride, a peptide linked to parathyroid hormone (PTHrp), a peptide fragment to N terminal (position 1-> 34) of the human PTH hormone, vasopressin or one of its derivatives, oxytocin, calcitonin, a calcitonin derivative having an activity similar to that of calcitonin, a peptide linked to calcitoni gene ne (CGRP), -6- glucagon, a glucagon-like peptide (GLP), gastrin, a gastrin-releasing peptide (GRP), secretin, pancreozymrine, cholecystokinin, angiotensin, human placenta lactogen , human chorionic gonadotropin (HCG), enkephalin, an enkephalin derivative, colony stimulating factor (CSF), endorphin, kyotorphin, interleukins, e.g. Interleukin 2, tuftsin, thymopoietin, thymosthymline, humoral thymic factor (THF), serum thymic factor (FTS), a serum thymic factor (FTS) derivative, thymosin, thymic factor X, tumor necrosis factor (TNF), motilin , bombesin or one of its derivatives as described in US Pat. No. 5,552,520 (this patent itself includes a list of other patents describing bombesin derivatives which are incorporated by reference into the present application), prolactin, neurotensin, dynorph ine, caeruleine, substance P, urokinase, asparaginase, bradykinin, kallikrein, nerve growth factor, blood clotting factor, polymixin B, colistin, gramicidin, bacitracin, peptide stimulating protein synthesis, an endothelin antagonist or a salt or derivative thereof, a vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH) or a fragment thereof, a platelet-derived growth factor (PDGF) , a bone morphogenetic protein (BMP), a pituitary adenylatecyclase activating polypeptide (PACAP), neuropeptide Y (NPY), peptide YY (PYY), a gastric inhibitor polypeptide (GIP), and polynucleotides, in particular double RNA strand (dsRNA) such as those described in patent application EP 0 300 680 or French patent No. 2 622 586. Any other water-soluble active substance, or one of its salts or precursors, may also be

  used by a person skilled in the art if he deems it useful.

  By dsRNA, is preferably meant polyadenylic acid complexed with polyuridylic acid, also called poly (A) -poly (U) or Poly-adenur. Other dsRNAs can be used for the invention, in particular a complex of polyinosinic acid with polycytidylic acid, also known as poly (I) -poly (C), as well as these same complexes modified by introduction uridylic acid in the acid chain

  polycytidylic, such as the Ampligen product from the company HEMISPHERx (for a description of

  these products, refer in particular to European patent application EP 0 300 680). The dsRNA used can for example be a mixture of dsRNA as defined above. Preferably,

  DsRNAs are prepared according to the method described in French patent No. 2,622,586.

  Salts of active substances which can be used for compositions according to the invention especially comprise the salts obtained from organic acids such as acetic, malic acids,

  tartaric, oxalic, fumaric, citric, lactic, stearic, pamoic, methanesulfonic or p-

  toluenesulfonic, or from inorganic acids such as hydrochloric, sulfuric, phosphoric or hydrobromic acids. Preferably, we will use a product

  water-soluble, obtained by salification in the form of a cation, with, for example, acetic acid.

-7-

  It is however possible to use an insoluble salt, for example a pamoate, as indicated above.

  By peptide and / or protein is meant both the peptide and / or the protein themselves as well as

  fragments, salts or pharmacologically active derivatives of these peptides or proteins.

  The water-soluble active substance as used to manufacture microcapsules or implants according to the invention, and in particular triptorelin acetate, lanreotide acetate, octreotide acetate, goserelin, leuprorelin, buserelin or their salts, is preferably obtained by a process mainly comprising two stages: - a lyophilization stage comprising rapid quenching of a dilute solution of the water-soluble substance in a medium of temperature below -70 C; - possibly a grinding step; preferably, this step will include ultrasonic grinding. By dilute solution of the active substance is meant a solution having a concentration of said active substance less than half the saturation concentration, and preferably less than a quarter of said saturation concentration when the latter is at least equal to 200 g / 1l. This process makes it possible to obtain an active substance having a high specific surface. By rapid quenching is meant contact with a medium at low temperature.

  causing an instant freezing of the water-soluble substance solution.

  For freeze-drying, we can for example freeze the solution in a bathing tray

  in a tank of liquid nitrogen, before proceeding with the lyophilization proper.

  Preferably, in order to obtain a maximum specific surface, the rapid quenching of the solution will be preceded by micronization of the solution of active substance. When the solution of active substance is micronized beforehand, the temperature of the medium at low temperature

  may be less than -50 C only.

  For example, to obtain a very high specific surface, one could choose to atomize the solution by spraying it through an atomizer on a metal plate at very low temperature. The temperature of the plate will preferably be less than -50 C, and more preferably less than -70 C or even -80 C or -120 C. This temperature can be reached for example by soaking a metal plate in a medium at very low temperature such as for example liquid nitrogen. According to a preferred variant of the invention, the metal plate is hollow and the solution is sprayed using an atomizer. inside

of said plate.

2776516 '

  Other freezing techniques can be envisaged, for example the atomization of the solution of active substance in a bath of nonsolvent of said active substance beforehand.

  refrigerated. As non-solvent, a liquefied gas such as liquid nitrogen is preferred.

  Another possibility is to freeze the solution of active substance on a refrigerated turntable ("drum-freezing"). As indicated previously, this freezing will be

  preferably preceded by micronization of the active substance solution.

  When the freezing method in a tray is applied to an active substance to prepare microcapsules or prolonged-release implants according to the invention, the specific surface of the active substance, after lyophilization but before grinding, will preferably be greater than 2 m2 / g. More preferably, the specific surface of the active substance

  will be greater than 3 m2 / g, or even 5 m2 / g.

  If a specific surface greater than 10 m2 / g is necessary, it will be preferable to use the process including a micronization step. Preferably, the specific surface obtained for the active substance after lyophilization will be greater than 15 m2 / g. Even more

  preferably, this specific surface will be greater than 20 m2 / g, or even 30 m2 / g.

  To vary the specific surfaces obtained, the freezing conditions of the solution of active substance can be varied by varying different parameters such as by

  example the freezing speed or the concentration of the solution.

  The specific surface area of the active substance is a favorable factor for obtaining a release over a prolonged period, in particular in the case of microcapsules. Indeed, as already mentioned, particles of an active substance of the same size but of specific surfaces

  different will give completely different results with the same polymeric excipient.

  The invention therefore also relates to the process as described above applied to a biologically active water-soluble substance. It also relates to the biologically active water-soluble substance as obtained by the process, which has a high specific surface. As indicated above, the compositions according to the invention find their use preferably in the pharmaceutical field. The pharmaceutical compositions can be administered to a patient by different routes; however, the preferred route is the subcutaneous or intramuscular injection route. The microcapsules according to the invention can firstly be suspended in a suitable vehicle intended for injection, such as an aqueous solution of sodium chloride

  or an aqueous solution of mannitol.

  Unless they are defined in another way, all the technical and scientific terms used here have the same meaning as that commonly understood by an ordinary specialist in the field to which this invention belongs. Likewise, all publications, patent applications, all patents and all other references mentioned herein are incorporated by reference. The following examples are presented to illustrate the above procedures and should not be

  no case be considered as a limit to the scope of the invention.

EXAMPLES:

  For all these examples, the inherent viscosities (IV) were measured according to the conventional methods for measuring the flow time as described for example in "European Pharmacopoeia", 1997, pages 17-18 (capillary tube method). The specific surface area of the active substance, when it was measured, was determined by the method known as the B.E.T. (absorption of a monolayer of nitrogen on the active substance), well method

known to those skilled in the art.

  For the following examples, the term “modified” peptide will mean a peptide which has undergone the lyophilization process according to the invention, in contrast to the “unmodified” peptide, which is lyophilized from

  classic way (without brutal quenching at low temperature).

Example 1:

  16.620 g of "unmodified" triptorelin acetate are dissolved in 554 ml of water. The solution

  is frozen in a tray bathed in a tank of liquid nitrogen, then lyophilized.

  , 18 g of "modified" triptorelin acetate are thus obtained with a yield of 91.34%.

  This compound has a specific surface of 4.7 m2 / g against 0.8 m2 / g before lyophilization.

  Then the ultrasonic grinding of the triptorelin acetate is carried out: 15 minutes are sufficient to obtain particles less than 10 μm for the modified peptide (whereas

  30 minutes are necessary to obtain such a particle size with the unmodified peptide).

  The encapsulation step is then carried out according to the coacervation method as described in European patents EP 52 510 and American patent US 3,773,919; from 3.378 g of this modified and ground triptorelin acetate and from a 7.30% solution of DL-PLGA (DLPLGA composed of 75% DL-lactide and 25% glycolide, viscosity inherent in the

  chloroform = 0.70 dl / g, acid number = 1.61 meq KOH / g) in dichloromethane.

  390 ml of silicone oil were added to form microcapsules by the process of

- 10 -

  coacervation. These microcapsules are recovered after immersion in a heptane bath

  (22 1) and filtration on 10 gm membrane.

Example 2:

  0.338 g of unmodified triptorelin acetate, with a particle size equal to 8 gm after ultrasonic grinding for 30 minutes, were added with stirring to a 7.30% solution

  DL-PLGA in dichloromethane (PLGA equivalent to that described in Example 1).

  ml of silicone oil were added to form microcapsules which are by the

  then precipitated in a heptane bath (2 1) then filtered on a 10 pam membrane.

  Examples 3 to 6: 10.338 g of triptorelin acetate modified according to the conditions described in Table No. 1 below, were added, after ultrasonic grinding, to a 7.30% solution of a mixture 33 , 3% / 33.3% / 33.3% of three DL-PLGAs (with the characteristics described in Table No. 2 below) in dichloromethane. 40 ml of silicone oil were added to form microcapsules which are subsequently precipitated in a bath

  of heptane (2 1) then filtered through a 10 p.m. membrane.

Table No. 1

  Example Concentration Quantity Quantity Surface g / l specific water acetate triptorelin (ml) m2 / g (g)

3,200 3,154 150 3 20 4.7

100 3 30 4.8

6 50 3 60 7.3

  The specific surface area of the starting triptorelin acetate (unmodified) is 0.8 m2 / g.

  -11- The physico-chemical characteristics of the three mixed polymers are brought together in Table No. 2 below

Table No. 2

  Characteristics PLGA No. 1 PLGA No. 2 PLGA No. 3 Lactide / glycolide ratio DL PLGA 50:50 DL PLGA 75:25 DL PLGA 75:25 Inherent viscosity in CHCI3 (dl / g) 0.47 0.61 0.70 Acid number (meq KOH / g) 2.68 2.08 1.61

Example 7:

  22.560 g of unmodified lanreotide acetate are dissolved in 752 ml of water. The solution is frozen in a tray bathed in a tank of liquid nitrogen, then lyophilized. 21.75 g of modified lanreotide acetate, with a specific surface area of 4.4 m2 / g, are obtained with a

yield of 96.41%.

  i0 The encapsulation step is then carried out according to the coacervation method as described in European patents EP 52 510 and American patent US 3,773,919; from 7.5 g of this modified and ground triptorelin acetate and from a 3.7% solution of DL-PLGA (DL-PLGA composed of 50% DL-lactide and 50% glycolide, viscosity inherent in HFIP = 0.55 dl / g) in dichloromethane. 650 ml of silicone oil were added to form microcapsules by the coacervation process. These microcapsules are recovered

  after immersion in a heptane bath (30 l) and filtration on a 10 gOm membrane.

  Examples 8 and 9: Triptorelin acetate microcapsules were made with DL-PLGA (DL-PLGA composed of 75% DL-lactide and 25% glycolide) of different weight average molecular weights (Mw). The manufacturing was carried out according to the process described in

  Example 1 with a triptorelin acetate with a specific surface area equal to 4.7 m2 / g.

  The physico-chemical parameters of Examples 8 and 9 are collected in the table below: Example Mw THF IV CHC13 (dl / g) Acid index (meq KOH / g)

8 58 400 0.61 2.08

9 132 650 0.93 1.80

-12-

Example 10:

  Microcapsules were produced according to the process described in Example 1 with DL-PLGA (DL-PLGA composed of 75% DL-lactide and 25% glycolide; molecular weight determined in THF: 80100; viscosity in chloroform: 0.75 dl / g, acid number = 0.40 meq KOH / g) with hydrophobic tendency.

Example 11:

  Microcapsules were manufactured according to the method described in Example 1, from an L-PLGA (L-PLGA composed of 75% L-lactide and 25% glycolide; molecular mass in THF: 99260; viscosity in chloroform: 0.78 dl / g, acid number = 1.31 meq

KOH / g) with crystal tendency.

Example 12:

  Four parts, by weight, of powder of D, L-PLGA (PLGA composed of 75% lactide and% glycolide; molecular weight determined in THF: 103810; inherent viscosity

  in chloroform: 0.82 dl / g) a part, by weight, of triptorelin acetate is added.

  The lumps are destroyed by sieving through a 400 gim mesh, mixing for 20 minutes at 42 revolutions per minute and the mixture is extruded at 120 ° C. with a screw extruder, through a die of 1 mm opening. Then cooled in air and calibrated by drawing (drawing) to a

final diameter of 0.85 mmn.

  The richness of the mixture per unit of length (mmn) is determined and the microimplants are assayed

  3 mg of triptorelin by cutting the extrudate rods to calculated lengths (here, 24 min).

  Finally, the weight of each microimplant is controlled.

  Examples 13 and 14: The same protocol is used for these two examples: g of lanreotide acetate are dissolved in water to give the chosen concentration to the

  solution (for example, to obtain the concentration of 30 g / l, 167 ml of sterile water are added).

  This solution is atomized using a 500 ml sprayer, the jet of which is adjusted so as to obtain the finest droplets possible. The droplets obtained are projected onto a tray, the bottom of which dips in liquid nitrogen. Two temperature sensors are

  previously introduced into the tray in order to follow the evolution of the product temperature.

  Once the product has been frozen, the tray is placed in a freeze dryer, the plate of which is

about -54 C.

- 13 -

  The temperature of the products and of the plate is allowed to equilibrate for 1 hour. Then we go to the sublimation phase (the plate temperature is fixed at 20 C and the pressure in the tank at 100 gbar). This phase lasts about 30 hours. The average final product temperature is 13 C. The following secondary desiccation (pressure in the tank 50 gbar) lasts approximately 24 hours. The average final temperature of the product is 20 C. The characteristics of the reactants used and the products obtained are summarized in the table below: Characteristics Example 13 Example 14 Mass of lanreotide acetate used (g) 5.00 5.00 Concentration of the solution (g / l) 30 10 Mass of lanreotide acetate recovered 4.54 4.10 Specific surface accacc obtained (m2 / g) 36 43 Lanreotide acetate with specific surface 43 m2 / g obtained previously (example 14) is incorporated into microcapsules according to the following method: 0.782 g of lanreotide acetate are weighed in a glass tube. 15 ml of dichloromethane are added to the peptide salt. The crushing of the peptide is carried out by ultrasound using an ultrasonic generator equipped with an amplifier and a probe with flat or plunging end.

  (frequency = 50 Hz, power 250 W; grinding for approximately 15 min).

  The encapsulation step is then carried out according to the coacervation method as described in European patents EP 52 510 and American patent US 3,773,919 using the 0.782 g of ground lanreotide acetate and a solution of 4 g of D, L- PLGA 50:50 (IV = 0.48 dl / g in CHCl3) in 35 ml of dichloromethane. 34.2 ml of silicone oil were added to form microcapsules by the coacervation method. These microcapsules are recovered

  after immersion in a heptane bath (2.5 l) and filtration through a 10 4m membrane.

  The microspheres obtained can then be dried under vacuum, distributed in bottles, and lyophilized with excipients (for example ballast or a surfactant) in order to be stored

  in good conditions and facilitate the suspension of the microcapsules.

  - 14- Study of the release profiles of microcapsules according to the invention In order to illustrate the advantage of microcapsules according to the invention, the study of their release profiles

in vitro was performed.

  For each of Examples 1 to 11, the release of three test portions of approximately 25 mg of mricrocapsules, placed in 4 ml of 0.9% sodium chloride solution, is measured. It is extracted after 1 hour, 1 day and 4 days of release in the solution maintained at 37 C. The triptorelin is assayed by high performance liquid chromatography (HPLC) with respect to a calibration range in gradient mode in the trifluoroacetic acid system. (TFA). To obtain the standard calibration range, a TI solution is prepared as follows: a test sample close to 7.5 mg of reference triptorelin acetate is placed in a 50 ml flask; make up to 50 ml with a 0.1% acetic acid solution. From the solution T1, the solutions T2 and T3 are prepared by proceeding as follows: for T2, we take mnl of TI solution and make up to 20 ml with the 0.1% acetic acid solution. For solution T3, take 1 ml of Tl solution and make up to 50 ml with the acid solution

0.1% acetic.

  The amount of triptorelin acetate or lanreotide acetate released is determined as a percentage relative to the amount of triptorelin acetate or lanreotide acetate

  initially present (100%), which serves as a reference.

  The results of the in vitro tests are summarized by the table below. Examples Cumulative release rate (%)

Examples

1 hour 1 day 4 days

1 8 16 27

2 9 47 57

3 10 45 67

4 10 37 65

9 41 66

6 8 30 55

7 3 14.5 28.3

8 1il 40 67

9 2 4 6

5 12 14

1i 1 I 2 2

- 15 -

  The results of in vivo tests are perfectly correlated with those of in vitro tests. By way of example, microcapsules from Example 1, at a dose of 1.2 mg / kg, were injected intramuscularly into rats. A plasma assay revealed that the level of triptorelin remained consistently above 0.1 ng / ml over a period of more than 90 days. The same studies carried out on microcapsules of Example 2 showed that the testosterone level remained

  consistently less than 1 ng / ml over a period of more than 90 days.

  The microimplants of Example 12 were tested in vivo as follows: a total dose of 3 mg of triptorelin was injected intramuscularly into 6 Beagles dogs (weight of approximately 12 kg) in a muscle of the hind paw of each of the animals. A plasma assay revealed that the level of triptorelin remained constantly above 0.1 ng / ml over a period of

more than 90 days.

- 16 -

Claims (15)

  1. Composition in the form of microcapsules or implants comprising an excipient or a mixture of biodegradable polymer or copolymer excipients with intrinsic viscosity of between 0.5 dl / g and 1.6 dl / g, preferably less than 1.2 dl / g in CHC13 and an active substance or a mixture of active substances, said microcapsules being able to release the active substance or the mixture of active substances over a prolonged period up to three months.   2. Composition in the form of microcapsules or implants according to claim 1, characterized in that the polymer or biodegradable copolymer excipient has a   intrinsic viscosity greater than 0.6 dl / g in CHC13.   3. Composition in the form of microcapsules or implants according to one of the   Claims 1 or 2, characterized in that the biodegradable polymer is a   lactide and glycolide copolymer (PLGA).   4. Composition in the form of microcapsules or implants according to claim 3, characterized in that the PLGA is prepared from 75% lactide and 25% glycolide. 5. Composition in the form of microcapsules or implants according to one of the   Claims 1 to 3, characterized in that the biodegradable polymer is a copolymer L-lactide and glycolide.   6. Composition in the form of microcapsules or implants comprising at least one excipient or a mixture of polymeric excipients or biodegradable copolymers and at   minus a water-soluble active substance with a high specific surface.   7. Composition in the form of microcapsules or implants according to one of the   Claims 1 to 6, characterized in that the specific surface of the active substance is   greater than 2 m2 / g, and preferably greater than 3 m2 / g.   8. Composition in the form of microcapsules or implants according to claim 7, characterized in that the specific surface of the active substance is greater than 5 m2 / g,   and preferably greater than 10 m2 / g. - 17 -   9. Composition in the form of microcapsules or implants according to claim 8, characterized in that the specific surface of the active substance is greater than   m2 / g, and preferably more than 30 m2 / g.   1 0. Composition in the form of microcapsules or implants according to any one of   Claims 1 to 9, characterized in that the active substance is a protein.   1 1. Composition in the form of microcapsules or implants according to any one of   Claims I to 9, characterized in that the active substance is a peptide.   1 2. Composition in the form of microcapsules or implants according to claim 11, characterized in that the peptide is chosen from the group consisting of the following substances: triptorelin or one of its salts, in particular triptorelin acetate, lanreotide or one of its salts, in particular lanreotide acetate, octreotide or one of its salts, in particular octreotide acetate or pamoate, a compound having LH-RH activity such as triptorelin, goserelin, leuprorelin, buserelin or their salts, an LH-RH antagonist, a GPIIb / IIIa antagonist, a compound having an activity similar to a GPllb / IIIa antagonist, erythropoietin (EPO) or one of its analogs , the different interferons a, interferon 3 or y, somatostatin, a somatostatin derivative, a somatostatin analog, insulin, a growth hormone, a growth hormone releasing factor (GRF), a hormone-releasing peptide growth hormone (GHRP), epidermal growth factor (EGF), melanocyte-stimulating hormone (MSH), thyrotropin-releasing hormone (TRH) or one of its salts or derivatives, a thyroid stimulating hormone (TSH), a luteinizing hormone (LH), a follicle stimulating hormone (FSH), a parathyroid hormone (PTH) or a derivative thereof, lysozyme hydrochloride, a peptide linked to parathyroid hormone (PTHrp), a peptide fragment to N terminal (position 1-> 34) of the human hormone PTH, vasopressin or one of its derivatives, oxytocin, calcitonin, a derivative of calcitonin having an activity similar to that of calcitonin, a peptide linked to calcitonin gene (CGRP), glucagon, a glucagon-like peptide (GLP), gastrin, a gastrin-releasing peptide (GRP), secretin, pancreozymine, cholecystokinin, angiotensin, human placenta lactogen , human chorionic gonadotropin (H CG), enkephalin, a derivative of enkephalin, colony stimulating factor (CSF), endorphin, kyotorphin, interleukins, e.g. Interleukin 2, tuftsin, thymopoietin, thymosthymline, humoral thymic factor (THF), serum thymic factor (FTS), a derivative of serum thymic factor (FTS), thymosin, thymic factor X, tumor necrosis factor (TNF), motilin, or one of its derivatives, prolactin, neurotensin, dynorphin, caerulein, substance P, urokinase, asparaginase, - 18 -   bradykinin, kallikrein, nerve growth factor, blood clotting factor, polymixin B, colistin, gramicidin, bacitracin, peptide stimulating protein synthesis, an endothelin antagonist or one of its salts or derivatives , a vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH) or a fragment thereof, a platelet-derived growth factor (PDGF), a bone morphogenetic protein (BMP), a polypeptide activating pituitary adenylate cyclase (PACAP) ), neuropeptide Y (NPY), peptide YY (PYY), an inhibitory polypeptide   gastric (GIP), and polynucleotides, including double-stranded RNA.   13. Composition in the form of microcapsules or implants according to one of   Claims 1 to 9, characterized in that the active substance is triptorelin acetate.   14. Composition in the form of microcapsules or implants according to one of   Claims 1 to 9, characterized in that the active substance is lanreotide acetate or octreotide acetate.   15. A process for the preparation of a water-soluble substance having a high specific surface, comprising the following steps: a freeze-drying step comprising rapid quenching of a dilute solution of said water-soluble substance in a medium of temperature below -70 ° C .;   - possibly a grinding step.   1 6. Method according to claim 15, characterized in that the grinding step comprises a ultrasonic grinding.   1 7. Method according to claim 15 or 16, characterized in that the diluted solution is a   solution at a concentration less than half the saturation concentration.   1 8. Process according to claim 15 or 16, characterized in that the water-soluble substance has a saturation concentration of at least 200 g / l and that the diluted solution is   a solution at a concentration less than a quarter of the saturation concentration.   1 9. Method according to one of claims 18 to 21, characterized in that the rapid quenching is   performed after a micronization step of the active substance solution.   2 0. Method according to claim 22, characterized in that the micronization step consists   by passing the solution through an atomizer.   2 1. Active substance as obtained by a process according to one of claims 15 to 20.   2 2. Protein according to claim 21. 2776516. -19 -   23. Peptide according to claim 21.   24. Peptide according to claim 23, characterized in that it is chosen from the group consisting of the following substances: triptorelin or one of its salts, in particular triptorelin acetate, lanreotide or one of its salts, in particular lanreotide acetate, octreotide or a salt thereof, in particular octreotide acetate, a compound having LH-RH activity such as triptorelin, goserelin, leuprorelin, buserelin or their salts, LH-RH antagonist, a GPIIb / IIIa antagonist, a compound having an activity similar to a GPIIb / IIIa antagonist, erythropoietin (EPO) or one of its analogs, the different cc interferons, interferon 3 or y , somatostatin, a somatostatin derivative, a somatostatin analog, insulin, a growth hormone, a growth hormone releasing factor (GRF), a growth hormone releasing peptide (GHRP), a epidermal growth factor (EGF), a melanocyte-stimulating hormone (MSH), a thyrotropin-releasing hormone (TRH) or one of its salts or derivatives, a thyroid stimulating hormone (TSH), a luteinizing hormone (LH), a follicle stimulating hormone (FSH), a parathyroid hormone (PTH) or a derivative thereof, a peptide linked to the parathyroid hormone (PTHrp), a fragment of N-terminal peptide (position 1-> 34) of the human PTH hormone, vasopressin or one of its derivatives, oxytocin, calcitonin, a calcitonin derivative having an activity similar to that of calcitonin, a peptide linked to the calcitonin gene (CGRP), glucagon, a peptide similar to glucagon (GLP), gastrin, a gastrin-releasing peptide (GRP), secretin, pancreozymine, cholecystokinin, angiotensin, lactogen in the human placenta, human chorionic gonadotropin (HCG), enkephalin, an enkephalin derivative, colony stimulating factor (CSF), endo rphine, kyotorphine, interleukins, for example Interleukin 2, tuftsin, thymopoietin, thymosthymline, humoral thymic factor (THF), serum thymic factor (FTS), a derivative of serum thymic factor (FTS), thymosin, thymic factor X, tumor necrosis factor (TNF), motilin, bombesin or one of its derivatives, prolactin, neurotensin, dynorphin, caerulein, substance P, urokinase, asparaginase, bradykinin, kallikrein, nerve growth factor, blood clotting factor, polymixin B, colistin, gramicidin, bacitracin, peptide stimulating protein synthesis, an endothelin antagonist or one of its endagonists salts or derivatives, a vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH) or a fragment thereof, a platelet-derived growth factor (PDGF), a bone morphogenetic protein (BMP), an adenyl-activating polypeptide pituitary atecyclase (PACAP), neuropeptide Y (NPY), peptide YY (PYY), a polypeptide   gastric inhibitor (GIP), and polynucleotides, including double-stranded RNA. - 20 -   2 5. triptorelin acetate as obtained by a process according to one of claims 15 to   20. 2 6. Lanreotide acetate or octreotide acetate as obtained by a process according to one of claims 15 to 20.   27. Double stranded RNA as obtained by a process according to one of claims 15 to 20.   2 8. Double stranded RNA according to claim 27, characterized in that it is a complex   polyadenylic acid with polyuridylic acid.