FR2649319A1 - FORMULATIONS WITH PROLONGED RELEASE OF WATER-SOLUBLE PEPTIDES - Google Patents

Abstract

The present invention relates to a sustained release formulation of a peptide, preferably a somatostatin such as octreotide, for example pamoate. The active principle is present in a polymer vehicle, preferably poly (lactic-co-glycolic acid). The formulation is preferably a depot formulation in the form of microparticles. The invention also comprises an improved process for preparing microparticles of active ingredient in a biodegradable and biocompatible vehicle.

Description

The present invention relates to -

  sustained release formulations of drugs.

  In particular, the invention relates to

  sustained-release formulations of hydrophilic peptides

  soluble compounds, for example somatostatin or a somatostatin analogue, such as octreotide, in a biodegradable and biocompatible polymeric vehicle, for example a matrix or an envelope, for example in the form of an implant or preferably in the form of microparticles (also known as microcapsules

or microspheres).

  The invention also relates to such formulations having satisfactory peptide release profiles for a particular period of time. The peptide drugs have, after their administration, whether orally or parenterally, a low bioavailability in the blood due, for example to their high biodegradability in the body. When administered orally or nasally, they often also have poor resorption through mucosal membranes. It is difficult to obtain therapeutically active blood levels during a

long period of time.

  It has been proposed to administer the peptide drugs parenterally, namely in the form of a formulation deposited in a biodegradable polymer, for example in the form of microparticles or an implant, to allow their prolonged release after a time of presence in the polymer, the latter

  protecting the peptide against enzymatic influences

  and hydrolytics of biological fluids.

  Although certain depot formulations intended for parenteral administration of peptide drugs in a microparticle or implant polymer are known, in practice a satisfactory release of the peptides is obtained only in very few cases. Special measures to ensure constant release of the peptides must therefore be taken to obtain therapeutically serum drug levels.

  assets and, if necessary, to avoid

  too high a drug in the serum

  cause undesirable side effects.

  The release of the peptides is a function of various factors, in particular of the type of peptide, and of its presentation, for example in free form or in another form, for example in salt form, which may have an effect on its solubility in the water. 'water. Another important factor is the choice of the polymer among the

  list of possibilities described in the literature.

temperature.

  Each polymer has its own bio-speed

  degradation. Carboxy groups can be formed which influence the pH value in the polymer and also have an effect on water solubility

  of the peptide and therefore on its release.

  Other factors that may influence the release of peptides are the

  concentration of the peptide in the vehicle, its

  in the polymer and the shape in the case of a

implant and its dimension.

  So far, no somatostatin-based composition in a sustained release form for parenteral administration has been placed on the market, perhaps because no composition with blood levels

satisfactory could not be obtained.

  Pharmaceutical compositions which are based on polymers, and which make it possible to obtain a prolonged or delayed release of the active ingredient,

are known in the art.

  No. 3,773,919 describes controlled-release formulations of active principle in which the active ingredient, for example a water-soluble peptide, is dispersed in a linear polymer of lactic acid or in a linear co-polymer of the acid. lactic and glycolic acid, biodegradable and biocompatible. The document, however, does not give any data on drug release and somatostatin is not mentioned. The patent

  US 4,293,539 discloses anti-

  bacterial microparticles.

  U.S. Patent No. 4,675,189 discloses sustained release formulations of nafarelin, an LHRH-like decapeptide, and other LHRH analogs in copolymers of lactic acid and glycolic acid, but gives no data. sure

the release of the active ingredient.

  T. Chang, in J. Bioneng., Vol. 1, pages -32, 1976, describes the extended release of products

  biological agents, enzymes and vaccines from micro-

  particles. U.S. Patent Nos. 3,991,776,

  4,076,798 and 4,118,470 describe polymers and copolymers.

  lactic acid polymers and compositions containing them for surgical use and

  sustained release by biodegradation of the poly-

  mother. European Patent Application No. 203,031 discloses a series of octapeptides analogous to somatostatin, for example RC160 having the formula

* *

  D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NE2 having a bridge between Cys- residues. Claim

  18 mentions the possibility of presenting somatos-

  tatines in the form of microcapsules with a copolymer of lactic acid and glycolic acid, but does not describe how to obtain constant serum levels

  that are therapeutically active.

  U.S. Patent No. 4,011,312 indicates that

  a continuous release of a medi-

  antimicrobial material, for example water-soluble polymyxin B, from a matrix, in the form of an implant, a lactic acid copolymer and glycolic acid with a low molecular weight (less than 2000) and with a content relatively high in units of glycolic acid, when the implant is inserted into the breast canal of a cow. The active ingredient is released in a very short time, because of the high content of glycolic acid units and the low molecular weight of the polymer, which promotes a rapid biodegradation of the polymer and therefore a rapid release of the active ingredient. A relatively high content of active ingredient

  also promotes a quick release of the latter.

  However, that patent does not mention any somatostatin and does not provide any data on the

release of the active ingredient.

  European Patent No. 58481 mentions that a continuous release of a water-soluble peptide from a polylactic acid implant is stimulated by lowering the molecular weight of at least a portion of the polymer molecules, by introducing

  of glycolic acid units in the molecule of the poly-

  mother, by increasing the sequential nature of the poly-

  when using a copolymer of lactic acid and glycolic acid, and by increasing the active ingredient content of the polymer matrix and the size of the implant. Although somatostatins are mentioned as water-soluble peptides, the document does not disclose a somatostatin release profile and no indication is given of how to combine all these parameters to obtain, for example, constant somatostatin levels in the serum. for at least a week, for example a month. European Patent No. 92918 indicates that a continuous release of peptides, preferably hydrophilic peptides, can be obtained for a prolonged time, when the peptide is incorporated into a

  hydrophobic polymer matrix, for example

  lactic acid, which is made more accessible to water by introducing into its molecule a hydrophilic unit, for example polyethylene glycol, polyvinyl alcohol, dextran or polymethacrylamide. The hydrophilic character of the amphiphilic polymer comes from groups

  ethyleneoxy in the case of a polyethylene unit

  glycol, free hydroxy groups in. the case of a polyvinyl alcohol unit or a dextran unit, and amide groups in the case of a polymethacrylamide unit. Due to the presence of a hydrophilic unit in the polymer molecule, the implant will have the properties of a hydrogel after absorption of water. Somatostatin is mentioned as a hydrophilic peptide, but the document only provides

  no release profile and gives no indica-

  the preferred type of polymer for this peptide and

  the molecular weight and the number of hydro-

  philes that the polymer should have.

  British Patent Application 2,145,422 indicates that it is possible to obtain a prolonged release of drugs of various types, for example vitamins, enzymes, antibiotics and antigens, when the active ingredient is incorporated in a matrix, for example in the form of microcapsules or implant, consisting of a polymer of a polyol, for example glucose or mannitol, comprising one or more ester groups with a polylactic acid, preferably at least 3. The remains of the polylactic acid

  preferably contain glycolic acid units.

  lic. However, peptides, for example somatostatin, are not mentioned as drugs that can be used in such a form to

sustained release.

  The invention thus relates to sustained-release formulations, for example microparticle formulations, a drug, especially a somatostatin or a somatostatin analogue, such as octreotide, which is water-soluble and has hormonal activity, which provide satisfactory plasma levels in

  active ingredient, for example in a bio-polymer

  compatible and biodegradable, for example in a polymer matrix obtained by microencapsulation. The polymer matrix may be a synthetic or natural polymer. The microparticles of the invention can be prepared according to any of the techniques

  usual, for example according to the technique of

  organic phase, the spray drying technique or the triple emulsion technique, in which the polymer is precipitated with the active ingredient, and then the resulting product is cured when

  phase separation technique or triple emulsion.

  If necessary, the release formulations

  prolonged can be in the form of an implant.

  The Applicant has found a particularly useful modification of the phase separation technique for preparing microparticles of any size.

which active ingredient.

  The invention therefore also relates to a process for the preparation of microparticles comprising a

  active principle in a biodegradable and biologically

  compatible method according to which a) dissolving the polymeric material in a suitable solvent in which the active ingredient is not soluble, b) in the solution of step a), adding and dispersing a solution of the active ingredient in a coated solvent, for example an alcohol, which is not a solvent for the polymer,

  c) a phase inducing agent is added to the

  persion of step b), to cause the formation of microparticles, d) an oil-in-water emulsion is added to the mixture of step c) to cure the microparticles, and

  e) the microparticles are recovered.

  The Applicant has also found a particularly useful modification of the technique

  triple emulsion for the preparation of micro-

  particles of any active ingredient.

  The invention thus relates to a process for the preparation of microparticles, wherein (i) a water-in-oil emulsion formed from an aqueous medium and a water-immiscible organic solvent, containing in

  phase the active ingredient and in the other phase a poly-

  biodegradable and biocompatible mother, with an excess of an aqueous medium containing an emulsifying substance or

  a protective colloid, to form a water-emulsion

  in-oil-in-water, without adding to the water-in-oil emulsion of substance retaining the active ingredient or without applying an intermediate step increasing the viscosity, (ii) subjecting the organic solvent to a desorption and

  (iii) the resulting microparticles are isolated and dried

aunts.

  The invention also relates to micro-

  particles obtained by these methods.

  The invention also relates to a) a sustained release formulation comprising a peptide in an ester of a polyol with a poly (40/60 to 60/40 lactic-co-glycolic acid), the

  polyol remaining being selected from the alcohols containing

  C 3 -C 6 hydrocarbon chain and 3 to 6 hydroxy groups and mono- and di-saccharides, and the esterified polyol having at least 3 poly (lactic-co-glycolic acid) chains, b) sustained release comprising a peptide which is a calcitonin, lypressin or somatostatin, in a linear / 60 to 60/40 copolymer of lactic acid and glycolic acid having a molecular weight Mw between 25,000 and 100,000 and a polydispersity Mw / Mn between 1.2 and 2, in a concentration between 0.2 and 10%, preferably between 2 and% by weight of peptide, c) a sustained-release formulation comprising octreotide or one of its derivatives, or a salt of the compound

  in a biodegradable polymer vehicle and bio-

  compatible. The Applicant has found that a new salt of octreotide, pamoate, is very stable in

such formulations.

  The invention thus relates to (i) octreotide pamoate and (ii) a method for preparing octreotide pamoate, wherein octreotide is reacted with pamoic acid (or a derivative thereof).

reagent of this compound).

  The invention also relates to formulas

  mentioned above for use in the

  treatment of acromegaly or breast cancer.

  The active ingredients used in the processes of the invention are preferably water-soluble active ingredients, for example peptides. The peptides used in the methods and formulations of the invention may be a calcitonin, such as salmon calcitonin, lypressin and somatostatin of natural origin, and

its synthetic analogues.

  Somatostatin of natural origin which is

  one of the preferred compounds is a tetradecapeptide

  with Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp formula

I I

Cys-Ser-Thr-Phe-Thr-Lys

  This hormone is produced by the hypo-

  thalamus and other organs, for example the gastrointestinal tract.Somatostatin intervenes at the pituitary level to inhibit the secretion of

  hGH (somatotropic hormone) -and TSH (thyroid

  stimulating hormone). At the digestive level, it inhibits many secretory processes (in the pancreas, stomach,

duodenum and small intestine).

  By the expression "analogous or derived from the

  somatostatin "means any of the polyps

  cyclic or straight chain derivatives of natural somatostatin in which one or more

  amino acid residues have been deleted and / or

  killed by one or more other amino acid residues

  and / or in which one or more functional groups

  These have been replaced by one or more other functional groups and / or in which one or more groups have been replaced by one or more other isosteric groups. In general, the term encompasses all modified derivatives of a biologically active peptide that exert a qualitatively similar

  that of unmodified somatostatin.

  Agonist analogs of somatostatin are therefore useful for replacing somatostatin of natural origin in its action on regulation.

physiological functions.

  Preferred known somatostatin compounds are: a) (D) Phe-CysPhe- (D) Trp-Lys-Thr-Cys-Throl (naming octreotide) b) (D) PheCys-Tyr (D) TrpLysValCysThrNH2 c) (D) ) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-TrpNH2 d) (D) Trp-Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH2 e) (D) Phe-Cys -Phe- (D) Trp-LysThr-Cys-ThrNH 2 f) 3 - (2 - (Naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-CysThrNH 2 g) (D) Phe-Cys- Tyr- (D) Trp-Lys-Val-Cys-B-Nal-NH 2 h) 3- (2-naphthyl) Alaz-Cys-Tyr- (D) Trp-Lys-Val-Cys-O-Nal- NH 2 i) (D) Phe-Cys-B-Nal- (D) Trp-LysVal-Cys-Thr-NH 2 o in each compounds a) to i) there is a gap between the labeled amino acids of an asterisk (*) as

  indicated in the formula below.

  The other preferred somatostats are as follows:

I I

  H-Cys-Phe-Phe- (D) Trp-Lys-Thr-Phe-Cys-OH [see Vale et al., Metabolism, 27, Supp. 1, 139,

(1978)]

  Asn-Phe-Phe- (D) Trp-Lys-Thr-Phe-Gaba [see European Patent Application No. 1295]

* *

  MeAla-Tyr- (D) Trp-Lys-Val-Phe [see Verber et al., Life Sciences, 34, 1371-1378 (1984) and European Patent Application No. 70,021) also known as

  (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe).

* *

  NMePhe-His- (D) Trp-Lys-Val-Ala [see R.F. Mutt et al., Klin. Wochenschr. (1986) 64 (Supplement VII)] * - * H-Cys-His-His-Phe-Phe- (D) Trp-Lys-Thr-Phe-ThrSer-Cys-OH (see European Patent Application 200,188 )

* *

  X-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NIH2 and

* *

  X-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol in which X signifies a cationic attachment especially

* *

  Ac-hArg (Et2) -Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-NE2 (see European Patent Application 363589) and in the above formulas, a bridge being present

  between amino acids marked with an asterisk (*).

  The content of these publications is incorporated

  to this application for reference.

  The derived expression also includes the

  corresponding compounds having a carbohydrate residue.

  When the somatostatin has a carbohydrate moiety, the latter is preferably coupled to an N-terminal amino group and / or to at least one amino group present in the side chain of the peptide, more preferably to an N-terminal amino group. Such compounds and their preparation are described for example

  in international application WO 88/02756.

  The expression derived from octreotide includes derivatives comprising a remainder

* *

D-Phe-Cys-Phe-Trp-Lys-Thr-Cys

  in which the Cys remains are connected by a bridge.

  Particularly preferred derivatives are

  N "- [c-glucosyl- (1-4-deoxyfructosyl) DPhe-Cys-Phe-

  DTrp-Lys-Thr-Cys-Thr-ol and Na- [O-deoxyfructosyl] -

  DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol, each comprising a bridge between the -Cys- residues, preferably in the form of acetate, and described respectively in the examples

  2 and 1 of the application mentioned above.

  The somatostats may be, for example, in free form, in salt form, or in the form of complexes. Acid addition salts

  can be formed for example with organic acids

  polymers, and mineral acids. The acid addition salts include, for example, hydrochloride and acetate. The complexes are formed for example from somatostatin by addition of mineral substances, for example mineral salts or hydroxides such as Ca and Zn salts and / or by

  addition of organic polymeric substances.

  Acetate is the preferred salt for such formulations, especially for microparticles causing a reduced peak plasma. The invention also relates to pamoate which is useful in

especially for implants.

  Pamoate can be obtained according to the

  conventional methods, for example by reacting the embonic acid (pamoic acid) with octreotide, for example in free base form. The reaction can be carried out in a polar solvent, for example

ambient temperature.

  Somatostats are indicated for a

  use in the treatment of disorders for

  which a long-term application of the drug is envisaged, for example for the treatment of disorders having an etiology comprising or associated with an excess of secretion of growth hormone, for example in the treatment of acromegaly, for a

  use in the treatment of gastrointestinal disorders

  eg peptic ulcers, enterocutaneous and pancreatocutaneous fistulas, irritable bowel syndrome, dumping syndrome, aqueous diarrhea, acute pancreatitis and gastrointestinal hormone-producing tumors (eg, vipoma, tumors secreting GRF, glucagonome, insulinoma, gastrinoma and tumors

  carcinoids) and gastrointestinal bleeding.

  tin, breast cancer and diabetes-related complications. The polymeric carrier may be prepared from biocompatible and biodegradable polymers, such as linear polyesters, branched polyesters consisting of linear chains developing from a polyol residue, for example glucose; the

  other esters are polylactic acid, poly-

  glycolic acid, polyhydroxybutyric acid, poly-

  caprolactone, polyalkylene oxalate, esters of a polyalkylene glycol with Krebs cycle acids, for example citric acid and the like, and their copolymers. Preferred polymers of the invention are linear polyesters and chain polyesters

  branched. Linear polyesters may be

  prepared by condensation of α-hydroxycarboxylic acids, for example lactic acid and glycolic acid, or by polymerization of the lactone (dimer of the acid)

  (see for example US Patent No. 3,773,919).

  The linear copolymers of lactic acid and of glycolic acid that are preferred for use according to the invention advantageously have a molecular weight of between 25,000 and 100,000 and a polydispersibility of Mw / Mn, for example between

1,2 and 2.

  Preferred branched polyesters for use according to the invention can be prepared using polyhydroxy compounds, for example polyols, such as glucose and mannitol, as initiators. These polyol esters are known and described in British Patent Application No. 2,145,422. The polyol contains at least 3 hydroxy groups and can have a molecular weight of up to 20,000 and at least 1, preferably at least 2 for example, an average of 3 hydroxyl groups of the polyol being in the form of an ester with a polylactic acid or a poly (lactic-co-glycolic acid). We use

  usually 0.2% glucose to initiate the polymer.

  authorization. The structure of the branched polyesters is star-shaped. Polyester chains in linear and star-shaped polymeric compounds

  preferably used in formulations of the invention.

  are preferably copolymer chains of carboxylic acids, lactic acid and

  glycolic, or their cyclic dimers (lactones).

  The molar ratios of lactic acid to glycolic acid are between about 75:25 and 25:75, for example between 60:40 and 40:60, the ratios between 55:45 and 45:55, for example between 55:45 and 50:50

  being particularly preferred.

  The star-shaped polymers are preferably obtained by reacting a polyol with the dilactide and preferably also with the diglycolide at a high temperature and in the presence of a catalyst, which makes possible the opening of the

  lactone cycle for polymerization.

  An advantage of the star-shaped polymer in the formulations of the invention is that its molecular weight can be relatively high, which confers a physical stability, for example a certain hardness, to the implants and the microparticles and prevents them from agglomerating. while having relatively short chains of polylactic acid or copolymer of lactic acid; this allows a controllable biodegradation rate of the polymer, ranging from a few weeks to one or two months and a corresponding sustained release of the peptide, the formulations obtained with these polymers being appropriate,

  example, for a release over a month.

  The star-shaped polymers preferably have a molecular weight (M ") of between about 10,000 and 200,000, more preferably between 1,000 and 100,000, especially between 35,000 and 10,000, and a polydispersity of, for example, between

  1.7 and 3.0, for example between 2.0 and 2.5. The visco-

  The intrinsic properties of 35,000 and 60,000 molecular weight star polymers are

  0.36 and 0.51 dl / g respectively in chloro-

  form. A star-shaped polymer having a molecular weight of 52,000 has a viscosity of

0.475 dl / g in chloroform.

  The expressions microspheres, microcapsules

  and microparticles are international expressions

  changeable according to the invention and mean the micro-

  encapsulation of the peptides by the polymer, the peptide being preferably distributed through the polymer,

  which then becomes a template for the peptide.

  In this case, the expression

  microspheres or more generally that of micro-

  particles. By using the phase separation technique of the invention, it is possible, for example, to prepare the formulations of the invention by dissolving the polymer carrier in a solvent which is not a solvent for the peptide, and adding and dispersing a peptide solution in the composition consisting of the polymer and the solvent. A phase inductor, for example a silicone fluid, is then added to enable the

  microencapsulation of the peptide with the polymer.

  The peak plasma effect can be greatly reduced by in situ precipitation of ultra-fine particles of the active ingredient by adding a solution of the active ingredient to the polymer solution before the separation phase. In the method described in the state of the art, the dry particles are added

  directly to the polymer solution.

  The duration of peptide release and the therapeutic activity of the peptide may be

  increased by a hardening / washing step of the micro-

  particles with a buffer / heptane emulsion. The method of the state of the art comprises a curing step not followed by a subsequent wash or

  followed by a separate aqueous wash step.

  An oil-type emulsion can be used

  in-water to wash and cure the microparticles and remove the unencapsulated peptide. The washing serves to remove the unencapsulated peptide from the surface of the microparticles. The removal of excess peptide from the microparticles decreases the initial peak plasma that characterizes many common formulations obtained by microencapsulation. Thus, a greater release of the active ingredient in a given time interval is made possible by the formulations of the invention. The emulsion also serves to eliminate the

  residual solvent of the polymer and the silicone fluid. The emulsion can thus be added to the mixture of poly-

  mother and peptide, or the mixture can be added to

  the emulsion. However, the second solution is preferred.

  The oil-in-water emulsion may be

  prepared using an emulsifier such as mono-

  sorbitan oleate (Span 80, ICI corp.) and the like, to form a stable emulsion. The emulsion can be buffered with a buffer that does not harm the peptide and the polymer matrix. The pH of the buffer can be between 2 and 8, preferably pH 4. The buffer can be prepared from acidic buffers such as phosphate buffer, acetate and the like.

  replace the buffer with water alone. We can also

  use heptane, hexane etc ... as

organic phase of the emulsion.

  The emulsion may contain agents of

  dispersion such as silicone oil.

  A preferred emulsion comprises heptane, pH 4 phosphate buffer, silicone oil and sorbitan mono-oleate. When an initial release of the active ingredient is desired, the hardening / washing step can be replaced by a hardening step with an organic solvent such as

heptane, hexane etc ...

  As another alternative to oil-emulsion

  in-water for the hardening of microparticles, one

  can use for example a solvent-plus an emul-

  to harden the microparticles without washing, and a solvent plus an emulsifier for curing

  followed by a separate washing step.

  The oil-in-water emulsion can be used

  without dispersing agent. However, the dispersing agent avoids the aggregation of the dry particles of the microparticles caused by electricity

  static and reduces the solvent content

  siduel. Examples of solvents for

  polymer matrix, mention may be made of the chloride of

  thylene, chloroform, benzene, ethyl acetate, etc. The peptide is preferably dissolved in an alcoholic solvent, for example methanol, which is

* miscible with the solvent of the polymer.

  Phase inducers (co-binding agents)

  vation) are miscible solvents with the active ingredient / polymer mixture and cause the formation of embryonic microparticles before curing; As phase inducers, silicone oils are preferred. The oil-in-water emulsion can be prepared according to the usual methods, using

  heptane, hexane, etc., for the organic phase.

  We can also prepare the micro-

  particles of the invention according to the known method of nebulization drying. According to this method, the somatostatin or a solution of this peptide is intimately mixed in an organic solvent, for example methanol, in water or in a buffer, for example of pH 3-8, with a solution of the polymer in a solvent. organic, immiscible with the first, for example the

methylene chloride.

  The solution is then sprayed, suspended

  sion or emulsion formed in a stream of air,

  hot air preference. The micro-organisms are then collected.

  particles formed, for example in a cyclone, and, if necessary, washed, for example in a buffer solution of pH between 3.0 and 8.0, preferably pH 4.0, or in water distilled, and dried under vacuum, for example at a temperature between -400C. The washing step can be applied when the particles have a peak plasma in vivo and when the plasma peak is not desired to be of excessive importance. As a buffer, one can use a

acetate buffer.

  It is therefore possible to obtain microparticles having an improved profile of release of

somatostatin in vivo.

  The invention therefore also relates to the -

  microparticles prepared according to this process. The invention

  further relates to a sustained release formulation

  liner prepared by mixing somatostatin or somatostatin solution in methanol, water or pH 3-8 buffer with a solution of a copolymer of lactic and glycolic acids in methylene chloride. The resulting solution, emulsion or suspension of somatostatin is then sprayed into a hot solution of hot air, the microparticles are collected and washed in a buffer solution of pH between 3.0 and 8, 0 or in distilled water and dried under

  vacuum at a temperature between 20 and 40 C.

  Compared with the microparticles prepared according to the phase separation technique, the microparticles thus obtained do not contain silicone oil, even in the form of traces, the silicone oil not being

  not used in the nebulized drying technique

  tion. The formulations of the invention may also be prepared using the triple emulsion method. According to this method, the peptide, for example octreotide, is dissolved in a suitable solvent, for example water, and is thoroughly emulsified in a solution of the polymer, for example poly (D, L-) acid. lactic acid-co-glycolic acid) glucose (50/50) in a solvent in which the peptide is

  not soluble, for example methylene chloride.

  As examples of suitable solvents for the polymer matrix, mention may be made of methylene chloride,

  chloroform, benzene, ethyl acetate etc ...

  The resulting water-in-oil emulsion is emulsified in an excess of water containing an emulsifier, for example anionic or nonionic surfactant or lecithin, or a protective colloid, for example gelatin, dextrin, carboxymethylcellulose polyvinylpyrrolidone or polyvinyl alcohol, which

  continuously generates a triple emulsion (water-in-water

  oil-in-water). The microparticles are formed by spontaneous precipitation of the polymer and are cured by evaporation of the organic solvent. Gelatin is used to prevent agglomeration of microparticles. After sedimentation of the microparticles, the supernatant is decanted and the microparticles washed with water and then with an acetate buffer. Then filter and

the microparticles are dried.

  The peptide can also be dispersed directly into the polymer solution and then the resulting suspension mixed with the aqueous phase.

containing gelatin.

  The triple emulsion is known from US Pat. No. 4,652,441. According to this patent, a solution of the active principle (1) in a solvent, for example somatostatin in a solvent, is thoroughly mixed in a first step. water (column 2, lines

  31-32), with an excess of a poly-acid solution

  (lactic-co-glycolic) (2) in another solvent where the first solvent is insoluble, for example methylene chloride, to give a water-in-oil emulsion (3) consisting of fines

  droplets of the solution (1) in the solution (2).

  In solution (1), a substance retaining the active ingredient (column 1, line 31) is also dissolved, for example gelatin, albumin, pectin or agar. In a second step, we

  increases the viscosity of the internal phase (1) by using

  appropriate means, for example by heating,

  cooling, pH adjustment, addition of metal ions

  for example gelatin with an aldehyde. In a third step, the water-in-oil emulsion (3) is intimately mixed with an excess of water (column 7, lines 52-54), which gives a

  three-phase emulsion of the water-in-oil type

  in water. If necessary, an agent called emulsifier may be present in the excess water (column 7, line 56), chosen for example from anionic or nonionic surfactants, polyvinylpyrrolidone, polyvinyl alcohol and gelatin. In the

  fourth step, the water-in-oil emulsion is

  in-water to a "drying in water" (line 52). This means that the organic solvent in the oil layer is desorbed to give the microparticles. The desorption of the solvent is carried out according to the known methods (column 8, lines 3-5), for example by reducing the stirring pressure (column 8, lines 5-7) or by bubbling nitrogen through the oily layer ( for example methylene chloride) (line 19). The formed microparticles are recovered by centrifugation or filtration (lines 26-27) and the components which have not been incorporated into the polymer are removed by washing with water (line 29). If necessary, the microparticles are heated under reduced pressure in order to better remove the water and the solvent (for example methylene chloride from

  the wall of microparticles) (lines 30-32).

  Although the above process is satisfactory

  making for the preparation of formulations of the in-

  tion, the substance containing the active ingredient mentioned above, for example gelatin, albumin, pectin or agar, is always

  included in the resulting microparticles.

  The Applicant has found that by omitting the addition, in solution a), of the substance retaining the active ingredient and the step for increasing the viscosity of the internal phase and that maintaining the addition of a emulsifying substance or a protective colloid, such as gelatin, in

  the excess water of the triple water-in-oil emulsion

  in-water, one can still obtain satisfactory microparticles. In addition, the microparticles contain no substance retaining the active ingredient and only a very small amount of methylene chloride. The invention therefore relates to a process for the preparation of microparticles, a method in which the following are intimately mixed: a) a solution of an active ingredient, preferably a somatostatin, especially octreotide, in an aqueous medium, preferably water or a buffer, preferably in a weight / volume ratio of 0.8 to 4.0 g / l to 120 ml, especially 2.5 / 10, and in a pH 3-8 buffer, especially an acetate buffer with b) a solution of a polymer, preferably a poly (lactic-co-glycolic) acid as mentioned above, in an organic solvent immiscible with

  the aqueous medium, for example methyl chloride

  lene, preferably in a weight / volume ratio of g / 90 to 400 ml, especially 40/100, so that the weight ratio of active ingredient to polymer is preferably 1/10 to 50, especially 1/16, and the volume / volume ratio of the aqueous medium to the organic solvent is 1: 1.5 to 30, especially 1/10, the water-in-oil emulsion of a) in b) thus obtained is intimately mixed with c) a excess of an aqueous medium, preferably water or a buffer, for example an acetate or phosphate buffer, preferably at pH 3-8, containing an emulsifying substance or a protective colloid, preferably in a concentration of between 0, 01 and 15.0%, in particular gelatin, especially in a concentration of between 0.1 and 3%, in particular 0.5% by weight, the mixture being preferably carried out at a speed such that the volume / volume of ab) / c) from 1/10 to 100, especially 1/40, without adding to the water-in-oil emulsion de-substance retaining the active ingredient or without performing an intermediate step to increase the viscosity, the embryonic microparticles are hardened in the emulsion formed water-in-oil-in water, by desorption, preferably by evaporation of the organic solvent, preferably methylene chloride, and

  we isolate, possibly we wash and dry the micro-

generated particles.

  The invention also relates to a variant of the process, according to which the active principle is dispersed directly in the solution of the polymer, and the resulting dispersion is then mixed with the phase

aqueous solution containing gelatin.

  The invention also relates to micro-

  particles prepared by these methods. Just like the microparticles prepared by the nebulizing technique, they do not contain silicone oil. Compared to the microparticles prepared according to the known triple emulsion method, they do not

  contain no protective colloid.

  Sustained-release formulations may also be prepared according to other known methods, for example when the peptide is stable enough for the preparation of an implant, by heating the peptide microparticles, by

  somatostatin in a poly (lactic acid)

  co-glycolic), especially as described above,

  or by heating a mixture of the peptide and the poly-

  mother, at a temperature for example between 70 and 1001C, and by extrusion and cooling of the compact mass. The extrusion product is then

  cut and optionally washed and dried.

  The formulations of the invention are advantageously prepared under aseptic conditions. The formulations of the invention can be used in a deposit form, for example in the form of

  injectable microparticles or implants.

  They can be administered according to

  usual methods, for example by sub-injection

  dermal or intramuscular, for example depending on the known indications for the drug they contain. The sustained-release formulations containing octreotide may be administered for all known indications of octreotide or its derivatives, for example those described in British Patent Application 2 199 829, as well as for

  the treatment of acromegaly or breast cancer.

  The microparticles of the invention may have a diameter of between about 1 and 250 microns; they preferably have a diameter of between 10 and 200, especially between 10 and 130, for example between 10 and 90 microns. The implants may be for example about 1 and 10 mm3. The amount of active principle, that is to say the peptide present in the formulation, depends on the necessary dose that must be

  released per day and therefore the biodegradation of the poly-

  mother serving to encapsulate it. The exact amount of

  peptide can be determined by bioassay

  availability. The formulations may contain the peptide in an amount of at least 0.2, preferably

  from 0.5 to 20% by weight with respect to the poly-

  mother, preferably between 2.0 and 10, especially between

3.0 and 6% by weight.

  The release time of the peptide from the microparticles can be between 1 or 2

weeks to about 2 months.

  The sustained-release formulation is advantageously a formulation comprising a somatostatin, for example octreotide, in a biodegradable and biocompatible polymeric carrier, which formulation, when administered to a rat subcutaneously at a dose of 10 mg of somatostatin per kg, gives a somatostatin plasma concentration of at least 0.3 ng / ml and preferably less than 20 ng / ml for 30 days, or advantageously for 60 days, or, when administered to a rabbit intramuscularly at a dose of 5 mg / kg gives a plasma somatostatin concentration of at least 0.3 ng / ml for 50 days and advantageously less than

at 20 ng / ml.

  Other preferred properties of the formulas

  The resulting depot compositions containing somatostatin, for example octreotide, are as follows, depending on the preparation method used: Phase separation technique

  Rabbit: 5 mg somatostatin / kg, intravenously

  muscular Delay (0-42 days) 76% Mean plasma level (cp, ideal) (042 days) 4 ng / ml AUC (0-42 days) 170 ng / ml x days Nebulization drying technique Rat: 10 mg somatostatin / kg, subcutaneously Delay (0-42 days)> 75% Mean plasma level (cp, ideal) (0-42 days) 4-6 ng / ml AUC (0-42 days) 170-210 ng / ml x days

  Rabbit: 5 mg somatostatin / kg, intravenously

  Muscle Delay (0-43 days)> 75% Mean plasma level (cp, ideal) (0-43 days) 4-6 ng / ml AUC (0-43 days) 200-240 ng / ml x days Emulsion technique triple Rat: 10 mg somatostatin / kg, subcutaneously Delay (0-42 days)> 75% Mean plasma level (cp, ideal) (0-42 days) 4-6.5 ng / ml AUC (0) -42 days) 170-230 ng / ml x days

  Rabbit: 5 mg somatostatin / kg, intravenously

  muscle Delay (0-42-43 days)> 74% Mean plasma level

(cp, ideal) (0-42-43 days) 3,5-

  6.5 - ng / ml AUC (0-42-43 days) 160-270 ng / ml x days The invention thus also relates to

  formulations comprising somatostatin, preferably

  The invention also provides for the following properties: 1. A delay of at least 70%, preferably at least 74%, for example at least 75%, 80%, 88%. % or at least 89% for a period ranging from 0 to 42 or 43 days and / or 2. A mean plasma concentration (ideal cp) in the rat of 2.5-6.5, preferably 4-6 , 5 ng / ml for a period ranging from 0 to 42 days, after administration of 10 mg of somatostatin per

  subcutaneous route and / or plasma concentration

  in the rabbit of 3.5-6.5, for example 4-6.5 ng / ml for a period ranging from 0 to 42 or 43 days, after administration of 5 mg of somatostatin intramuscularly and / or 3. An area under the curve for a period ranging from 0 to 42 days of at least 160, preferably between 160 and 230 ng / ml x days in the rat, after administration of 10 mg of somatostatin -cutaneous and / or an area under the curve for a period ranging from 0 to 42 or 43 days of at least 160, preferably between 160 and 275, for example between 200 and 275 ng / ml x days in the rabbit, after administration of 5 mg of

  somatostatin intramuscularly.

  To characterize quantitatively the

264931 9

  sustained-release formulations described above, using the Area Deviation (AD) method described by F. Nimmerfall and J. Rosenthaler;

  Intern. J. Pharmaceut. 32, 1-6 (1986).

  This method calculates the surface deviations of the experimental plasma profile compared to the ideal profile which is a plasma concentration.

  mean constant (- ideal cp) obtained in

  weaving into a rectangle of the same area the area under the curve (AUC) of plasma levels as a function of time obtained experimentally. The delay in% is calculated as follows:% delay - 100 x (1 AD / AUC) According to this method, the total plasma profile measured for a pre-determined time is

  characterized by a single numerical index.

  In Proc. Natl. Acad. Sci. USA, 85 (1988) 5688-5692, FIG. 4 shows a plasma plasma profile in the rat of a somatostatin analogue octapeptide, corresponding to the formula

* *

  However, a clear comparison can not be established with the plasma levels reported above and obtained in the rat with the compositions of the present invention.

  the invention, since the profile of the plasma

  described in this document is based on another method of administration, namely intramuscular injection and that, much more importantly, there is no clear indication of

  the concentration of active principle in the micro-

  capsules and on the dose administered, the document merely indicating a concentration of active principle in the microcapsules of between 2 and 6% and a dose of 25 to 50 mg of microcapsules for 30 days, although the evaluations concerned at least 45 days. In addition, the type of poly (DL-lactic-co-glycolic acid) used is not specified. Given the lack of data, this document can not therefore be

  no way be considered as giving a sign-

  In particular, any of the well-defined formulations of the invention have the advantages already mentioned, especially plasma levels.

as previously reported.

  The following examples illustrate the present invention without in any way limiting its scope. In these examples, Mw means the average molecular weight of the polymer determined by exclusion chromatography (GPC) using polystyrene as the reference substance.

EXAMPLE 1

  1 g of poly (D, L-lactic acid) is dissolved

  co-glycolic) (50/50 molar, Mw - 45,000;

  dispersity - about 1.7) in 15 ml of methylene chloride with magnetic stirring and 75 mg

  of octreotide acetate dissolved in 0.5 ml of methanol.

  To the polymer-peptide mixture is added 15 ml of silicone oil (Dow 360 Medical Fluid, 1000 cs). The resulting mixture was added to a stirred emulsion containing 400 ml of n-heptane, 100 ml of pH 4 phosphate buffer, 40 ml of Dow 360 Medical Fluid, 350 cs and 2 ml of Span 80 (emulsifier). The mixture is stirred for at least 10 minutes. The resulting microparticles are recovered by vacuum filtration and dried overnight under vacuum. The yield is about 90% in microparticles of a

  dimension between 10 and 40 microns.

  The microparticles are suspended

  in a vehicle and are administered intra-

  at a dose of 4 mg octreotide to rabbits (New Zealand). Blood samples taken at regular intervals indicate plasma levels of 0.5 to 1.0 ng / ml for 30 days (determined by

  radioimmunoassay method).

EXAMPLE 2

  In 25 ml of ethyl acetate with stirring

  1 g of poly (D, L-lactic acid) was dissolved in the

  glycolic co-acid) -glucose prepared according to the process described in British Patent Application 2,145,422 (Mw-45,000; 55/45 molar; polydispersity - about 1.7; prepared from 0.2% glucose) and mg of octreotide acetate dissolved in 3 ml of methanol is added. To this polymer-peptide mixture is added 25 ml of silicone oil (Dow 360 Medical Fluid, 1000 cs). The resulting mixture is added to the emulsion described in Example 1. The mixture is stirred for a further period of at least 10 minutes. The resulting microparticles are recovered by vacuum filtration and dried overnight under vacuum. A yield of more than 80% is obtained in microparticles of

  dimension between 10 and 40 microns.

  The microparticles are suspended

  in a vehicle and are administered intra-

  at a dose of 4 mg octreotide to rabbits (New Zealand). Blood samples taken at regular intervals indicate plasma levels of 0.5 to 2 ng / ml for 21 days (determined by

  radioimmunoassay method).

EXAMPLE 3

  A solution of 1.5 g of octreotide acetate in 20 ml of methanol is added with stirring.

  a solution of 18.5 g of poly (D, L-lactic acid-co-

  glycolic acid) glucose (50:50 molar, Mw 45,000) in 500 ml of methylene chloride. The phase separation is carried out by adding 500 ml of Dow 360 Medical Fluid (1000 cs) and 800 ml of Dow 360 Medical.

  Fluid (350 cs) to the suspension of peptide and poly-

  mother. The resulting mixture is added to a stirred emulsion comprising 1800 ml of n-heptane, 2000 ml of sterile water and 40 ml of Span 80. After stirring for 10 minutes, the microparticles are collected.

by vacuum filtration.

  The half of the product is dried overnight under vacuum at 37 ° C. The residual chloride

methylene is 1.2%.

  While stirring, the other half of the product is washed with 1000 ml of ethanol containing 1 ml of Span 80. After stirring for 1 hour, the ethanol is decanted and the microparticles are stirred in 1000 ml of n-heptane containing 1 ml. After stirring for 1 hour, the microparticles were collected by vacuum filtration and dried overnight under vacuum at 37 ° C. The residual methylene chloride level of the microparticles washed in this manner was

reduced from 1.2 to 0.12%.

  The combined yield of the product is 91%

  (18.2 g) in microparticles containing 5.6% of

  tide; the average diameter is 24 microns and the rate in

residual heptane is 1.5%.

  The microparticles are suspended

  in a vehicle and are injected intra-

  at a dose of 5 mg / kg octreotide to white rabbits. Blood samples taken at regular intervals indicate plasma levels of 0.3 to 7.7 ng / ml for 49 days (determined by

  radioimmunoassay method).

EXAMPLE 4

  With magnetic stirring, 1 g of

  poly (D, L-lactic acid-co-glycolic acid) glucose (M.

  - 46,000; 50/50 molar, produced according to the process described in British Patent Application 2,145,422; polydispersity - about 1.7; prepared from 0.2% glucose) in 10 ml of methylene chloride and 75 mg of octreotide acetate dissolved in 0.133 ml of methanol are added. For 1 minute, is intimately mixed, for example using an UltraTurax, at 000 rpm, which gives a suspension of very fine crystals of octreotide in the polymer solution. The suspension is sprayed with a high speed turbine (Niro Atomizer) and the fine droplets are dried in a stream of hot air generating the microparticles. These are collected by means of a cyclone and dried for

  overnight at room temperature under vacuum.

  The microparticles were washed with 1/15 molar acetate buffer at pH 4.0 for 5 minutes and dried again at room temperature.

  under vacuum. After 72 hours, the microwells are sifted

  particles (sieve 0.125 mm) to obtain the final product. The microparticles are suspended

  in a vehicle and are administered intra-

  at a dose of 5 mg / kg octreotide to white rabbits (chinchilla bastards) and

  subcutaneously at a dose of 10 mg / kg to male rats.

  Blood samples taken at regular intervals indicate plasma levels of 0.3 to 0 ng / ml in rabbits and 0.5 to 7.0 ng / ml in rats for 42 days (determined by

radioimmunoassay).

EXAMPLE 5

  Proceeding as described in Example 4,

  prepares microparticles by nebulized drying

  the only difference being that octreotide acetate is suspended directly in the solution of the

  polymer, without using methanol.

  The microparticles are suspended

  in a vehicle and are administered under the

  at a dose of 10 mg / kg octreotide to male rats. Blood samples taken at regular intervals indicate plasma levels of 0.5 to 0 ng / ml in rats for 42 days (determined

  by the radioimmunoassay method).

EXAMPLE 6

  2.5 ml of methylene chloride are dissolved in poly (D, L-lactate-co-glycolic acid) glucose (Mw 46,000, 50/50 molar) prepared according to the process of the British patent application. The polydispersity - about 1.7, prepared from 0.2% glucose), 75 mg of octreotide acetate dissolved in 0.125 ml of demineralized water are added and the mixture is thoroughly mixed, for example with using an Ultra-Turax,. for 1 minute at 20,000 rpm (phase

internal water-in-oil).

  1 g of gelatin A is dissolved in 200 ml of deionized water at 50 ° C. and the solution is cooled to 20 ° C. (external aqueous phase). The water-in-oil phase and the aqueous phase are intimately mixed. The internal water-in-oil phase separates in the form of fine droplets which are dispersed homogeneously in the external aqueous phase. The resulting triple emulsion is slowly stirred for 1 hour. Methylene chloride evaporates and droplets from the phase

  internal harden in the form of microparticles.

  After sedimentation of the microparticles, the supernatant is aspirated, the microparticles are recovered by vacuum filtration and rinsed with water for

remove the gelatin.

  Dry, sieve, wash and dry the microparticles again as described in

example 4.

  The microparticles are suspended

  in a vehicle and are administered intra-

  at a dose of 5 mg / kg octreotide to white rabbits (chinchilla bastards) and

  subcutaneously at a dose of 10 mg / kg to male rats.

  Blood samples taken at regular intervals indicate plasma levels of 0.3 to 0 ng / ml in rabbits and 0.5 to 8.0 ng / ml in rats for 42 days (determined by

radioimmunoassay).

EXAMPLE 7

  Using the procedure described in Example 6, microparticles are prepared according to the triple emulsion technique, but with the following modifications: 1. Using 0.25 ml of acetate buffer at pH 4.0 instead of 0.125 ml of water, to prepare the phase

internal water-in-oil.

  2. After collecting the microparticles, rinse with 1/45 molar buffer

  acetate at pH 4.0, and not with water.

  3. Subsequent washing of the microwaves is

particles.

EXAMPLE 8

  Microparticles are prepared according to the triple emulsion technique by proceeding as described in

  example 7, but by preparing the internal phase water-

  in-oil with water containing 0.7% sodium chloride (w / v) and not with acetate buffer.

EXAMPLE 9

  By proceeding as described in Example 6, microparticles are prepared, but with the difference that the active ingredient is dispersed directly in the solution of the polymer and that the resulting dispersion is mixed with the aqueous phase containing gelatin.

EXAMPLE 10

  Octreotide Pamoate In 1 liter of a water / dioxane (1: 1) mixture, 10.19 g of octreotide base and 3.88 g of

  of embonic acid (10mM). The mixture is filtered

  and freeze-dried to give a yellow powder [t] 20D - +7.5 (c-0.35 in DMF) of octreotide pamoate hydrate. Factor - 1, 4 (the factor means the weight of the lyophilisate in relation to the weight

of octreotide it contains).

  Octreotide pamoate may be used in place of the acetate in the microparticles of Examples 1 to 9; it is characterized by excellent stability.

EXAMPLE 11

  With stirring, a solution of 1 g of poly (D, L-lacquerceco-glycolic) acid (50/50 molar, M.-36 100) in 20 ml of methylene chloride is added to a solution of 100 mg of calcitonin. in 1.5 ml of methanol. Phase separation is carried out by addition of 20 ml of silicone fluid (Dow 360 Medical Fluid, 1000 cs). The resulting mixture was added to a stirred emulsion comprising 100 ml pH 4 phosphate buffer, 400 ml n-heptane, 4 ml Span 80 and 40 ml silicone fluid (Dow 360 Medical Fluid, 1000 cs). After stirring for 10 minutes, the microparticles are collected by vacuum filtration and dried overnight under vacuum at 37 ° C. 1.1 g of microparticles containing 5.9% of calcitonin are obtained.

EXAMPLE 12

  At 100 mg of lypressin, a

  solution of 9.9 g of poly (D, L-lactic-co-

  glycolic) (50/50 molar, Mw - 44,300) in 140 ml of methylene chloride. The dispersion is stirred with a magnetic stirrer for 1 hour before adding 140 ml of silicone fluid (Dow 360 Medical Fluid, 1000 cs) and 2.5 ml of Span 80. The mixture is added to 2000 ml of heptane and it is stirred for 10 minutes. The microparticles are collected by vacuum filtration, washed 3 times with heptane and filtered for 10 minutes to dry. Half of the sample is washed with stirring in water for 10 minutes. We do not wash the other half. The two samples are then dried overnight at 30 ° C. under vacuum. 10.65 g of microparticles are obtained. The washed sample contains 0.5% lypressin and the unwashed sample with water contains 0.6% lypressin (determined

by analysis).

26493 19

Claims (20)

  1. A process for preparing microorganisms   particles comprising an active ingredient in a biodegradable and biocompatible vehicle, characterized in that a) dissolving the polymeric material in a suitable solvent in which the active ingredient is not soluble, b) in the solution of step a), a solution of the active ingredient is added and dispersed in a suitable solvent which is not a solvent for the polymer,   c) a phase inducing agent is added to the   persion of step b), to cause the formation of microparticles, d) an oil-in-water emulsion is added to the mixture of step c) to cure the microparticles, and   e) the microparticles are recovered.   2. A process for preparing microorganisms   particles comprising an active ingredient in a biodegradable and biocompatible vehicle, characterized in that (i) intimately mixing a water-in-oil emulsion formed from an aqueous medium and an immiscible organic solvent in the water, containing in a   phase the active ingredient and in the other phase a poly-   biodegradable and biocompatible mother, with an excess of an aqueous medium containing an emulsifying substance or   a protective colloid, to form a water-emulsion   in-oil-in-water, without adding to the water-in-oil emulsion of substance retaining the active ingredient or without applying an intermediate step increasing the viscosity, (ii) subjecting the organic solvent to desorption and   (iii) the resulting microparticles are isolated and dried aunts.   3. A process for preparing microorganisms   particles comprising an active ingredient in a poly-   biodegradable and biocompatible mother, characterized in that (i) intimately mixing a suspension formed from an active ingredient and an organic solvent   immiscible in water containing a bio-polymer   degradable and biocompatible, with an excess of an aqueous medium containing an emulsifying substance or a   protective colloid, to form a water-emulsion   in-oil, the active ingredient being dispersed in   oil, without adding any substance   or without applying an intermediate viscosity-increasing step, (ii) the organic solvent is desorbed, and   (iii) the resulting microparticles are isolated and dried aunts   4. A process for preparing microorganisms   characterized in that a) a solution of an active ingredient in an aqueous medium is intimately mixed with b) a solution of a polymer in an organic solvent immiscible with the aqueous medium, the water-in-water emulsion is intimately mixed with in-oil of a) in b) thus obtained with -   c) an excess of an aqueous medium containing a protective colloid, without adding to the water-in-oil emulsion of substance retaining the active principle or without performing an intermediate step to increase the viscosity, the embryonic microparticles are hardened in the emulsion formed in oil-in-water, by   desorption, and the microparticles generated are isolated.   5. A method according to claim 4, characterized in that the active ingredient is a somatostatin. 6. A method according to claim 4, characterized in that the active ingredient is octreotide.   7. A process for preparing microorganisms   particles, characterized in that a solution of a somatostatin in water or in a buffer, in a weight / volume ratio of 0.8 to 4.0 g / l to 120 ml, is intimately mixed with   b) a solution of a poly (lactic-co-glyco) acid   lique) in an organic solvent immiscible with the aqueous medium, in a weight / volume ratio of g / 90 to 400 ml, so that the weight ratio of the active ingredient to the polymer is 1/10 to 50 and the volume / volume ratio. from the aqueous medium to the organic solvent of from 1: 1.5 to 30, the water-in-oil emulsion of (a) is thoroughly mixed in (b) with (c) an excess of water or a buffer containing a protective colloid, at a rate such that the volume / volume ratio of ab) / c) is 1/10 to 100, without adding to the water-in-oil emulsion the substance retaining the active ingredient or without effecting intermediate step for increasing the viscosity, the embryonic microparticles are hardened in the emulsion formed in oil-in-water, by evaporation of the organic solvent, and   the microparticles generated are isolated.   8. A sustained-release formulation, characterized in that it comprises octreotide or one of its derivatives, or a salt of the compound, in a   biodegradable and biocompatible polymeric vehicle.   9. A sustained-release formulation, characterized in that it comprises a peptide in an ester of a polyol with a poly (40/60 to 60/40 lactic-co-glycolic acid), the polyol residue being chosen from alcohols containing a hydrocarbon chain   C3-C6 and from 3 to 6 hydroxy groups and mono- and di-   saccharides, and the esterified polyol having at least 3   poly (lactic-co-glycolic acid) chains.   10. A sustained-release formulation, characterized in that it comprises a calcitonin, lypressin or somatostatin, in a 40/60 to 60/40 linear copolymer of lactic acid and glycolic acid having a molecular weight Mw inclusive. between 000 and 100,000 and a polydispersity Mw / Mn between 1.2 and 2, in a concentration of between 0.2 and 10% by weight of peptide.   11. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rat by subcutaneous   at a dose of 10 mg / kg gives a plasma concentration of at least 0.3 ng / ml and ng / ml for 30 days.   12. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rabbit intravenously   at a dose of 5 mg / kg, gives a concentration   plasma concentration of at least 0.3 ng / ml and below at 20 ng / ml for 50 days.   13. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rabbit intravenously   muscle at a dose of 5 mg / kg gives a delay of at least 70% for a period ranging from 0 to 42 or 43 days. 14. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rat by subcutaneous   at a dose of 10 mg / kg gives a mean plasma concentration (ideal cp) of between 2.5 and   6.5 ng / ml for a period of 0 to 42 days.   15. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rabbit intravenously   at a dose of 5 mg / kg, gives a concentration   mean plasma concentration (ideal cp) between 3.5 and 6.5 ng / ml for a period ranging from 0 to 42 days. 16. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rat by subcutaneous   at a dose of 10 mg / kg gives an area under the curve of between 160 and 230 ng / ml x days   a period ranging from 0 to 42 or 43 days.   17. A sustained release formulation   according to any one of claims 8 to 10, which   when administered to a rabbit intravenously   at a dose of 5 mg / kg, gives an area under the curve of between 160 and 275 ng / ml x days   for a period ranging from 0 to 42 or 43 days. 18. Octreotide pamoate.   19. A sustained-release formulation, characterized in that it comprises a peptide selected from a calcitonin and lypressine and the pharmaceutically acceptable salts of these compounds, in   a biodegradable and biocompatible polymer matrix.   20. A formulation according to claim 8 for use in the treatment of   acromegaly or breast cancer.