NO302928B1 - Process for the preparation of microparticles - Google Patents

Description

The invention relates to a method for producing microparticles comprising a drug in a biodegradable, biologically compatible polymer carrier.

The medication includes particularly water-soluble peptides, e.g. somatostatin or somatostatin analogues, such as octreotide. The formed microparticles can also be termed microcapsules or microspheres.

Peptide medicines often show a low bioavailability in blood after oral or parenteral administration, which e.g. is due to their short biological half-lives caused by their metabolic instability. If they are administered orally or nasally, they also often show poor resorption through the mucous membrane. A therapeutically relevant level in blood over an extended period of time is difficult to achieve.

The parenteral delivery of peptide drugs as a depot preparation in a biodegradable polymer, e.g. as microparticles or implants, have been proposed and enable prolonged release after a residence time in the polymer that protects the peptide against enzymatic and hydrolytic action of the biological medium.

Although some parenteral depot preparations of peptide drugs in a polymer in the form of microparticles or an implant are known, in reality satisfactory peptide release profiles are only achieved in very few cases. Special measures must be taken for continuous peptide release to achieve a therapeutically active drug serum level and, if desired, to avoid overly high drug serum concentrations, which produce unwanted pharmacological side effects.

The release process of the peptide drug depends on a number of factors, e.g. of the peptide type, and e.g. whether it is present in its free form or in another form, e.g. in the form of a salt, which can affect its solubility in water. Another important factor is the choice of polymer, based on the many possibilities described in the literature.

Each type of polymer has its characteristic rate of biological degradation. Free carboxyl groups can be formed which contribute to the pH value in the polymer and thus further affect the peptide's water solubility and thus its release process.

Other factors that can affect the release course of a depot preparation are the drug content in its polymeric carrier, how this is distributed in the polymer, particle size and, in the case of an implant, additionally its shape. Furthermore, this applies to the place in the body where the preparation is supposed to work.

To date, no extended-release somatostatin preparation for parenteral administration is available on the market, perhaps because it has not been possible to obtain a preparation with a satisfactory serum level profile.

Polymer preparations with drugs designed to provide a prolonged or delayed release of the drug are known in the art.

US-PS 3,773,919 deals with preparations with controlled release of the drug in which the drug, e.g. a water-soluble peptide drug, is dispersed in a biodegradable and biocompatible linear polylactide or a polylactide-co-glycolide polymer. However, no pattern for drug release has been described and there is no reference to a somatostatin. US-PS 4,293,539 describes antibacterial preparations in the form of microparticles.

US-PS 4,675,189 describes sustained release preparations of the LHRH analogue decapeptide nafarelin and related LHRH analogues in polylactide-co-glycolide polymers. There is no pattern described for release.

T. Chang, J. Bioeng., vol. 1, pages 25-32, 1976, describes sustained release of biological compounds, enzymes and vaccines from microparticles.

Polymers/copolymers of lactic acid and lactide/glycolide copolymers and related compositions for surgical use and for sustained release and biodegradability are described in US-PS 3,991,776, 4,076,798 and 4,118,470.

EP patent application 0 2 03 031 describes a number of somatostatin octapeptide analogues, e.g. compound RC-160 with the formula:

* *

The possibility of microencapsulation of somatostatins with polylactide-co-glycolide polymer is mentioned in claim 18, but no guidance is given as to how to achieve a continuous therapeutically active serum level.

US-PS4,011,312 describes that one can achieve a continuous release of an antimicrobial drug, e.g. the water-soluble polymyxin B from a polylactide-co-glycolide matrix with a low molecular weight (below 2000) and a relatively high glycolide content in the form of an implant, when the implant is introduced into the teat canal of a cow. The medication is released within a short period of time due to the high glycolide content and the polymer's low molecular weight, both of which stimulate a rapid biological breakdown of the polymer and thus a correspondingly rapid release of the medication. A relatively high drug content further contributes to rapid drug release. No somatostatins are mentioned and no pattern of release is described therein.

EP patent 58,481 relates that a continuous release of a water-soluble peptide from a polylactide polymer implant is stimulated by reducing the molecular weight of at least some of the polymer molecules, by introducing glycolide units into the polymer molecule, by increasing the block polymer character of the polymer when the polylactide-co-glycolide molecules are used, by increasing the content of drug in the polymer matrix and by increasing the surface area of the implant.

Although somatostatins are mentioned as water-soluble peptides, somatostatin release profiles are not described, nor are indications given as to how all these parameters can be combined to achieve e.g. a continuous somatostatin serum level for at least one week, e.g. one month.

EP patent 92,918 describes that a continuous release of peptides, preferably hydrophilic peptides, over an extended period of time can be achieved when the peptide is incorporated into a conventional hydrophobic polymer matrix, e.g. of a polylactide, which is made more accessible to water by introducing a hydrophilic unit into its molecule, e.g. of polyethylene glycol, polyvinyl alcohol, dextran, polymethacrylamide. The hydrophilic contribution to the amphipathic polymer is given by all the ethylene oxide groups in the case of a polyethylene glycol unit, by the free hydroxyl groups in the case of a polyvinyl alcohol unit or a dextran unit and by the amide groups in the case of a polyurethane unit. Due to the presence of the hydrophilic unit in the polymer molecules, the implant will acquire hydrogel properties after absorption of water. Somatostatin is mentioned as a hydrophilic peptide, but no release profile is described, and it is not indicated which type of polymer is preferred for this peptide and which molecular weight and how many hydrophilic groups it should have.

GB patent 2,145,422 B describes that a sustained release of drugs of a variety of types, e.g. vitamins, enzymes, antibiotics, antigens, can be obtained over an extended period of time when the drug is incorporated into an implant, e.g. of microparticle size, which is constituted by a polymer of a polyol, e.g. glucose or mannitol, with one or more, preferably at least three, polylactide ester groups. The polylactide ester groups preferably contain e.g. glycolide units. No peptides, e.g. somatostatins, are mentioned as drugs and serum drug levels are not described either.

The present invention relates to a method for the production of microparticles comprising a drug in a biodegradable, biologically compatible polymer carrier. The obtained microparticles, which may be in the form of a microparticle preparation, have prolonged release of a drug, in particular of a hormonally active water-soluble somatostatin or a somatostatin analogue such as octreotide, which provides a satisfactory drug plasma level. The biodegradable, biocompatible polymeric carrier can e.g. be an encapsulating polymer matrix. The polymer matrix can be a synthetic or natural polymer.

Microparticles can generally be produced by a number of applied techniques, e.g. by an organic phase separation technique, a spray drying technique or a triple emulsion technique, in which the polymer is precipitated together with the drug, followed by curing of the obtained product, when the phase separation technique or the triple emulsion technique is used.

The above-mentioned preparation with extended release can, if desired, be in the form of an implant.

According to the invention, it has now been found that a method comprising modification of the phase separation technique is applicable for the production of microparticles comprising a drug.

Accordingly, the present invention provides a method for the preparation of microparticles comprising a drug in a biodegradable, biocompatible carrier comprising the steps of: a) dissolving the polymeric carrier material in a suitable solvent in which the drug compound is not

soluble,

b) adding and dispersing a solution of the drug compound in a suitable solvent which is a non-solvent for the polymer, in the solution in step a), c) adding a phase inducing agent to the dispersion in step b) to induce microparticle formation, d) adding an oil-in-water emulsion to the mixture in step c) to harden the microparticles, and

e) extraction of the microparticles.

It has also been found that a method which includes

modification of the triple emulsion technique is applicable for the production of microparticles comprising a drug.

Accordingly, the present invention provides a method for the production of microparticles comprising a drug in a biodegradable, biocompatible carrier characterized by: (i) intimate mixing of a water-in-oil emulsion formed from an aqueous medium and an organic solvent which is immiscible with water containing the drug in one phase and a biodegradable, biocompatible polymer in the other phase, with an excess of an aqueous medium containing an emulsifying substance or a protective colloid to form a water-in-oil-in- water emulsion, without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step,

ii) separation of the organic solvent therefrom,

iii) isolation and drying of the obtained microparticles.

The present invention further provides a method for the production of microparticles comprising a drug compound in a biodegradable, biocompatible polymer which is characterized by the steps of: (i) incorporating a drug compound suspension formed from a drug compound and a water-immiscible organic solvent containing a biodegradable, biocompatible polymer, with an excess of aqueous medium containing an emulsifying substance or a protective colloid, to form an oil-in-water emulsion, wherein the drug compound is dispersed in the oil component, and without the addition of any drug-retaining substance or the use of any intermediate viscosity-increasing step,

(ii) separating the organic solvent therefrom,

(iii) isolation and drying of the obtained microparticles.

Finally, the invention also provides a method for the production of microparticles, which is characterized by the fact that it comprises a thorough mixture of: a) a solution of a somatostatin in water or a buffer in a weight/volume ratio from 0.8 to 4.0 g/ l to 120 ml, and b) a solution of a polylactide-co-glycolide in an organic solvent which is immiscible with the aqueous

medium, in a weight/volume ratio from 40 g/90 to 400 ml in such a way that the weight/weight ratio between the drug and the polymer is from 1/10 to 50, and the volume/volume ratio of the aqueous medium/organic solvent is 1/1.5 to 30, including mixing of the water-in-oil emulsion from

a) ib) together with

c) an excess of water or a buffer containing a protective colloid at a volume/volume mixing rate ratio of ab)/c) from 1/10 to 100,

without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step, with hardening of the embryonic microparticles in the formed water-in-oil-in-water emulsion by evaporation of the organic solvent and isolation of the formed microparticles.

The methods according to the invention also enable the provision of: a) A preparation with extended release in the form of microparticles as mentioned above comprising a

peptide drug compound in a 40/60 to 60/40 polylactide-co-glycol diester of a polyol, the polyol-

the het is selected from the group of a (C3-6) carbon chain-containing alcohol with three to six hydroxyl groups and a mono- or disaccharide, and the esterified polyol has

at least three polylactide-co-glycolide chains.

b) A preparation with extended release in the form of microparticles as mentioned above comprising a

peptide drug compound selected from the group of a calcitonin, lypressin or a somatostatin in a 40/60 to 60/40 polylactide-co-glycolide polymer with linear chains having a molecular weight M^, between 25,000 and 100,000, a polydispersity Mw/Mn between 1.2 and 2 in a concentration from 0.2 to

10% by weight of the peptide drug compound therein.

c) A preparation with extended release in the form of microparticles as mentioned above comprising octreotide

or a salt or derivative thereof in a biodegradable, biocompatible polymer carrier.

The above concentration is preferably from 2 to 10% by weight.

It has also been found that a new salt of octreotide which is the pamoate thereof is very stable in such preparations.

The drugs used in the method according to the invention are preferably water-soluble drugs, e.g. peptides.

The peptides used in the methods according to the invention can be a calcitonin, such as salmon calcitonin, lypressin, and the naturally occurring somatostatin and synthetic analogues thereof.

The naturally occurring somatostatin is one of the preferred compounds and is a tetradecapeptide with the structure:

This hormone is produced by the hypothalamus gland as well as by other organs, e.g. gastrointestinal tract, and mediates, together with GRF, the neuroregulation of pituitary growth hormone release. In addition to inhibiting GH release from the pituitary gland, somatostatin is a potent inhibitor of a number of systems, including the central and peripheral nervous system and gastrointestinal and vascular smooth muscle. It also inhibits the release of insulin and glucagon.

The term "somatostatin" includes its analogs or derivatives. Derivatives and analogues mean straight-chain, bridged or cyclic polypeptides in which one or more amino acid units have been omitted and/or replaced by one or more other amino radicals and/or in which one or more functional groups have been replaced by one or more other functional groups and/or or one or more groups are replaced by one or more other isosteric groups. In general, the term covers all modified derivatives of a biologically active peptide which exhibit a qualitatively similar effect to the unmodified somato-statin peptide.

Agonist analogs of somatostatin can thus be used to replace native somatostatin in its effects on the regulation of physiological functions.

Preferred known somatostatins are:

wherein in each of the compounds a) to i) there is a bridge between the amino acids marked with a<*> as indicated in the next formula.

Other preferred somatostatins are:

wherein in the above amino acids there is a bridge between the amino acids marked with a<*>.

The term derivative also includes the corresponding derivatives that carry a sugar residue.

When somatostatins carry a sugar residue, this is preferably linked to an N-terminal amino group and/or to at least one amino group present in a peptide side chain, more preferably to an N-terminal amino group. Such compounds and their preparation are indicated in e.g. WO 88/02756.

The term octreotide derivatives includes those with the moiety

Particularly preferred derivatives are Na- [oc-glucosyl-(1,4-deoxyfructosyl]-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol and Na<->[S-deoxyfructosyl-DPhe-Cys- Phe-DTrp-Lys-Thr-Cys-Thr-ol, each of which has a bridge between the -Cys moieties, preferably in acetate salt form and which are respectively described in Examples 2 and 1 of the above application.

The somatostatins can e.g. exist in free form, in salt form or in the form of complexes thereof. Acid addition salts can be formed with e.g. organic acids, polymeric acids and inorganic acids. Acid addition salts include e.g. the hydrochloride and the acetates. Complexes are formed e.g. from somatostatins by adding inorganic substances, e.g. inorganic salts or hydroxides such as Ca and Zn salts and/or by the addition of polymeric organic substances.

The acetate salt is a preferred salt for microparticles which leads to a reduced initial drug release. A pamoate salt can also be used, especially for implants, and it can be obtained in the usual way, e.g. in that embonic acid (pamoic acid) is reacted with octreotide in e.g. free base form. The reaction can be carried out in a polar solvent, e.g. at room temperature.

The somatostatins are indicated for use in the treatment of disorders where administration of the drug is expected over a long period of time, e.g. disorders with an etiology extensive or associated with excessive GH secretion, e.g. in the treatment of acromegaly, for use in the treatment of gastrointestinal disorders, e.g. in the treatment or prophylaxis of peptic ulcer, enterocutaneous and pancreaticocutaneous fistula, irritable bowel, gastric emptying, watery diarrhea, acute pancreatitis and gastroenterophatic endocrine tumors (eg vipomas, GRFomas, glucagonomas, insulinomas, gastrinomas and carcinoid tumors) as well as gastrointestinal bleeding, breast cancer and complications related to diabetes.

The polymeric carrier can be made from biocompatible and biodegradable polymers, such as linear polyesters, branched polyesters which are linear chains radiating from a polyol moiety, e.g. glucose. Other esters are those of polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polycaprolactone, polyalkylene oxalate, polyalkylene glycol esters of acids from the Krebs cycle, e.g. the citric acid cycle and the like, and copolymers thereof.

The preferred polymers are the linear polyesters and the branched polyesters. The linear polyesters can be prepared from α-hydroxycarboxylic acids, e.g. lactic acid and glycolic acid, by condensation of the 1-actone dimers, see e.g. US-PS 3,773,919.

Linear polylactide-co-glycolides which are preferably used in the method according to the invention suitably have a molecular weight between 25,000 and 100,000, and a polydispersibility Mw/Mn between 1.2 and 2.

The branched polyesters which are preferably used in the method according to the invention can be produced by using polyhydroxy compounds, e.g. polyol, e.g. glucose or mannitol as the initiator. These esters of a polyol are known and described in GB patent 2,145,422 B. The polyol contains at least three hydroxy groups and has a molecular weight of up to 20,000, with at least one, preferably at least two, e.g. with an average of three of the hydroxy groups in the polyol in the form of ester groups, which contain polylactide or co-polylactide chains. 0.2% glucose is typically used to initiate polymerization. The structure of the branched polyesters is star-shaped. The preferred polyester chains in the linear and star-shaped polymer compounds which are preferably used are copolymers of the cc-carboxylic acid parts, lactic acid and glycolic acid, or of the lactone dimers. The molar ratio of lactide to glycolide is from 75:25 to 25:75, e.g. from 60:40 to 40:60, with a ratio from 55:45 to 45:55, e.g. 55:45 to 50:50 is most preferred.

The star-shaped polymers can be produced by reacting a polyol with a lactide and preferably also a glycolide at an increased temperature in the presence of a catalyst, which enables ring-opening polymerization.

It has now been found that an advantage of the star polymer type is that its molecular weight can be relatively high, which provides physical stability, e.g. a certain hardness, to an implant and to microparticles that causes them not to stick together even if relatively short polylactide chains are present, leading to a controllable biodegradation rate of the polymer ranging from several weeks to one or two months, and leading to a correspondingly prolonged release of the peptide which makes a depot preparation prepared from it suitable for e.g. one month release.

The mineral polymers preferably have a main molecular weight M,^ in the range from 10,000 to 200,000, preferably from 25,000 to 100,000, especially from 35,000 to 60,000, and a polydispersibility, e.g. from 1.7 to 3.0, e.g. from 2.0 to 2.5. The limiting viscosities for the star polymers with M^, 35,000 and M^, 60,000 are respectively 0.36 and 0.51 dl/g in chloroform. A mineral polymer with 52,000 has a viscosity of

0.475 dl/g in chloroform.

The terms microsphere, microcapsule and microparticle can be used interchangeably in connection with the invention and indicate encapsulation of the peptides by means of the polymer, preferably with the peptide distributed through the polymer, which is then a matrix for the peptide. In the preferred case, the term microsphere and more generally microparticle is used.

When using the phase separation technique according to the invention, the formulations can be prepared by dissolving the polymer carrier material in a solvent which is a non-solvent for the peptide followed by the addition and dispersion of a solution of the peptide in the polymer solvent mixture. A phase inducing agent, e.g. a silicone fluid, is then added to induce encapsulation of the peptide by the polymer.

Release or "burst" of the drug can be reduced considerably by producing ultrafine drug particles in situ by adding a drug solution to the polymer solution before phase separation. The previously known method involves adding dry particles directly to the polymer solution.

The therapeutic duration of peptide release can be increased by curing/washing the microparticles with a buffer/heptane emulsion. The previously known method comprises a curing step followed by either no subsequent washing or by a separate washing step with water.

An oil-in-water emulsion (= o/w) is used to harden the microspheres. It can also be used to wash them. The washing causes the removal of non-encapsulated peptide from the surface of the microspheres. Removal of excess peptide from the microspheres reduces the initial drug release that is characteristic of many conventional encapsulated preparations. A more uniform drug delivery over a period of time is thus possible with the present microsphere formulations.

The emulsion also supports the removal of the remaining polymer solvent and the silicone fluid. The emulsion is added to the polymer peptide mixture. The mixture can also be added to the emulsion. The o/w emulsion can be prepared using an emulsifier such as sorbitan monooleate (Span 80 ICI Corp.) and the like to form a stable emulsion. The emulsion can be buffered with a buffer that is not harmful to the peptide and the polymeric matrix material. The buffer can have a pH from 2 to 8, with pH 4 being preferred. The buffer can be prepared from acidic buffers such as phosphate buffer, acetate buffer and the like. Water alone can replace the buffer.

Heptane, hexane and the like can be used as the organic phase in the buffer. The emulsion may contain dispersants such as silicone oil.

A preferred emulsion may comprise heptane, phosphate buffer with pH 4, silicone oil and sorbitan monooleate. When an initial drug release is desired, a simple curing step without solvent can replace the emulsion curing. Heptane, hexane and the like can be used as solvent.

Other alternatives to the o/w emulsion can be used for curing, such as solvent plus emulsifier for curing microcapsules without washing, and solvent plus emulsifier for curing followed by a separate washing step.

The o/w emulsion can be used without a dispersant. The dispersant, however, avoids aggregation of the dry microparticles due to static electricity and reduces the level of residual solvent.

Examples of solvent for the polymeric matrix material include methylene chloride, chloroform, benzene, ethyl acetate and the like. The peptide is preferably dissolved in an alcohol solvent, e.g. methanol, which is miscible with the polymeric solvent.

The phase inducing agents ("coacervation" agents) are solvents which are miscible with the polymer drug mixture, and provide formation of embryonic microcapsules prior to curing, silicone oils being the preferred phase inducing agents.

The o/w emulsion can be prepared in the usual way by using heptane, hexane and the like for the organic phase.

The above-mentioned microparticles are also produced using a triple emulsion procedure as mentioned above. Typically, peptide dissolves, e.g. octreotide, in a suitable solvent, e.g. water, and is thoroughly emulsified into a solution of the polymer, e.g. 50/50 poly(D,L-lactide-co-glycolide)glucose in a solvent which is a non-solvent for the peptide, e.g. in methylene chloride. Examples of solvent for the polymeric matrix material include methylene chloride, chloroform, benzene, ethyl acetate and the like.

The obtained water-in-oil (w/o) emulsion is further emulsified in an excess of water containing an emulsifying substance, e.g. an anionic or non-ionic surfactant or lecithin, or a protective colloid such as gelatin, dextrin, carboxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, which provide continuous formation of the triple (w/o/w) emulsion. The microparticles are formed by spontaneous precipitation of the polymer and are hardened by evaporation of the organic solvent. Gelatin serves to prevent agglomeration of the microspheres. After sedimentation of the microparticles, the supernatant is decanted and the microparticles are washed with water and then with acetate buffer. The microparticles are then filtered and dried.

The peptide can also be dispersed directly in the polymer solution, after which the obtained suspension is mixed with the gelatin containing the water phase.

The triple emulsion procedure is known from US-PS 4,652,441. According to this patent, in a first step, a medicinal solution (1) in a solvent, e.g. somatostatin in water (column 2, lines 31-32), mixed thoroughly with an excess of a polylactide-co-glycolide solution (2) in another solvent, in which the first solvent is not soluble, e.g. methylene chloride, obtaining a water-in-oil type (w/o) emulsion (3) of fine drug-containing droplets of (1) in the solution (2). A so-called drug-retaining substance is further dissolved in the solution (1).

(column 1, line 31), e.g. gelatin, albumin, pectin or agar.

In another step, the viscosity of the internal phase (1) is increased by means of suitable means, such as heating, cooling, pH change, addition of metal ions or cross-linking of e.g. gelatin with an aldehyde.

In a third step, an excess of water is thoroughly mixed with the w/o emulsion (3), (column 7, lines 52-54), which leads to a w/o/w type emulsion with three layers. If desired, a so-called emulsifier can be present in the excess of water (column 7, line 56), selected from the group consisting of e.g. an anionic or nonionic surfactant, or e.g. polyvinyl pyrrolidone, polyvinyl alcohol or gelatin.

In a fourth step, the w/o/w emulsion is subjected to an "in-water drying" (line 52). This means that the organic solvent in the oil layer is desorbed to form microparticles. The desorption is carried out in a known manner (column 8, lines 3-5), by e.g. pressure reduction with stirring (column 8, lines 5-7), or by e.g. nitrogen gas is blown through the oil layer (e.g. methylene chloride) (line 19).

The formed microparticles are recovered by centrifugation or filtration (lines 26-27) and the components that are not incorporated into the polymer are removed by washing with water (line 29). If desired, the microparticles are heated under reduced pressure to achieve a better removal of water and solvent (e.g. methylene chloride from the microparticle wall) (line 30-32).

In the above-mentioned method, however, the so-called drug-retaining substance as mentioned above, e.g. gelatin, albumin, pectin or agar, still incorporated in the microparticles obtained.

It has now been found that when the addition of the drug-retaining substance (= in solution (1)) and the step of increasing the viscosity of the inner phase is avoided, and the measure of adding an emulsifying substance or a protective colloid, like gelatin, is maintained in the excess of water in the ternary w/o/w emulsion, satisfactory microparticles can still be obtained. In addition, the microparticles do not contain any drug-retaining substance and only a very small amount of methylene chloride.

In one aspect of the invention, it is also possible to produce microparticles which comprise a thorough mixture of: a) a solution of the drug somatostatin, particularly octreotide, in an aqueous medium, such as water or a buffer, in

a weight/volume ratio of from 0.8 to 4.0 g/l to 120 ml, in particular 2.5/10 and in a buffer with pH 3 to 8, in particular a

acetate buffer, and

b) a solution of the polymer polylactide-co-glycolide as mentioned above, in an organic solvent which is not

miscible with the aqueous medium, e.g. methylene chloride, in a weight/volume ratio from 40 g/90 to 400 ml, especially 40/100, in such a way that the weight/weight ratio between the drug and the polymer is from 1/10 to 50, especially 1/16, and volume/ the volume ratio between the aqueous medium and the organic solvent is from 1/1.5 to 30, in particular 1/10,

as the w/o emulsion of a) is thoroughly mixed in b).

with

c) an excess of an aqueous medium such as water or a buffer, e.g. an acetate or phosphate buffer, preferably with a

pH from 3 to 8, containing an emulsifying substance or a protective colloid, preferably in a concentration of from 0.01 to 15.0%, in particular gelatin, in particular in a concentration of from 0.1 to 3%, in particular 0.5 wt %, and in a volume/volume mixing speed ratio of ab)/c) from 1/10 to

100, especially 1/40,

without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step, with hardening of the embryonic microparticles in the formed w/o/w emulsion by desorption, preferably by evaporation of the organic solvent, preferably methylene chloride, and

with isolation, possibly washing and drying of the formed microparticles.

The drug can also be dispersed directly in the polymer solution, after which the resulting dispersion is mixed with the gelatin containing the water phase.

Like the microparticles produced according to the spray-drying technique, the microparticles produced by the above methods do not contain silicone oil. Compared to microparticles produced according to the known triple emulsion process, they do not contain any protective colloid.

A preparation in the form of microparticles prepared according to the invention is suitably prepared under aseptic conditions.

A preparation in the form of microparticles produced according to the invention can be used in depot form, such as e.g. injectable microspheres or implants.

It can be added in the usual way by e.g. subcutaneous or intramuscular injection for e.g. indications known for the drug contained therein.

The extended-release preparations containing octreotide can be administered for all known indications of octreotide or its derivatives, e.g. those specified in GB 2,199,829 A, pages 89-96, as well as for acromegaly and for breast cancer.

The microparticles produced according to the invention can have a diameter size ranging from 1 to 250 µm, preferably 10 to 200, especially 10 to 130, e.g. from 10 to 90 fim. Implants can e.g. be from 1 to 10mm<3>. The amount of drug, i.e. peptide present in the preparation depends on the desired daily released dose and thus the degree of biological degradation of the encapsulating polymer. The exact amount of peptide can be determined by bioavailability experiments. The preparations can contain peptide in an amount from at least 0.2, preferably from 0.5 to 20% by weight in relation to the polymer matrix, preferably from 2.0 to 10, in particular from 3.0 to 6% by weight.

The release time of the peptide from the microparticle can be from one or two weeks to about two months.

The sustained release preparation suitably comprises a somatostatin, e.g. octreotide in a biodegradable, biocompatible polymeric carrier which, when administered subcutaneously to a rat at a dose of 10 mg somatostatin per kg body weight of the animal, exhibits a somatostatin concentration in the blood plasma of at least 0.3 ng/ml and preferably less than 20 ng/ml during a period of 30 days, or suitably over a period of 60 days.

Alternatively, the sustained-release formulation suitably comprises a somatostatin, e.g. octreotide in a biodegradable, biocompatible polymer carrier which, when administered intramuscularly to a rabbit at a dose of 5 mg per kg body weight, exhibits a somatostatin concentration of at least 0.3 ng/ml over a 50-day period, and suitably a concentration of no more than 20 ng/ml.

Further preferred properties of the obtained somatostatin, e.g. depot preparations containing octreotide depend on the manufacturing procedures used.

Phase separation technique:

Triple emulsion technique:

The above-mentioned methods enable the production of a somatostatin preparation, preferably octreotide and octreotide analogue preparations, with the following properties: 1. a retardation of at least 70%, preferably at least 74%, e.g. at least 75%, 80%, 88% or at least 89% above one

period from 0 to 42 or 43 days and/or

2. an average plasma level (Cp, ideal) in a rat of 2.5 - 6.5, preferably 4 - 6.5 ng/ml over a period of from 0 to 42 days, when 10 mg of somatostatin is administered subcutaneously and/or an average plasma level in a rabbit of 3.5 - 6.5, e.g. 4 - 6.5 ng/ml over a period from 0 to 42 or 43 days when 5 mg of somatostatin is administered

intramuscularly and/or

3. an AUC over a period from 0 to 42 days of at least 160, preferably from 170 - 230 ng/ml x day, for the rat, when 10 mg of somatostatin is administered subcutaneously and/or an AUC over a period from 0 to 42 or 43 days of at least 160, preferably from 180 to 275, e.g. from 200 to 275 ng/ml x day for the rabbit, when 5 mg of somatostatin is administered intramuscularly.

For quantitative characterization of the extended-release preparations described above, the method of area deviation (AD) was used, published by F. Nimmerfall and J. Rosenthaler, Intern. J. Pharmaceut. 32, 1-6, 1986. Briefly, the AD method calculates the area deviations of the experimental plasma profile from an ideal profile which is a constant mean plasma level (= Cp, ideal) produced by transforming the experimental area under the plasma level-time curve (AUC) into a rectangle of equivalent area. From the percentage area deviation (referred to as AUC), the percentage retardation is calculated as follows:

Using this method, the entire plasma profile that has been measured over a predetermined time period is characterized with the help of a simple numerical index.

In Proe. Nati. Acad. Pollock. USA 85, 1988, 5688-5692, a plasma level profile in a rat of the octa-peptide analogue of somatostatin with the formula is described in Fig.4:

However, a clear comparison cannot be made with the plasma level data for the preparations produced according to the invention in the rat as mentioned above, as the described plasma level profile is based on a different delivery method (intramuscular injection) and, what is more important, the content level of the microcapsules (between 2 and 6%) and dose amount for administration (25 to 50 mg portions of microcapsules for 30 days, although determinations were carried out for at least 45 days were not precisely indicated. In addition, the type of poly-(D,L-lactide-co-glycolide) used was ) not precisely described.

The value stated in the above-mentioned publication is thus too low to be a pre-publication that interferes with what is achieved in connection with the present invention.

The following examples illustrate the invention.

My of the polymers is the average molecular weight as determined by GLPC using polystyrene as a standard.

EXAMPLE 1:

1 g of poly(D,L-lactide-co-glycolide) (50/50 molar, N^= 45,000, polydispersity approx. 1.7) was dissolved in 15 ml of methylene chloride with magnetic stirring followed by the addition of 75 mg of octreotide acetate dissolved in 0.5 ml of methanol. 15 ml of silicone oil (brand Dow 360 Medical Fluid 1000 cs) (silicone fluid) was added to the polymer-peptide mixture. The resulting mixture was added to a stirred emulsion containing 400 ml n-heptane, 100 ml pH 4 phosphate buffer, 40 ml Dow 360 Medical Fluid, 350 cs and 2 ml Span 80 (emulsifier). Stirring was continued for at least ten minutes. The microparticles obtained were recovered by vacuum filtration and dried overnight in a vacuum oven. The yield was about 90% microparticles in the size range 10 to 40/zm.

The microparticles were suspended in a vehicle and administered intramuscularly in a 4 mg dose of octreotide to white New Zealand rabbits. Blood samples were taken at regular intervals, and indicated plasma levels of 0.5 to 1.0 ng/ml for 30 days, as measured by radioimmunoassay (RIA) analyses.

EXAMPLE 2:

1 g of poly(D,L-lactide-co-glycolide)glucose (M„ = 45,000, 55/45 molar prepared according to the process described in GB 2,145,422 B, polydispersity approx. 1.7, prepared from 0.2 % glucose) was dissolved in 25 ml of ethyl acetate with magnetic stirring followed by the addition of 75 mg of octreotide dissolved in 3 ml of methanol. 25 ml of silicone oil (brand Dow 360 Medical Fluid, 1000 cs) was added to the polymer-peptide mixture. The resulting mixture was added to the emulsion described in Example 1. Stirring was continued for at least ten minutes. The microparticles obtained were recovered by vacuum filtration and dried overnight in a vacuum oven. The yield was greater than 80% microparticles in the size range 10 to 40/xm.

The microparticles were suspended in a vehicle and administered intramuscularly in a 4 mg dose of octreotide to white New Zealand rabbits. Blood samples were taken at regular intervals and indicated plasma levels from 0.5 to 2 ng/ml for 21 days, as determined by RIA.

EXAMPLE 3:

A solution of 1.5 g of octreotide acetate in 20 ml of methanol was added with stirring to a solution of 18.5 g of poly(D,L-lactide-co-glycolide) glucose (50:50 molar, M^, 45,000) in 500 ml methylene chloride. Phase separation was accomplished by adding 500 ml of Dow 3 60 Medical Fluid (1000 cs) and 800 ml of Dow 3 60 Medical Fluid (350 cs) to the peptide-polymer suspension. The resulting mixture was added to a stirred emulsion containing 1800 ml of n-heptane, 2000 ml of sterile water and 40 ml of Span 80. After stirring for ten minutes, the microspheres were collected by vacuum filtration.

Half of the product was dried overnight in a vacuum oven at 37°C. The residual methylene chloride level was 1.2%.

The other half of the product was washed with 1000 ml of ethanol containing 1 ml of Span 80. After stirring for one hour, the ethanol was decanted and the microparticles were stirred with 1000 ml of n-heptane containing 1 ml of Span 80. After stirring for one hour, the microparticles were collected by vacuum filtration and dried overnight in a vacuum oven at 37°C. The residual methylene chloride level in the microparticles washed in this way was reduced from 1.2% to 0.12%.

The combined yield of product was 18.2 g (91%) of microparticles containing 5.6% octreotide, with an average diameter of 24 µm, and with 1.5% residual heptane.

The microparticles were suspended in a vehicle and injected intramuscularly at a 5 mg/dose of octreotide into white rabbits. Blood samples were taken at fixed intervals, which indicated plasma levels from 0.3 to 7.7 ng/ml for 49 days, as measured by RIA.

Reference example 1:

1 g of poly(D,L-lactide-co-glycolide)glucose (N^46,000, 50:50 molar, prepared according to the process in GB 2,145,422 B, polydispersity approx. 1.7, prepared from 0.2% glucose) was dissolved in 10 ml of methylene chloride with magnetic stirring followed by the addition of 75 mg of octreotide dissolved in 0.133 ml of methanol. The mixture was thoroughly mixed e.g. using an Ultra-Turax for one minute at 20,000 rpm, obtaining a suspension of very small crystals of octreotide in the polymer solution.

The suspension was atomized using a high-speed turbine (Niro Atomizer) and the small droplets were dried in a stream of hot air to form microparticles. The microparticles were collected using a "cyclone" and dried overnight at room temperature in a vacuum oven.

The microparticles were washed with 1/15 molar acetate buffer, pH

4.0, for five minutes and dried again at room temperature in a vacuum oven. After 72 hours, the microparticles were sieved (mesh size 0.125 mm) to give the final product. The microparticles were suspended in a carrier and administered intramuscularly in a 5 mg/kg dose of octreotide to white rabbits (chinchilla bastard) and subcutaneously in a 10 mg/kg dose to male rats. Blood samples were taken at regular intervals indicating plasma levels from 0.3 to 10.0 ng/ml (5 mg dose) in the rabbits and from

0.5 to 7.0 mg/ml in the rats for 42 days, as measured by radioimmunoassay (RIA) analyses.

EXAMPLE 4:

1 g poly(D,L-lactide-co-glycolide)glucose (M„ 46,000, 50:50 molar prepared according to the process in GB 2,145,422 B, polydispersity approx. 1.7, prepared from 0.2% glucose ) was dissolved in 2.5 ml of methylene chloride followed by the addition of 75 mg of octreotide dissolved in 0.125 ml of deionized water. The mixture was thoroughly mixed using e.g. an Ultra-Turax for one minute at 20,000 rpm (inner w/o phase). 1 g of gelatin A was dissolved in 200 ml of deionized water at 50°C and the solution was cooled to 20°C (external w-phase). The w/o and w phases were mixed thoroughly. Thereby, the inner w/o phase was separated into small droplets which were dispersed homogeneously in the outer w phase. The triple emulsion obtained was stirred slowly for one hour. The methylene chloride was thereby evaporated and the microcapsules were hardened from the droplets of the inner phase. After sedimentation of the microparticles, the supernatant was aspirated and the microparticles were recovered by vacuum filtration and washed with water to remove gelatin. Drying, sieving, further washing and a second drying of the microparticles were carried out as described in reference example 1. The microparticles were suspended in a carrier and administered intramuscularly in a 5 mg/kg dose of octreotide to white rabbits (chinchilla bastard) and subcutaneously in a 10 mg/kg dose for male rats. Blood samples were taken at regular intervals indicating plasma levels from 0.3 to 15.0 ng/ml (5 mg dose) in rabbits and 0.5 to 8.0 ng/ml in rats over 42 days, as measured by radioimmunoassay (RIA) analyses.

EXAMPLE 5:

Microparticles were prepared by the triple emulsion technique in the same way as described in Example 4 with three changes: 1. 0.25 ml of acetate buffer, pH 4.0, was used instead of 0.125 ml of water to prepare the inner w/o phase, 2 . washing after collection of the microparticles was carried out with 1/45 molar acetate buffer, pH 4.0, instead of water, 3. further washing of the microparticles was omitted.

EXAMPLE 6:

Microparticles were prepared using the triple emulsion technique in the same manner as described in Example 5, with the only change being that the inner w/o phase was prepared using water containing 0.7% (w/v) sodium chloride instead of acetate buffer.

EXAMPLE 7:

Microparticles were prepared in the same way as described in example 4, with the only difference that the drug compound is dispersed directly in the polymer solution after which the obtained dispersion is mixed with the gelatinous water phase.

Reference example 2:

Qctreotide pamoate.

10.19 g of octreotide as free base (10 mM) and 3.88 g of embonic acid (10 mM) are dissolved in 1 liter of water/dioxane (1:1). The reaction mixture is filtered and freeze-dried to give a yellow powder [α]<20>D=+ 7.5° (C = 0.35, in DMF) of octreotide pamoate hydrate. Factor = 1.4 where the factor is equal to weight of freeze-dried powder/weight of octreotide contained therein.

The pamoate can replace the octreotide acetate present in the microparticles in Examples 1-7 and has excellent stability.

EXAMPLE 8:

A solution of 1 g of poly(D,L-lactide-co-glycolide) (50:50 molar, M,^ = 36,100) in 20 ml of methylene chloride was added with stirring to a solution of 100 mg of calcitonin in 1.5 ml of methanol . Phase separation was carried out by adding 20 ml of silicone fluid (Dow 360 Medical Fluid, 1000 cs). The resulting mixture was added to a stirred emulsion consisting of 100 ml of pH 4 phosphate buffer, 400 ml of n-heptane, 4 ml of Span 80, and 40 ml of silicone fluid (Dow 360 Medical Fluid, 1000 cs). After stirring for ten minutes, the microspheres were collected by vacuum filtration and dried overnight in a vacuum oven at 37°C. The yield was 1.1 g of microspheres containing 5.9% calcitonin.

EXAMPLE 9:

A solution of 9.9 g of poly(D,L-lactide-co-glycolide) (50/50 molar, M , = 44,300) in 140 ml of methylene chloride was added to 100 mg of lypressin. The dispersion was magnetically stirred for one hour before the addition of 140 ml of silicone fluid (Dow 360 Medical Fluid, 1000 cs) and 2.5 ml of Span 80. The mixture was added to 2000 ml of heptane and stirred for ten minutes. The microcapsules obtained were collected by vacuum filtration, washed three times with heptane, and dried for ten minutes under suction. Half of the sample was washed by stirring in water for ten minutes, and the other half was not washed. Both samples were dried overnight in a vacuum oven at 30°C. The total yield was 10.65 g of microcapsules. Analyzes of the washed sample showed 0.5% lypressin and 0.6% for the sample not washed with water.

Claims (9)

1.Procedure for the production of microparticles comprising a drug in a biodegradable, biologically compatible polymer carrier, characterized by the steps of: a) dissolving the polymer carrier material in a suitable solvent in which the drug compound is not soluble, b) adding and dispersing a solution of the drug compound in a suitable solvent which is a non-solvent for the polymer, in the solution in step a) , c) adding a phase-inducing agent to the dispersion step b) to induce microparticle formation, d) adding an oil-in-water emulsion to the mixture in step c) to harden the microparticles, and e) recovering the microparticles. 2. Method for the production of microparticles comprising a drug in a biodegradable, biologically compatible carrier, characterized by the steps with: (i) intimate mixing of a water-in-oil emulsion formed from an aqueous medium and an organic solvent that is not miscible with water containing the drug in one phase and a biodegradable, biologically compatible polymer in the other phase, with an excess of an aqueous medium containing an emulsifying substance or a protective colloid to form a water-in-oil-in-water emulsion, without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step, (ii) separation of the organic solvent therefrom, (iii) isolation and drying of the microparticles obtained. 3. Process for the production of microparticles comprising a drug compound in a biodegradable, biocompatible polymer, characterized by the steps of: (i) intimate mixing of a drug compound suspension formed from a drug compound and a water-immiscible organic solvent containing a biodegradable, biocompatible polymer, with an excess of aqueous medium containing an emulsifying substance or a protective colloid, to form an oil-in-water emulsion, the drug compound being dispersed in the oil component, and without the addition of any drug-retaining substance or the use of any intermediate viscosity-increasing step, (ii) separation of the organic solvent therefrom, (iii) isolation and drying of the obtained microparticles. 4. Method for the production of microparticles as stated in claim 2, characterized in that it comprises thorough mixing of: a) a solution of a drug in an aqueous medium, and b) a solution of a polymer in an organic solvent which is not miscible with the aqueous medium, the water-in-oil emulsion of a ) and b) are thoroughly mixed with c) an excess of an aqueous medium containing a protective colloid, without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step, curing the embryonic microparticles in the formed water-in-oil-in-water emulsion by desorption and isolation of the formed microparticles. 5. Method as stated in claim 4, characterized in that the drug is a somatostatin. 6. Process for the production of microparticles, characterized in that it comprises the thorough mixing of: a) a solution of a somatostatin in water or a buffer in a weight/volume ratio from 0.8 to 4.0 g/l to 120 ml, and b) a solution of a polylactide-co-glycolide in an organic solvent which is not miscible with the aqueous medium, in a weight/volume ratio from 40 g/90 to 400 ml in such a way that the weight/weight ratio between the drug and the polymer is from 1/10 to 50, and the volume/volume ratio of the aqueous medium/organic solvent is 1/1.5 to 30, including mixing of the water-in-oil emulsion from a) in b) together with c) an excess of water or a buffer containing a protective colloid at a volume/volume mixing rate ratio of ab)/c) from 1/10 to 100, without the addition of any drug-retaining substance to the water-in-oil emulsion or the use of any intermediate viscosity-increasing step, with hardening of the embryonic microparticles in the formed water-in-oil-in-water emulsion by evaporation of the organic solvent and isolation of the formed microparticles. 7. Method as stated in one or more of claims 1-6, characterized in that a preparation with extended release in the form of microparticles as mentioned above comprises octreotide or a salt or derivative thereof in a biodegradable, biologically compatible polymer carrier. 8. Method as stated in one or more of claims 1-6, characterized in that a preparation with extended release in the form of microparticles as mentioned above comprises a peptide drug compound in a 40/60 to 60/40 polylactide-co-glycol ester of a polyol, wherein the polyol unit is selected from the group of a (C3-6) carbon chain-containing alcohol with three to six hydroxyl groups and a mono- or disaccharide, and the esterified polyol has at least three polylactide-co-glycolide chains. 9. Method as set forth in one or more of claims 1-6, characterized in that a preparation with extended release in the form of microparticles as mentioned above comprises a peptide drug compound selected from the group of a calcitonin, lypressin or a somatostatin in a linear 40/ 60 to 60/40 polylactide-co-glycolide polymer with chains having a molecular weight IVL^ between 25,000 and 100,000, a polydispersity M^/l^ between 1.2 and 2 in a concentration of 0.2 to 10% by weight of the peptide drug compound therein.