Sustained release formulations of water soluble peptides

(54) (56) (57)

OMBUD AWAPATENT AB

NAME The delayed release preparations of water-soluble peptides

CALLED PUBLICATIONS:

EP A2 145 240 (A61K 9/52), GB A 2 209 937 (A61K 9/22)

SUMMARY:

Delayed-release preparation of a peptide drug compound, preferably a somatostatin, such as octreolide, e.g. in the form of a pamoate salt.

The drug compound is present in a polymeric carrier, preferably a polylactide co-glycolide, in particular a poly (lactide co-glycolide> glucose. The preparation is preferably a depot preparation in the form of a monolithic microparticle.

The numbers In parentheses, the international identification code, the INID code, indicates. Letters in clamps indicate international document code.

512 992

BACKGROUND OF THE INVENTION

The present invention relates to delayed release formulations (depot preparations) of drugs, in particular water-soluble peptides, e.g. somatostatin or somatostatin analogs, such as octreotide, in a biodegradable and biocompatible polymeric carrier, for example, a matrix or coating, e.g. in the form of an implant or preferably a microparticle (also known as microcapsule or microsphere).

The invention also relates to such preparations having satisfactory peptide release profiles for a particular period of time.

Following oral or parenteral administration, peptide drugs often show poor blood bioavailability, e.g. due to their short biological half-lives caused by their metabolic instability. Furthermore, if administered orally or nasally, they often exhibit poor resorption through the mucous membranes. A therapeutically relevant blood level over a long period of time is difficult to achieve.

Parenteral administration of peptide drug1 as a depot preparation in a biodegradable polymer, e.g. such as microcapsules or implants, have been proposed to enable delayed release thereof after a residence time in the polymer which protects the peptide from enzymatic and hydrolytic action of the biological media in question.

Although some parenteral depot preparations of peptide drugs in a polymer in the form of microparticles or an implant are

512 992 known, satisfactory peptide-free loading profiles can in practice be obtained only in very few cases. Special measures must be taken to achieve continuous peptide release for a therapeutically active drug serum level and, if desired, to avoid excessive drug serum concentrations, causing undesirable pharmacological side or side reactions.

The peptide drug release pattern is dependent on numerous factors, e.g. peptide type and, for example, whether it is present in its free or any other form, e.g. salt form, which may affect its water solubility. Another important factor is the choice of polymer from the very extensive list of possibilities described in the literature.

Each polymer type has its own characteristic biodegradation rate. Free carboxyl groups can be formed which contribute to the pH of the polymer and thus additionally affect the water solubility of the peptide and consequently its release pattern.

Other factors which may affect the release pattern of the depot preparation are by how much amount the polymeric carrier is filled with drug, the way of its distribution in the polymer, the particle size and, in the case of an implant, also its shape. Furthermore, the place of preparation in the body is important.

To date, no somatostatin composition in a delayed-release form for parenteral administration has appeared on the market, probably because no composition exhibiting satisfactory serum level 1 profile has been obtained.

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DESCRIPTION OF THE PRIOR ART

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

US Patent 3,773,919 describes controlled release formulations of the drug, wherein the drug, e.g. a water-soluble peptide drug, is dispersed in a biodegradable and biocompatible linear polylactide or polylactide co-glycolide polymer. However, some drug release patterns have not been described, and there is no reference to a somatostatin. U.S. Patent 4,293,539 describes antimicrobial formulations in microparticle form.

US Patent 4,675

189 describes delayed formulations of it

LHRH analog decapeptide nafareline and analog LHRH relatives in polylactide co-glycolide polymers. No release pattern has been described.

T. Chang, J. Bioeng., Vol. 1, pp. 25-32, 1976 have described delayed release of biological compounds, enzymes and vaccines from microparticles.

Polymers / copolymers of lactic acid and lactide / glycol copolymers and related compositions for use in surgical applications and for delayed release and biodegradation have been unpublished in US Patents Nos. 3,991,776, 4,076,796 and 4,116,470.

European Patent Application 0 203 031 discloses a series of somatostatin octapeptide analogs, e.g. compound RC-160 of the formula D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 with a bridge between the -Cys units in columns 15-16.

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The possibility of microencapsulating somatostatins with polylactide co-glycolide polymer has been mentioned in claim 18, but no instructions or details have been given on how to obtain continuous therapeutically active serum levels.

U.S. Patent 4,011,312 discloses continuous release of an antimicrobial drug, e.g. the water-soluble polymyxin B from a low molecular weight polylactide co-glycolide matrix (during 2000) and relatively high glycolide content in the form of an implant can be obtained when the implant is inserted into the teat canal of a cow. The drug is released in a short time due to the high glycolide content and the low molecular weight of the polymer, both of which stimulate rapid polymer biodegradation and thus the corresponding rapid release of the drug. Furthermore, a relatively high content of drugs contributes to rapid drug release. No somatostatin and no drug release patterns have been described.

European Patent No. 58,481 discloses that continuous release of a water-soluble peptide from a polylactide polymer implant is stimulated by lowering the molecular weight of at least a portion of the polymer molecules by introducing glycolide units into the polymer molecule, thereby increasing the segment polymer character of the polymer molecule, thereby increasing the polymer character of the polymer. of drugs in the polymer matrix is ​​increased, thereby increasing the surface of the implant.

Although somatostatins are referred to as water-soluble peptides, some somatostat release profiles have not been described and no indication has been given on how to combine all of these parameters to obtain, for example, continuous somatostatin serum levels for at least one week, e.g. one month.

European Patent No. 92,918 discloses that continuous release of peptides, preferably hydrophilic peptides, for long periods of time

512 992 time period can be obtained if the peptide is incorporated into a conventional hydrophobic polymer matrix, e.g. of a polylactide which is made more accessible to water by incorporating into its molecule a hydrophilic moiety, for example of polyethylene glycol, polyvinyl alcohol, dextran, polymethacrylamide. The hydrophilic contribution to the amphipathic polymer comes from all ethylene oxide groups in the case of the free hydroxyl groups with a polyethylene glycol unit, from a polyvinyl alcohol case or a dextran unit and from a polymethacrylamide unit.

Depending on the amide groups in the case of the presence of the hydrophilic moiety in the polymer molecules, the implant obtains hydrogel properties after water absorption.

Somatostatin is referred to as a hydrophilic peptide, but no release profile has been described and no information has been provided as to the type of polymer preferred for this peptide or its molecular weight and how many hydrophilic groups it should have.

The British patent writing

GB 2 145 422 B discloses that delayed release of drugs of various types, e.g. of vitamins, enzymes, antibiotics, antigens, where long periods of incorporation of the drug can be obtained in an implant,

e.g. of microparticle size, made of a polymer of one or mannitol, with one or more polylactic idester groups. The polylactide ester groups preferably contain, for example, glycolide as described.

best. No peptides, e.g. somatostatin, referred to as drug and no serum drug levels

SUMMARY OF THE INVENTION

The present invention relates to delayed release formulations, e.g. microparticle preparations, of a drug, in particular a hormonally active water-soluble somatostatin or a somatostatin analogue, such as octreotide, which provide satisfactory plasma drug levels and,

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e.g. in a biodegradable, biocompatible polymer, for example in an encapsulating polymer matrix. The polymer matrix may be a synthetic or natural polymer.

The microparticles of the present invention can be prepared by any conventional technique, e.g. an organic phase separation technique, a spray drying technique, or a triple emulsion technique, wherein the polymer is precipitated together with the drug, followed by a hardening or curing of the resulting product, when phase separation or triple emulsion technique is used.

If desired, the sustained release formulations may be in the form of an implant.

We have found a particularly useful modification of the phase-separation ion technique for the preparation of microparticles of any drug.

Accordingly, the present invention also provides a process for preparing a microparticle comprising a drug in a biodegradable, biocompatible carrier, which comprises the steps of:

a) dissolves 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, e.g. an alcohol which is a non-solvent for the polymer in the solution from adds a phase-inducing agent to the dispersion on step b) to induce or effect microparticle formation;

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d) adding an oil-in-water emulsion to the mixture of step c) to cure the microparticle or cure the microparticle, and

e) recovering the microparticle.

We have also found a particularly useful modification of the triple emulsion technique for the preparation of microparticles of any drug.

Accordingly, the present invention provides:

A process for the preparation of microparticles, comprising:

<td>(i)</td><td>intensively mixes a water-in-oil emulsion formed by</td>

an aqueous medium and an aqueous immiscible organic solvent, wherein the drug is present in one phase and a biodegradable, biocompatible polymer is present in the other, 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 any drug preservation or retention.

<td>ratio</td><td>substance is added to the water-in-oil emulsion or that</td>

<td>something</td><td>intermediate viscosity enhancement step was used,</td>

<td>(Ii)</td><td>desorb the organic solvent therefrom,</td>

<td>(ii i)</td><td>isolates and dries the resulting microparticles.</td>

The present invention further relates to the microparticles obtained by these processes.

The present invention also provides:

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a) a sustained release preparation comprising 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 containing an alcohol containing a (Cg_g) carbon chain and having from 3 to 6 hydroxyl groups and a mono- or di-saccharide, wherein the esterified polyol has at least 3 polylactide co-glycolide chains,

<td>b)</td><td>A sustained release preparation comprising one</td>

peptide drug compound selected from the group a calcitonin, lypressin or somatostatin in a 40/60 to 60/40 polylactide co-glycolide polymer of linear molecular weight M_w of between 25,000 and 100,000, a polydispersity M_w/ M_n of between 1.2 and 2 at a concentration of from 0.2 or preferably from 2 to 10% by weight of peptide drug preference.

<td>compound</td><td>in it.</td>

<td>c)</td><td>a sustained release preparation comprising</td>

octreotide or a salt or derivative thereof in a biodegradable, biocompatible polymeric carrier.

We have found that a new salt of octreotide is the pamoate, which is very stable in such preparations.

Accordingly, the present invention provides (i) octreotide pamoate and a process for producing octreotide pamoate, which comprises reacting octreotide with embonic acid (or a reactive derivative thereof).

Furthermore, the present invention provides:

A method of administering a peptide to an individual, comprising parenteral administration to an individual in need of such treatment by a depot preparation as defined above, particularly for the treatment of acromegaly or breast cancer.

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DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drugs used in the methods of the invention are preferably water-soluble drugs, e.g. peptides.

The peptides used in the methods and formulations of the present invention may be a calcitonin such as salmon calcitonin, lypressin and a naturally occurring somatostatin and synthetic analogs thereof.

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

Ala-Gly-Cy s-Ly s-Asn-Phe-Phe-Trp₍Cys-Ser-Thr-Phe-Thr-Lys

This hormone is produced by the hypothalamus gland and other organs, e.g. The Gl area, and mediates, together with GRF,

qv the neuroregulation of the pituitary release of growth hormone. In addition to inhibiting GH release of the pituitary gland, somatostatin is a potent or potent inhibitor of a number of systems, including central and peripheral neural, gastrointestinal and vascular smooth muscle. It also inhibits the release of insulin and glucagon.

The term somatostatin includes its analogs or derivatives thereof. By derivatives and analogs is meant straight-chain, bridged or cyclic polypeptides wherein one or more amino acid moieties have been omitted and / or replaced by one or more other amino radicals and / or wherein one or more functional groups have been replaced by one or more other functional groups and / or or several groups have

<td>replaced with</td><td>one or more other isostatic groups. Generally</td>

<td>seen covers</td><td>the term all modified derivatives of a biological</td>

<td>quite active</td><td>peptide having a qualitatively similar</td>

512 992 effect as that applicable to the unmodified somatostatin peptide.

Thus, agonistal scents of somatostatin are useful in replacing natural somatostatin in terms of its effect on the regulation of physiological functions.

Preferred known somatos tatins are:

a) (D) Phe-Cys-Phe- &lt;D) Trp-Lys-Thr-Cys-Thr-ol (Generic name Octreotide)

b) (D) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-ThrNH₂

c) &lt;D&gt; Phe-Cys-Tyr- (D) Trp-Lys-Va1-Cys-TrpNH₂

d) (D) Trp-Cys-Phe- (D&gt; Trp-Lys-Thr-Cys-ThrNH₂

e) (D) Phe-Cys-Phe- (D&gt; Trp-Lys-Thr-Cys-ThrNH₂

f) 3- (2- &lt;Naphthyl) - &lt;D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-ThrNH₂

g) (D&gt; Phe-Cys-Tyr- &lt;D&gt; Trp-Lys-Val-Cys-β-Na1-NH₂

h) 3- &lt;2-naphthyl) -Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys-β-Nal-NH₂

i) (D) Phe-Cys-β-Na1- (D) Trp-Lys-Val-Cys-Thr-NH₂ where in each of compounds a) to i) there is a bridge between the amino acids marked with an *, as indicated in the following formula.

Other preferred somatostatins are:

ride * ------------------------------ *

II

H-Cys-Phe-Phe- (D) Trp-Lys-Thr-Phe-Cys-OH

512 992 (See Vale et al., Metabolism, 27 Supp. 1, 139 (1978)).

* *

Asn-Phe-Phe- (D) Trp-Lys-Thr-Phe-Gaba (See European patent with publication no. 1295 and application no. 78100994.9).

* *

MeAla-Tyr- (D) Trp-Lys-Val-Phe (See Verber et al., Life Sciences, 34, 1371-1378 (1984) and

European Patent Application No. 82106205.6 (published as No. 70,021) also known as Cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe).

* *

NMePhe-Hi s- (D) Trp-Lys-Va1-Ala (See RF Nutt et al., Kl in.Wochenschr. (1986) 64 (Suppl.VII)

H-Cys-His-His-Phe-Phe- (D) Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (see EP-A-200 188)

X-Cy s-Phe-J) -Trp-Ly s -Thr-Cy s -Thr-NH 2 and

X-Cy s-Phe-D, -Trp-Ly s-Thr-Cy s-Thr-o 1 wherein X is a cationic anchor, especially

Ac-hArg (Et2&gt; ~ Gly-Cys-Phe-D-Trp-Lys-Thr-Cys-NH2 (see EP 0363589A2)

512 992 wherein in the above amino acids there is a bridge between the amino acids marked with a *.

The contents of all the above publications, including the specific compounds, are hereby specifically incorporated into the present text by the references in question.

The term derivative also includes the corresponding derivative bearing a sugar residue.

When somatostatin carries a sugar residue, it 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 described e.g. in WO 88/02756.

The term octreotide derivative includes those containing the unit * *

-D-Phe-Cys-Phe-DTrp-Lys-Thr-Cys with a bridge between the Cys residues.

Particularly preferred derivatives are N^A-Ca-glucosyl- (1-4-deoxifructosyl)-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol and N^A-tp-deoxifructosyl-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol, each of which has a bridge between the Cys units, preferably in acetate salt form and described in Examples 2 and 1, respectively, of the above application.

The somatostatin may exist, for example, in free form, salt form or in the form of complexes thereof. Acid addition salts can be formed with, for example, organic acids, polymeric acids and inorganic acids. Acid addition salts include e.g. hydrochloride id and acetates. Complexes, for example, are formed by somatostatin genome

512 992 addition of inorganic substances, e.g. inorganic salts or hydroxides, such as Ca and Zn salts, and / or addition of polymeric organic substances.

The acetate salt is a preferred salt for such preparations, especially for microparticles, as it results in reduced initial or initial drug failure. The present invention also provides the pamoate salt, which is useful especially for implants, and a method for its preparation.

The pamoate can be obtained in a conventional manner, e.g. by reacting embonic acid (pamoic acid) with octreotide, e.g. in the form of the free base. The reaction may be carried out in a polar solvent, e.g. at room temperature.

The somatostatin drugs are intended for use in the treatment of disorders or diseases where long-term application of the drug is desired or needed, e.g. condition with an etiology comprising or associated with excessive GH secretion,

e.g. in the treatment of acromegaly, for use in the treatment of gastrointestinal disorders or diseases, e.g. in the treatment or prophylaxis of peptic ulcers, enterocutaneous and pancreatic cutaneous fistula, irritable bowel syndrome, dumping syndrome, water diarrhea syndrome, acute pancreatitis and gastroenteropathic endocrine tumors (eg vipoma, GRF, glucagon, insulinoma, gastrinoma and carcinoma , cations associated breast cancer and complicated diabetes.

The polymeric carrier can be made of biocompatible and biodegradable polymers such as linear polyesters, branched polyesters which are linear polyol unit or core, chains emitting from a polymer of lactic acid, polyglycolic acid, polyhydrox in butter acid, polycaprolactone polyalkylene polyalkylene

512 992 of acids from Krebs cycle, e.g. the citric acid cycle and the like, and copolymers thereof.

The preferred polymers of the present invention are the linear polyesters and the branched chain polyesters. The linear polyesters can be prepared from the alpha hydroxycarboxylic acids, e.g. lactic acid and glycolic acid, by condensitone of the lactone dimers, see e.g. U.S. Patent No. 3,773,919.

Linear polylactide co-glycolides, which are preferably used in accordance with the invention, preferably have a molecular weight between 25,000 and 100,000 and a polydispersibility Mw / Mn of, for example, between 1.2 and 2.

The branched polyesters of the invention can be prepared using polyhydroxy compounds, e.g.

ex. polyol, or mannitol, as the initiator.

known and are disclosed in English patent GB 2 145 422 B.

The polyol contains at least three hydroxy groups and has a molecular weight of up to 20 00

0, wherein at least 1, preferably at least 2, e.g. average

3, of the hydroxy groups in the polyol are in the form of ester groups which contain polylactide or copolylactide chains. Typically, 0.2% glucose is used to initiate the polymerization. The structure of the branched polyesters is star-shaped. The preferred polyester chains in the linear polymer compounds and the star polymer compounds preferably used according to the invention are copolymers of alpha-carboxylic acid units, lactic acid and glycolic acid, or of the lactone dimers. The molar ratio lactide: glycolide is from about 75:25 to 25:75, e.g. 60:40 to 40:60, where from 55:45 to 45:55, e.g. from 55:45 to 50:50 are the most preferred.

The star polymers can be prepared by reacting a polyol with a glycolide also

512 992 elevated temperature in the presence of a catalyst, which allows a ring-opening polymerization.

We have found that an advantage of the star polymer type in the formulations of the present invention is that its molecular weight can be relatively high, imparting physical stability.

e.g. a certain hardness, to implants and to microparticles, which prevents them from sticking together or sticking together, although relatively short polylactide chains are present, leading to controllable or controllable biodegradation rate of the polymer in the range of several weeks to one or two months, and to the corresponding delayed release of the peptide, which makes a depot preparation made thereof suitable for, for example, a month's release.

The star polymers preferably have an average molecular weight M_win the range of about 10,000 to 200,000, preferably from 25,000 to 100,000, especially from 35,000 to 60,000, and a polyydispersibility of, for example, from 1.7 to 3.0, e.g. from 2.0 to 2.5. The intrinsic viscosity of star polymers with 35,000 and M_w 60,000 are 0.36 and 0.51 dl / g of chloroform, respectively. A star polymer with an M_w of 52,000 have a viscosity of 0.475 dl / g of chloroform.

The terms microsphere, microcapsule and microparticle may be considered equivalent or interchangeable with the invention and denote encapsulation of the peptides of the polymer, preferably with the peptide distributed in the polymer, which is thereby a matrix for the peptide. In this case, the terms microsphere or, more generally, microparticles are preferably used.

Using the phase separation technique of the present invention, the formulations can be read, for example, by dissolving the solvent in a peptide, followed by addition, and according to the invention, the non-solvent polymeric carrier material prepared to disperse a solution of

512 992 peptide in the polymer-solvent composition. A phase inducing agent, e.g. a silicon liquid, is then added to induce encapsulation of the peptide by the polymer.

The drug burst effect can be substantially reduced by precipitation in situ of ultrafine drug particles by adding a drug solution to the polymer solution prior to phase separation. The prior art method involves the addition of dry particles directly to the polymer solution.

The therapeutic duration of the peptide-free setting can be increased by curing / washing the microparticles with an emulsion of buffer / heptane. The prior art method comprises a curing step followed by either no subsequent washing at all or a separate water-based washing step.

An oil-in-water (= o / w) emulsion can be used to wash and cure the microspheres and remove unencapsulated peptide. The washing helps to remove unencapsulated peptide from the surface of the microspheres. The removal of excess peptide from the microspheres reduces the initial drug release, which is characteristic of many conventional encapsulation preparations. Thus, a smoother delivery of drugs over time is possible with the present microsphere preparations.

The emulsion also facilitates the removal of residual polymer solvent and silicone liquid. The emulsion may be added to the polymer peptide mixture, or the mixture may be added to the emulsion. It is preferred that the polymer peptide mixture 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 obtain a stable emulsion. The emulsion

512 992 can be buffered with a buffer, and the polymer matrix material. from pH 2 to be prepared from which is not deleterious to the peptide. The buffer may be Θ, with pH 4 being preferred. This buffer can acidify buffer substances such as phosphate buffer, acetate buffer and stirring buffer.

organic phase in the like. Only water can replace the heptane, hexane and the like can be used as the raw buffer.

The emulsion may contain dispersants, such as silicone oil.

A preferred emulsion may include heptane, pH 4, silicone oil and sorbitan monooleate.

phosphate buffer

When an initial step with non-solvent replace the emulsion cure. Heptane, hexane and the like can be used as solvents.

Other alternatives to the o / w emulsion can be used to cure the microcapsules, or to harden them, such as:

Solvents plus emulsifiers for curing the microcapsules without washing and solvents plus emulsifiers for curing followed by a separate washing step.

The 0 / w emulsion can be used without the dispersant. However, with the aid of the dispersant, aggregation of the dry microcapsule particles due to static electricity is avoided, and it also helps to reduce the level of residual solvent.

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

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1β

The phase inducers (coacervatives) are solvents miscible with the polymer-drug mixture and cause the embryonic microcapsules to form before curing or curing; silicone oils are the preferred phase induction agents.

The o / w emulsion can be prepared in a conventional manner using heptane, hexane and the like for the organic phase.

The microparticles of the present invention may also be prepared by the commonly known spray drying procedure. According to this net part

In the 3 part method, somatostatians or a solution are thoroughly mixed

e.g. methanol, in and one of the peptide in a water or in an organic solution buffer,

e.g. with pH solution of the polymer in an organic which is not miscible with the aforementioned, t.

methylene chloride.

The resulting solution is sprayed

The microparticles formed are collected, e.g. with a cyclone if desired, and, for example, pH 3.0 to 8

0, preferably at pH 4.0, or dried in vacuo or under pressure.

it. ex.

at a temperature of from 20 to 40 ° C.

applied if the particles exhibit so-called

drug shortage in vivo and the extent of drug shortage would be undesirable. As an buffer, an acetate buffer can be used

Microparticles can be obtained in this way, which exhibit improved somatostatin release profile in vivo.

The invention thus also relates to the microparticles prepared by this process. Accordingly, the invention provided 512,992 additionally pours a precipitate which is prepared statin or a solution of a water or a buffer with the pH time co-glycolide formed, statin in the polymer solution of the microspheres and with pH 3.0 to 8.0 in a vacuum or 20 to 40 ° C. Compared to the phase separation technique and this is not even used is not used in sustained release preparation of mixing a somatosomatostatin in methanol or and a solution of polylaccharide and injection of the suspension of somatovarm air, collecting in a buffered solution of water and drying a temperature of from prepared from silicone oil , say 1 icon oil

In the methylene chloride emulsion or in a stream of washing them or distilled under pressure with microparticles, they do not contain any trace amounts since any spray drying technique.

The formulations used according to the invention may also be prepared during a triple emulsion procedure. In a typical technique, peptide is dissolved,

e.g.

octreotide, in a suitable solvent, e.g.

water, and intensively emulsified in a solution of the polymer,

e.g. 50/50 poly &lt;D, L-lactide co-glycolide glucose in a solvent which is a non-solvent for the peptide, for example in methylene chloride. Examples of the solvent for the polymer matrix material are methylene chloride, chloroform, benzene, ethyl acetate and the like. The resulting water / oil (w / o) emulsion is further emulsified in an excess of water containing an emulsifying substance, e.g. an anionic or nonionic surfactant or lecithin or a protective colloid, e.g.

gelatin, dextrin, carboxymethyl cellulose, triple formation &lt;w / o / w) emulsion.

The microparticles are formed by spontaneous precipitation of the polymer and cured by evaporation of the organic solvent. Gelatin helps prevent agglomeration of the microspheres. After sedimentation of the microparticles, the supernatant is decanted and the microparticles washed with water and then with acetate buffer. The microparticles are filtered and then dried.

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

The triple emulsion procedure is known from US Patent No. 4,652,441. According to this patent, in a first step, a drug solution (1) is thoroughly mixed in a solvent, e.g. somatostatin in water (column 2, lines 31-32&gt;, with an excess of a polylactide-co-glycolide solution (2) in another solvent, wherein the first solvent is not soluble, e.g., methyl enchloride, giving an emulsion (3) water-in-oil (w / o) type of fine drug-containing drops of (1&gt; in solution (2)). In solution (1), a so-called drug-retaining substance (column 1, line 31), e.g. gelatin, albumin, pectin or agar.

In a second step, the viscosity of the inner phase (1) is appropriately increased, such as by heating, cooling, pH change, addition of metal ions or cross-linking of, for example, gelatin with an aldehyde.

of water with the w / o emulsion (3) (column 7, lines 52-54), leading to a ternary layer emulsion of w /_O/_w-type. In the excess of water, a so-called emulsifier may be present, if desired (column 7, line 56), which is selected from the group of, for example, anionic or nonionic surfactant or e.g.

polyvinylpyrrolidone, polyvinyl alcohol or gelatin.

In a fourth step, w /_O/_wthe in-water drying emulsion (line 52). This means that the organic solvent in the oil layer is desorbed to form microparticles. Desorption is accomplished in a manner known per se (column Θ, lines 3-5).

e.g. by lowering the pressure during agitation (column Θ, lines 5-7) or e.g. by blowing nitrogen gas through the oil layer (eg methylene chloride) (line 19).

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

Although the above process is satisfactory for the preparation of formulations according to the invention, however, the above-mentioned drug-retaining substance, e.g. gelatin, albumin, pectin or agar, still contained in the formed microparticles.

We have now found that, by avoiding the addition of the drug-retaining substance (= in solution (1)) and the step of increasing the viscosity of the inner phase, and in excess of water for the ternary w /_O/_wemulsion, and retains the action of adding an emulsifying substance or a protective colloid, such as gelatin, satisfactory microparticles can still be obtained. In addition, the microparticles do not contain any drug-retaining substance and only a very small quantity of methylene chloride.

The invention therefore provides a process for producing microparticles, which means that these microparticles are prepared by intensive mixing of:

a) a solution of a drug, 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, in particular 2 , 5/10, and in a buffer of pH 3-8, in particular an acetate buffer, and

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b) a solution of a polymer, preferably a polylactide co-glycolide, as mentioned above, in an organic solvent which is immiscible with the aqueous medium, e.g. methylene chloride, preferably in a weight / volume ratio of 40 g / 90 to 400 ml, in particular 40/100, preferably 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 the volume / volume ratio of aqueous medium / organic solvent is 1 / 1.5 to 30, in particular

1/10, and intensive mixing of the w / o emulsion of a) ib) together with an excess of an aqueous medium, preferably water or a buffer, e.g. an acetate or phosphate buffer

3-8, containing an emulsifying substance or a protective colloid, preferably at a concentration of from 0.01 to 15.0%, in particular a concentration of from 0.1 to gelatin, especially in weight percent, in particular 0.5 weight percent , preferably at a volume / volume mixing rate of ab) / c) of 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 enhancing step, the microparticles in the formed cure of the embryonic w /_{o / w}the emulsion by desorption, preferably by evaporation or evaporation, of the organic solvent, preferably methylene chloride, and the isolation, optionally washing and drying of the microparticles forming.

The invention also provides the process variant in which the drug is dispersed directly into the polymer solution, after which the resulting dispersion is mixed with the gelatin-containing aqueous phase.

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The invention also provides the microparticles produced by these processes. Like microparticles prepared according to the spray-drying technique, they do not contain any silicone oil. Compared to microparticles prepared according to the known triple emulsion process technique, they do not contain any protective colloid.

The delayed release formulations can also be prepared by other methods known per se.

e.g.

- if the peptide is sufficiently stable to produce an implant, by heating microparticles containing the peptide, e.g. a somatostatin in a polylactide co-glycolide, in particular as described above or a mixture thereof obtained by mixing the peptide and polymer, at a temperature of, for example, from 70 to 100 ° C and extrusion or extrusion and cooling of the compact mass, after which the extrudate is cut and possibly washed and dried.

The formulations of the invention are preferably prepared under aseptic conditions.

The formulations of the invention can be used in depot form, e.g. in the form of injectable microspheres or implants.

They can be administered in a conventional manner, e.g. subcutaneous or intramuscular injection, for example, for indications known for the drug contained therein.

The delayed-release preparations containing octreotide may be administered for any known indications for the octreotide or its derivatives, e.g. those described in GB 2 199 829A, pages 89-96, as well as for acromegaly and for breast cancer.

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The microparticles of the present invention may have a size range of about 1 to 250 μπι in diameter, preferably from 10 to 200, especially from 10 to 130, e.g. from 10 to 90, μπι. Implants can be, for example, from about 1 to 10 mrn The amount of drug present, i.e., peptide, in the formulation is dependent on the desired daily release dose and thus on the biodegradation rate of the encapsulating polymer. The exact amount of peptide can be determined by bioavailability tests. The formulations may contain peptide in an amount of at least 0.2, preferably from 0.5, to 20% by weight relative to the polymer matrix, preferably from 2.0 to 10, especially 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 2 months.

Conveniently, the sustained release preparation comprises an octreotide, in a biodegradable biosomatostatin, e.g.

compatible polymeric carrier which, when administered subcutaneously to the rat at a dose of mg somatostatin per kg body weight of the animal, exhibits a concentration of somatostatin in ng / ml and preferably less than 20 ng / ml over a 30-day period or preferably a 60-day period. long period.

Alternatively, the sustained release preparation preferably comprises a somatostatin, e.g.

octreotide, in a biodegradable biocompatible polymer carrier, rabbit intramuscularly at a dose of mg per kg of body weight provides a concentration of a somatostatin of at least 0.3 ng / ml over a 50 day period and preferably a concentration of no more than ng / ml.

Other withdrawn the depot preparations containing somatostatin, t. ex.

octreotide, is, depending on the preparation processes used:

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phase separation

Rabbit 5 mg somatostatin / kg, intramuscular deceleration &lt;0-42 days)

76% average plasma level (0-42 days) ng / ml (cp, ideal)

AUC (0-42 days)

170 ng / ml x days

Spray Drying Technology:

Rat 10 mg somatostatin / kg subcutaneously

<td>deceleration</td><td> (0-42</td><td>days)</td><td> &gt;75%</td><td></td>

<td>plasma levels of genome111</td><td></td><td></td><td></td><td></td>

<td>(cp, ideal)</td><td> (0-42</td><td>days r)</td><td colspan="2">4-6 ng / ml</td>

<td>AUC</td><td> &lt; 0-42</td><td>days)</td><td> 170-210</td><td>ng / ml</td>

<td>Rabbit 5 mg somatostatin / kg.</td><td colspan="3">intramuscular</td><td></td>

<td>deceleration</td><td> &lt;0-43</td><td>days)</td><td> &gt;75%</td><td></td>

<td>average plasma level</td><td></td><td></td><td></td><td></td>

<td>(cp, ideal)</td><td> &lt;0-43</td><td>days</td><td colspan="2">4-6 ng / ml</td>

<td>AUC</td><td> &lt;0-43</td><td>days)</td><td> 200-240</td><td>ng / ml</td>

<td>Triple Emulsion Technology:</td><td></td><td></td><td></td><td></td>

<td>Rat 10 mo somatoetatin / kg_r</td><td colspan="2">subcutaneous</td><td></td><td></td>

<td>deceleration</td><td> (0-42</td><td>days</td><td> &gt;75%</td><td></td>

<td>average plasma level</td><td></td><td></td><td></td><td></td>

<td>(cp, ideal)</td><td> (0-42</td><td>days)</td><td> 4-6,5</td><td>ng / ml</td>

<td>AUC</td><td> ( 0-42</td><td>days)</td><td> 170-230</td><td>ng / ml</td>

xx

x dgr dgr dgr

Rabbit 5 mg somatostatin / kg intramuscularly

<td>deceleration</td><td> (0-43/43</td><td>days)</td><td> &gt;74%</td>

<td>average plasma level</td><td></td><td></td><td></td>

<td>(cp, ideal)</td><td> (0,42/43</td><td>dgr)</td><td>3.5-6.5 ng / ml</td>

<td>AUC</td><td> (0,42/43</td><td>days)</td><td>160-270 ng / ml x dgr</td>

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Thus, the invention also provides somatostatin compositions, preferably octreotide and octreotide analogues, having the following characteristics:

1st a deceleration of at least 70%, preferably at least 74%,

e.g. at least 75%, 80%, 88% or at least 89% over a period of from 0 to 42 or 43 days and / or

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

3rd an AUC for a time period of from 0 to 42 days of at least 160, preferably 170-230, ng / ml x days, for rats when 10 mg of somatostatin is administered subcutaneously, and / or an AUC for a time period of 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 days for rabbit, when 5 mg somatostatin is administered intramuscularly.

For quantitative characterization of the delayed release preparations described above, we use the area deviation method (AD) published by F. Nimmerfall and J. Rosenthaler; Internal. J. Pharmacist. 32. 1 ~ 6 (1986).

Briefly, in the AD method, the area deviations of the experimental plasma profile are calculated from an ideal profile, which is a constant average plasma level (= cp, ideal) produced by converting the experimental area under the plasma level time curve (AUC) into a rectangle with

512 992 equal area. From the percentage area deviation (referred to as AUC), the percentage deceleration is calculated as follows:

% deceleration = 100 x (1 - AD / AUC)

Using this method, the entire plasma profile measured over a predetermined period of time is characterized by a single numerical index.

In Proc. Acad.Sci.USA 85 (1988) 5688-5692 discloses in Figure 4 a plasma level profile of the octapeptide analog of somatostatin of the formula

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 in rat.

However, no clear comparison can be made with the plasma level data for the compositions of the invention in the rat, as mentioned immediately above, since the described plasma level profile is based on a different method of administration (intramuscular injection) and - which is more important - the microcapsules' filling level (2). 6%) and the dosage amount for administration (25 to 50 mg servings of microcapsules for 30 days, although determinations were made at least for 45 days) are not exactly specified. In addition, the exact type of poly (D1-lacquer-co-glycolide) used is also not described.

Thus, the publication's value in terms of description is too low for it to be considered a prepublication which prejudices the invention.

The following examples illustrate the invention.

M_w for the polymers, the average molecular weight or average molecular weight is determined by GLPC using polystyrene as standard.

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EXAMPLE 1 ·.

One g of poly (D, L-lactide-co-glycolide) (50/50 molar, M_w=45

000; polydispersibility (1.7) was dissolved in 15 ml of methylene chloride by means of magnetic stirring followed by the addition of 75 mg of octreotide acetate dissolved in 0.5 ml of methanol. Fifteen ml of silicone oil (trade name 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 of n-heptane, 100 ml of phosphate buffer pH 4, 40 ml of Dow 360

Medical Fluid, 350 cs, and 2 ml Span 80 (emulsifier). Stirring was continued for at least 10 minutes. The microparticles formed were recovered by vacuum filtration and dried overnight in a vacuum oven. The yield was approximately 90% microparticles in the size range of 10 to 40 µm.

The microparticles were suspended in a carrier and administered IM in a 4 mg dose of octreotide to white New Zealand rabbits. Blood samples were taken periodically giving plasma levels of 0.5 to 1.0 ng / ml for 30 days as measured by radioimmunoassay (RIA).

EXAMPLE 2:

One g of poly (D, L-lactide-co-glycolide) glucose (M_w = 45,000; 55/45 molar prepared according to the process of GB 2 145 422 B; polydispersibility about 1.7; (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 (trade name 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 10 minutes. The resulting microparticles were extracted by vacuum filtration and dried overnight in a vacuum oven. The yield was more than 80% microparticles in the size range of 10 to 40 µm

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The microparticles were suspended in a carrier and administered IM at a dose of 4 mg octreotide to white New Zealand rabbits. Blood samples were taken periodically, giving plasma levels in the range of 0.5 to 2 ng / ml for 21 days measured with 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, Mw 45,000) in 500 ml of methylene chloride. . Phase separation is achieved by the addition of 500 ml Dow 360 Medical Fluid (1000 cs) and 800 ml Dow 360 Medical Fluid (350 cs) to the peptide-polymer suspension. The resulting mixture was added to a stirred emulsion consisting of 1800 ml of n-heptane, 2000 ml of sterile water and 40 ml of Span 80. After stirring for 10 minutes, the microspheres were collected by vacuum filtration.

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

The other half of the product was washed with stirring with 1000 ml of ethanol containing 1 ml of Span B0. 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 1 hour, the microparticles were collected by vacuum filtration and dried overnight in a vacuum oven at 37 ° C. The content of methylene chloride remaining for the microparticles washed in this way was reduced from 1.2% to 0.12%.

The combined yield of the product was 18.2 g (91%) of microparticles containing 5.6% octreotide, mean diameter 24 μπι, 1.5% residual heptane.

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The microparticles were suspended in a carrier and injected intramuscularly at a dose of 5 mg / kg of octreotide to white rabbits. Blood samples were taken periodically, giving plasma levels from 0.3 to 7.7 ng / ml for 49 days measured by RIA.

EXAMPLE 4:

One gram of poly (D, L-lactide-co-glycolide) glucose with M_w 46,000 (50:50 molar) prepared according to the process described in GB 2,144,422B, polydispersibility approximately 1.7, prepared from 0.2% glucose) were dissolved in 10 ml of methylene chloride under magnetic stirring followed by the addition of 75 mg of octreotide dissolved in 0.133 ml of methanol. The mixture was mixed intensively, e.g. using an Ultra-Turax, for one minute at 20,000 rpm, to give a suspension of very small crystals of octreotide in the polymer solution.

The suspension was sprayed using a high speed turbine (Niro Atomizer) and the droplets were dried in a stream of hot air to form microparticles. These microparticles were collected with 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 5 minutes and dried again at room temperature in a vacuum oven. After 72 hours, the microparticles (0.125 mm mesh size) were screened to form the final product. The microparticles were suspended in a carrier and administered in a dose of 5 mg / kg octreotide to white rabbits (chinchilla bastard) and sc in a dose of 10 mg / kg to male rats. Blood samples were taken periodically giving plasma levels of from 0.3 to 10.0 ng / ml (5 mg dose) in rabbits and from 0.5 to 7.0 ng / ml in rats for 42 days as measured by radioimmunoassay (RIA).

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EXAMPLE 5:

Microparticles were prepared by spray drying in the same manner as described for Example 4 with the only change that octreotide was suspended directly in the polymer solution without the use of methanol.

The microparticles were suspended in a carrier and administered sc at a dose of 10 mg / kg octreotide to male rats. Blood samples were taken periodically, giving plasma levels of from 0.5 to 10.0 ng / ml in rats for 42 days as measured by radioimmunoassay (RIA).

EXAMPLE 6.

One gave poly (D, L-lactide-co-glycolide&gt; glucose, M_w 46,000 &lt;50:50 molar prepared according to the process from GB 2,144,422B, polydispersibility (1.7, prepared from 0.2% glucose) was dissolved in 2.5 ml of methylene chloride followed by addition of 75 mg of octreotide dissolved in 0.125 ml of deionized water . The mixture was intensively mixed, e.g. using an Ultra-Turax, for one minute at 20,000 rpm (internal W / O phase).

One gram of Gelatin A was dissolved in 200 ml of deionized water at 50 ° C and the solution cooled to 20 ° C (outer W phase). The W / O and W phases were intensively mixed. Thereby, the inner W / O phase was separated into small, fine droplets which were homogeneously dispersed in the outer W phase. The resulting triple emulsion was stirred slowly for one hour. Thereby the methylene chloride was evaporated and the microcapsules were cured from the fine droplets of the inner phase. After sedimentation of the microparticles, the supernatant was aspirated and the microparticles were recovered by vacuum filtration and rinsed with water to remove gelatin. Drying, sieving, washing and secondary drying of the microparticles was carried out as described for Example 4. The microparticles were suspended in a carrier and administered in a dose of 5 mg / kg octreotide to white rabbits (chinchilla bastard) and sc at a dose of 10 mg / kg in male rats, blood samples were taken periodically, giving plasma levels of from 0.3 to 15.0 ng / ml (5 mg dose) in rabbits and from 0.5 to 8.0 ng / ml in rabbits. rats for 42 days measured by radioimmunoassay (RIA).

EXAMPLE 7:

Microparticles were prepared using the triple emulsion technique at the same changes:

as described in Example 6 but with three

0.25 ml of acetate buffer, pH 4.0, was used instead

0.125 ml of water to prepare the interior

W / O phase.

2nd Rinse after collection of the microparticles was performed with 1/45 molar acetate buffer, pH 4.0, instead of water.

3rd Further washing of the microparticles was omitted.

EXAMPLE 8:

Microparticles were prepared by the triple emulsion technique as described for Example 7, with the only change being that the internal W / O phase was prepared using water containing 0.7% (w / v) sodium chloride instead of acetate buffer.

EXAMPLE 9:

Microparticles were prepared in the same manner as described in Example 6, with the only difference being that the drug compound is dispersed directly into the polymer solution, whereupon the resultant

512 The 992 dispersion is mixed with the gelatin-containing aqueous Example IQ;

Oktreotidpamoat

10.19 g of free base octreotide (10 mM) and 3.88 embonoic acid (10 mM) are dissolved in 1 liter of water / dioxane (1: 1). The reaction mixture is a yellow powder, filtered and lyophilized to give LaI²⁰D = + 7.5 ° (C = 0.35, DMF), of octreo15 time pamoate hydrate.

Factor = 1.4, where the factor = weight of lyophilisate / weight of octreotide contained therein.

The pamoate can be replaced by octreotide acetate present in the microparticles of Examples 1-9 and has excellent stability.

EXAMPLE 11:

A solution of 1 g of poly (D, L-lactide-co-glycolide) (50:50 molar,

MW = 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 performed by adding 20 ml of silicone liquid (Dow 360 Medical Fluid, 1000 cs). The resulting mixture was added to a stirred emulsion consisting of 100 ml of phosphate buffer with pH 4, 400 ml of n-heptane, 4 ml of Span 80 and 40 ml of silicone liquid (Dow 360 Medical Fluid, 1000 cs). After stirring for 10 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 12:

A solution of 9.9 g of poly (D, L-lactide-co-glycolide) (50/50 molar, Mw - 44,300) in 140 ml of methylene chloride was added to 100 mg of lypressin. The dispersion was subjected to magnetic stirring during 1

512 992 ml in 1 fluid (Dow 360 Medical hour before addition)

Fluid, 1000 cs)

2000 ml of heptane microcapsules three times with ning.

under the protection of

140 ml

Span 80. The mixture was added and stirred for 10 minutes. The resulting were collected by vacuum filtration, washed minutes during suction Half of the sample was washed by stirring in water minutes; the other half was not washed. Both test overnight in a vacuum oven at 30 ° C.

dried

The total yield was the sample yielded 0.5 lypressin and 0.6% for the sample not washed with water.

in"

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