Biodegradable microparticles or implant containing insoluble peptide salts

(54) METHOD FOR THE PRODUCTION OF A CONTINUOUS AND CONTROLLED CHARGE OF A

NATURAL OR SYNTHETIC PEPTIDS OF SPECIFIC PHARMACEUTICAL COMPOSITION, ESPECIALLY IN THE FORM OF MICRO-CAPSULES OR IMPLANTS (57) A pharmaceutical composition, in particular in the form of microparticles or an implant, is produced. The pharmaceutical composition contains a biodegradable polymer selected from poly-1, 4-butylene succinate, poly-2,3-butylene succinate,

Poly-1, 4-butylene fumarate and poly-2,3-butylene fumarate.

As the active ingredient in the pharmaceutical composition is the embonate, tannate, stearate or palmitate of a natural or synthetic peptide preferably having 3 to 45 amino acids, such as such salt of LH-RH, somatostatin, GH-RH or caleitonin or one of its synthetic analogues or homologs.

The preparation comprises the dry mixing of the ingredients in powder form, the pre-compaction and the

QQ preheating the mixture and then extruding the precompressed and preheated mixture.

The product obtained by the extrusion can be pulverized and finally sieved.

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The invention relates to a process for the preparation of a pharmaceutical composition in the form of microparticles or an implant for the continuous and controlled release of a natural or synthetic peptide. Namely, the present invention relates to a process for producing a pharmaceutical composition ensuring the continuous and controlled delivery of a drug containing a biodegradable polyester-type copolymer such as a polysuccinate or a polyfumaiate, wherein the embonate, tannate, stearate or palmitate of a natural or synthetic peptide , in particular a peptide having 3 to 45 amino acids, is incorporated as an active ingredient.

In order to prepare compositions which ensure a continuous and controlled release of a drug, various solutions have been proposed to date which have utilized degradable implants, microencapsulations or biodegradable porous matrices, for example, obtained as microparticles of various sizes.

It is here for microencapsulations or for the production of implants or biodegradable porous matrices based on polylactides or Copolylactidglykoliden on EP-A2 0052510, further to EP-A1 0058481, US-PS 3,976,071, or British Pat. No. 2,209,937 and also to DE-OS 3,835,099, which disclose the use of polyesters, such as, for example, poly-1,4-butylene succinate or fumarate and poly-2,3-butylene succinate or fumarate for the preparation of pharmaceutical microcapsules or implants, in particular with a delayed and controlled release of polypeptides, also in the form of non-toxic, but unspecified salts. In all these methods according to the prior art, at least as far as in the case of DE-OS 3,835,099 also demonstrated by concrete embodiments - first the biodegradable polymer or copolymer used as a carrier is dissolved in an organic solvent; sometimes the drug itself is also dissolved. In such cases, if the dispersion of the active ingredient in the biodegradable polymer is satisfactory, there remains the problem that traces of the solvent will be left behind, which may jeopardize the use of such compositions in therapeutic applications. The selection of only low-toxicity solvents or the complete removal of traces of the solvent present can sometimes be difficult and expensive; furthermore, an unacceptable reduction in the purity of the product may also occur. In contrast, according to the invention, the use of such solvents is avoided for the use of certain peptide salts not mentioned above in connection with the special support material provided according to the invention, namely for embonates, tannates, stearates or palmitates.

It has also been proposed to mix dry a protein (bovine serum albumin, BSA) and a biodegradable copolymer of lactic acid and glycolic acid in powder form, ie to mix without the use of a solvent, and then to densify the mixture at the melting temperature, see JD Grosser and Koll "Biopolymeric Controlled Release System, Volume Π, page 136. However, this method has proved to be unsatisfactory, in particular succeeds so no homogeneous distribution of the protein (BSA) within the total product, and accordingly, no uniform release of the active ingredient is guaranteed.

US Pat. No. 3,978,203 discloses that it relates to implantable solid materials or articles, which are produced by means of two different processes, namely sintering or mulling. The claimed there composition is for active ingredients, such as ζ. B. the hydrocortisone used there according to examples quite so produced, but this is true in no case, if present as active ingredients peptides. In any case, it is completely impossible to describe the above-mentioned first route of sintering for peptides, since they would be destroyed by the temperature of 150-200 ° C. used for this purpose. As for the second way of "mulling", this method disclosed in Examples 4, 5, 7 and 8 of the US patent is not suitable for the task according to the invention and its solution since this technique is incapable of producing a compact product as it is needed to deliver. When this technique is applied to peptides, only the formation of a porous product can be achieved, which then does not have the properties of ensuring a continuous and regular release of the active ingredient.

Also, the subject matter of US Pat. No. 4,481,353 has little to do with the present invention, as it relates to new polyesters which are useful for making surgical articles such as sutures, ligatures, and microchannels for neural pathways. The demands made on such articles and problems, especially as regards their mechanical properties, are completely different from the problems in the subject invention. In addition, the polyester according to this U.S. patent is composed of 3 components, one of which is a hydroxycarboxylic acid.

The invention is therefore based on the problem to provide a method for producing a pharmaceutical composition which does not have the disadvantages indicated in the prior art. In particular, the problem underlying the invention is to provide a method by which pharmaceutical compositions can be prepared in which the active ingredient is uniformly distributed in the carrier material, with the result that it is continuously and controllably reused in therapeutic use

-2AT397 198 B is released.

It has surprisingly been found that the various difficulties described in the prior art can be overcome by

a) the biodegradable polymer and, as an active ingredient, the embonate tannate, stearate or palmitate of the peptide are mixed dry, both substances being used as microparticles with an average size of less than about 500 pm;

b) the powder mixture is gradually compressed and gradually heated to about 90 ° C;

c) subjecting the precompressed and preheated mixture to extrusion at a temperature between 90 and 100 ° C and cooling the extruded product; and optionally

d) the product obtained by the extrusion is pulverized at a reduced temperature, and finally the obtained microparticles are selected and collected.

According to the invention, the natural or synthetic peptides are used in the form of their salts, as embonates (usually referred to in the American literature as pamoate), tannates, stearates or palmitates, with embonate being the particularly preferred salt form. It should be emphasized at this point that these peptide salts are water-insoluble.

Both the abovementioned salts and also the abovementioned biodegradable polyesters are used as powders, in particular as microparticles with an average size of less than 500 μm. Good results are achieved with polymer microparticles in the range of 180 pm or less, and the particle size of the peptide salt can be even lower. The mixing of these compounds is carried out by mixing in any suitable apparatus, for example a ball mill, at room temperature (about 25 ° C) or at even lower temperatures, for example in the range of 5 to 10 ° C. The proportions of the powdered ingredients may vary considerably depending on the desired therapeutic effect, for example from 0.1 to 15% by weight for the peptide salt.

In accordance with the invention, when fully homogenized, the selected mixture is subjected to staged densification and, at the same time, staged heating prior to being extruded. Both process steps, as well as the transfer of the mixture into the precompression and bulk heating zone, can advantageously be carried out using a suitably sized screw or, if necessary, two cooperating screws. The rate of compaction may vary depending on many factors, such as extruder geometry or particle size of the powdered mixture. An important factor to be monitored is preheating and advancing as the mixture progresses. Depending on the nature of the products to be treated (polyesters, peptides), care should be taken that the temperature gradient is maintained at a maximum of about 90 ° C. The initial temperature of the powdery mixture may be 25 ° C or more or less depending on the circumstances.

The thus precompressed and preheated mixture is then extruded at a temperature which is between 90 and 100 ° C, the upper limit of the specified temperature range depending on the type of peptide, which must not be affected. The extrusion can be carried out in a wide pressure range between 50 and 500 bar, wherein it is important that the extrusion temperature and the pressure are adapted to the viscosity of the product. A suitable pressure and temperature are advantageous in order to ensure a completely homogeneous mixing of the constituents and, in particular, a uniform distribution of the peptide salt in the total amount of the biodegradable polymer.

The extrusion itself is carried out using a mold of conventional shape and size, the mold being located at the downstream end of said screw. Cooling of the extruded product can be accomplished by any suitable means, for example, by simply transferring the heat to a cooled sterile gas or cooled sterile air.

When the manufacturing process is terminated after this step, a composition in the form of an implant is obtained according to the invention. Such implants are simply collected by cutting sections of predetermined length from the product after it has left the extrusion mold

The shape of the recovered implant can be varied by changing the shape of the extruder die.

According to one embodiment of the invention, the correspondingly cooled extruded product is then subjected to grinding at a reduced temperature, for example at 0 ° C., or at even lower temperatures, for example at -30 ° C. Advantageously, the cryogenic pulverization method known per se is used for this purpose. According to the method according to the invention, the product thus comminuted is then classified on the basis of the average size of its microparticles, with particles of less than 200 μm, preferably less than or equal to

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180 pm, be withheld. The selection of microparticles can be done, for example, by sieving. The selected and collected microparticles are then ready to use.

According to the method of the invention, the above-mentioned steps are carried out in succession, without delays occurring between the steps. The advantage of the method according to the invention is the fact that it can be carried out continuously, wherein all process steps are carried out successively, simply in which the mixture to be processed is transported further.

According to the invention, poly-1,4-butylene succinate is preferably used as the biodegradable polyester. These polymers can either be prepared as described in the cited literature or can be obtained commercially.

Regardless of whether they are of natural or synthetic origin, the peptides incorporated into the polymer preferably contain from 3 to 45 amino acids. Particularly preferred are the salts of LH-RH (luteinizing hormone-regulating hormone), somatostatin, GH-RH (growth hormone-regulating hormone), calcitonin and their synthetic homologues and analogues.

Particular preference is given to the emulsions of LH-RH, somatostatin and their synthetic analogs and homologs, such as ι-1

D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH, ι - 1

D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Tip-NH₂, ι ι

D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂, ι ι

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂, l-1

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH₂, ι-1

AcPhe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH₂, l

AcPhe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH₂, (pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Arg-Pro-NHR ^, (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro NHR *, (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂, and (pyro) Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH₂in which R¹ is a lower alkyl radical

The microparticles obtained from the above-mentioned components according to the method of the invention are then used after appropriate sterilization for the preparation of injectable suspensions.

The following examples illustrate the invention.

Example 1 g of poly-1, 4-butylene succinate (inherent viscosity about 0.35 in HFIP) obtained as granules having a diameter of 3 to 5 mm was first milled at reduced temperature and then sieved, using microparticles having an average size of 500 μm or were received less.

To this pulverized mixture was added 0.445 g of finely pulverized D-Trp-6-LH-RH embonate. The peptide had the following formula:

-4AT 397 198 B (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 ·

This product consists of microparticles of about 10 pm and is of amorphous structure. The resulting mixture was homogenized in a mill at room temperature.

The resulting homogenized mixture was then placed in a device having a screw co-operating with a conventional extrusion die. The snail was about 25 cm long and had a diameter of about 1.5 cm. It has a first area since^- is used to move the mixture and adjacent to a second area in which is compressed and preheated.

During the movement of the mixture is heated from 25 ° C to about 90 ° C, the

Increasing rate is chosen so that this phase lasts about 5 minutes. The Exmittierung itself takes place at 98 ° C through an extrusion die with an opening of about 1 mm in diameter.

The strands thus obtained are cooled to room temperature, then cut into small sections and finally ground at -30 ° C. After a sieving step, the microparticles are collected with an average diameter of 180 pm or smaller.

The chemical analyzes carried out on the product samples after the exiting and grinding confirm the complete homogeneity of the active agent dispersion throughout the polymer.

The microparticles obtained above were then sterilized with gamma rays and then suspended in a suitable sterile vehicle

In vivo tests (determination of blood testosterone level in lines of male rats) confirm that the release of the active ingredient is maintained for at least 25 days, which results from the collapse of the

Testosterone levels on values, as can be observed in castrated animals, can be closed.

Example 2

The procedures of Example 1 were repeated to obtain microparticles of poly-1, 4-butylene succinate 25 (inherent viscosity about 0.35), the equivalent amounts of embonate one of the following

Dekapeptides contain:

(pyro) Glu-His-Tip-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH2, (pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg Pro-NR¹, or (pyro) Glu-His-Trp-Scr-Tyr-D-Thr-Leu-Arg-Pro-NR ^, wherein Rl is an ethyl group 35

Example 3

The procedures of Example 1 were repeated to prepare microparticles of the desired particle size using as starting material 18 grams of poly-1,4-butylene succinate (inherent viscosity about 0.35) and 2.85 grams of the embonate of a somatostatin analog having the following peptide formula were:

I-1

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2

The chemical analyzes carried out on the product samples after the exiting and grinding confirm the complete homogeneity of the active agent dispersion throughout the polymer.

In vivo tests further confirm that the delivery of the active ingredient (a somatostatin analog) is sustained over a period of at least 7 days.

Example 4

The procedures of Example 3 were repeated to obtain microparticles of poly-1,4-butylene succinate with 50 equivalent amounts of the embonate of one of the following octapeptides:

D-Phc-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH, ⁵⁵ .-.

D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Trp-NH 2,

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D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2,

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2,

AcPhe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2,

AcPhe-Cys-Tyr-D-Trp-Lys-Val-Cys-Tip-NH2 ·

The chemical analyzes carried out on the product samples after extrusion and grinding confirm the complete homogeneity of the active agent dispersion throughout the copolymer.

During the experiments described above, it has been found that the extruded strands, when cut into bars of suitable length, can be used immediately after sterilization as implants. These implants also ensure a continuous and controlled release of the active substance.