A method for preparing a sustained-release pharmaceutical peptide composition

(42) Date of commencement of the patent: 15. 7.1993 (45) Date of issue: 25. 2.1994

<td>(30) Priority:</td><td>(73) Patent owner:</td>

<td>28. 7.1989 CH 2829/89.</td><td>DEBIOPHARM SA CH-1006 LAUSANNE (CH).</td>

<td>(56) Documents:</td><td></td>

<td>GB-A-2209937 EP-A2-0211267 US-PS 3976071 US-PS 3978203</td><td></td>

CQ

AT 397 197 (54) PROCESS FOR PREPARING PHARMACEUTICAL PREPARATION IN THE MICROPARTICLE FORM

FOR THE PREPARATION OF INJECTABLE SUSPENSIONS FOR THE DELAYED AND CONTROLLED DELIVERY OF A PEPTIDE (57) A process is disclosed for the preparation of a pharmaceutical preparation in the form of microparticles for the sustained and controlled delivery of a natural or synthetic peptide using a copolymer of lactic acid and glycolic acid and the active Peptides as a pamoate, Tannat, Stearate or palmitate, which microparticles are used for the preparation of injectable suspensions, stated which is characterized that he

a) the copolymer of lactic acid and glycolic acid and the peptide salt, both used in the form of microparameters having an average particle size of less than about 200 microns, dry mixed together;

b) the resulting powder mixture is progressively compressed and progressively increased to about 80 ⁰ Heated C;

c) the precompressed and preheated mixture at a temperature between 80 and 110 ⁰ C extruded and the extruded product cools, and

d) the extruded product at a temperature of less than 0 ⁰ C or even lower, eg at -10 ⁰ C or -30 ⁰ C, pulverized and the microparticles with a particle size below about 200 microns selects and collects.

BW 0078013

AT397 197 B

The invention relates to a process for the preparation of a pharmaceutical preparation in the form of microparticles for the preparation of injectable suspensions.

Namely, the invention relates to a process for the preparation of a pharmaceutical preparation which is directed to a sustained and controlled delivery of a medicament and is obtained in the form of microparticles of a copolymer of lactic acid and glycolic acid and which contains as active substance the pamoate, tannate, stearate or palmitate of a natural or synthetic peptide, in particular a peptide having 3 to 45 amino acids.

Various solutions have been proposed to produce preparations that provide sustained and controlled release of drugs; These solutions are based on the production of biodegradable implants, on the microencapsulation or on the production of porous, biodegradable matrices, for example in the form of microparticles of different particle size. In this connection, EP-A-0052510 (microencapsulation) or EP-A-0058481 or US-A-3 976071, which relates to the manufacture of implants or of porous, biodegradable matrices in living tissues. In all of these methods, generally, the biodegradable polymer or copolymer used as a carrier is first dissolved in an organic solvent, and then, if necessary, the drug itself. Even if the distribution of the active substance in the biodegradable mass is satisfactory, there are always problems with trace amounts of residual solvent which may interfere with the use of such formulations in therapeutic applications. The selection of solvents with a low toxicity or the thorough removal of traces of solvent can sometimes be complicated and expensive or can lead to an unacceptable loss of product purity. It is noted in US-A-3,976,071, especially Example 1, that the preparation of the solids treated materials there is substantially similar to the step of melting the polymer to form a "solid solution" at a relatively high temperature also to US-A-3 978 203, which also implantable solid materials or Articles are produced by means of two different processes, namely by sintering or by milling (mulling). The claimed there composition is for active ingredients such. B. the hydrocortisone used according to the examples, quite so producible, but in no case if there are peptides as active ingredients. It is in any case completely impossible, the o. a. The first route of sintering for peptides, since they would be destroyed by the applied temperatures of 150 - 200 ° C. As for the second way of "mulling", this method disclosed in Examples 4, 5, 7 and 8 of US-A is not suitable for the object of 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 property of ensuring a continuous and regular release of active ingredient in the form of injection suspensions to be administered microparticles.

It has also been proposed that a proteinaceous substance (bovine serum albumin) and a biodegradable copolymer of lactic acid and glycolic acid in the form of dry powders, d. H. without any solvent, to mix together and then to compress the resulting mixture at its melting temperature (J. D. Gresser et al., Biopolymeric Controlled Release System, Vol. II, page 136). This method was unsatisfactory, especially with regard to the homogeneity of the distribution of the protein substance (RS A) in the mass.

In contrast, it has now surprisingly been found that these difficulties can be overcome using the method according to the invention, if as starting materials biodegradable polymers or copolymers and certain salts of natural or synthetic peptides, such as octa, nona or decapeptides, in particular of peptides with 3rd to 45 amino acids, can be used.

Pamoates, tannates, stearates or palmitates, preferably pamoates, are used according to the invention as salts of the natural or synthetic peptides. These peptide salts are insoluble in water.

The combination of such peptide salts with copolymers of lactic acid and glycolic acid to obtain pharmaceutical preparations with delayed and controlled release of active ingredient in the form of microparticles or their further processing into injectable suspensions is known per se from British Pat. No. 2,209,937 Process the disadvantages of a solvent additive described above.

According to the invention, the above-mentioned peptide salts and the copolymers of lactic acid (L- or DX-lactic acid) and glycolic acid are used in the form of a powder in the form of microparticles having a mean particle size below about 200 pm. Good results were obtained with microparticles from the copolymer a grain size on the order of 180 pm or less, and the peptide salt may even have a smaller particle size. These substances are in a suitable device, for example in a ball mill, at room temperature (about 25 ° C) or at a lower temperature, for. B. at 5 to 10 ° C, dry mixed together The proportion of powdered components can be within a wide range

-2AT 397 197 Β

Range, for example, from 0.1 to 15 wt .-% for the peptide salt, which depends on the desired therapeutic effects.

The process of the present invention for the preparation of a pharmaceutical composition for the sustained and controlled delivery of a natural or synthetic peptide in the form of microparticles of a copolymer of lactic acid and glycolic acid using the active peptide as pamoate, tannate, stearate or palmitate which uses microparticles for the preparation of injectable suspensions are, therefore, summarized in that one

a) the copolymer of lactic acid and glycolic acid and the peptide salt, both of which are used in the form of microparticles having an average particle size of less than about 200pm, dry blended together;

b) progressively compressing the resulting powder mixture and progressively heating to about 80 ° C;

c) the precompressed and preheated mixture at a temperature between 80 and 110 ^ÖC extruded and the extruded product cools, and

d) the extruded product at a temperature of less than 0 ° C or even lower, z. At -10 ° C or -30 ° C, and selects and collects the microparticles having a particle size below about 200pm

Thus, according to the invention, once sufficiently homogenized, the particular mixture undergoes progressive compression and, at the same time, progressive heating before it is extruded. These two process steps as well as the transport of the mixture into the pre-compression and preheating zone can be carried out expediently with a correspondingly dimensioned extruder screw. The compression ratio may be affected by various factors, e.g. B. depend on the geometry of the device or on the grain size of the powdered mixture. The control or Regulation of preheating and its change in the course of mixing is more critical: depending on the nature of the products to be treated (copolymer, peptide), a temperature gradient of not more than about 80 ° C should be maintained as far as possible. The starting temperature to which the powdered mixture is exposed may, depending on the circumstances, be 25 ° C or lower or higher.

The thus precompressed and preheated mixture is then extruded at a temperature generally between 100 and 110 ° C, the upper limit of this temperature range being determined by the type of drug (peptide) which is not to be decomposed. The extrusion can be carried out at a pressure which can vary within wide limits in the range of about 50 to 500 bar, the main concern being that the extrusion temperature and the extrusion pressure be matched to the viscosity of the product. Obviously, a suitable pressure and temperature will promote perfect homogenization of the ingredients, especially the uniform distribution of the peptide salt in the bulk of the copolymer.

The extrusion is carried out in practice by means of the standard nozzle and standard dimensions located at the downstream end of the above mentioned screw. The cooling of the extruded product may be carried out in any suitable manner, e.g. As mitkalter, sterile air or gas, or simply by the natural heat loss done.

According to the invention, the sufficiently cooled extruded product is subsequently pulverized at the above-mentioned low temperatures. For example, it can employ known cryogenic pulverization. The product thus pulverized is then classified with regard to the average particle size of the microparticles, the particles having a particle size below 200 μm, preferably &lt;180 μm, being retained according to the invention.

The classification of the microparticles according to step d can be done for example by sieving. The classified microparticles are collected and ready to use.

According to the method according to the invention, the steps described above can be carried out successively without longer delays between two successive stages. An advantage of this method is that it can also be carried out continuously, with all process steps being carried out successively, simply by further transporting the treated mixture.

According to the invention, in stage a, as the copolymer of lactic acid and glycolic acid, one can use all kinds of biodegradable copolymers based on this; It is preferable to use a copolymer of L- or DT-lactic acid with 45 to 90 mol% of lactic acid units and 55 to 10 mol% of glycolic acid units. These polymers can be easily prepared according to the literature mentioned above, or they can be obtained from specialized companies.

The natural or synthetic peptide salts used in step a as active substances incorporated into the bulk of the copolymer in this way are preferably salts of peptides of 3 to 45 amino acids, in particular salts of LH-RH (luteinizing hormone-releasing hormones) of Somatostatin, GH-RH (Growth Hormone - Releasing Hormone) or calcitonin or their synthetic homologs or

-3AT 397 197 B

Analog.

In particular, according to step a, the pamoate is selected from LH-RH, from somatostatin or from one of its homologues or analogues

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

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

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

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

D-Phe-Cys-Tyr-D-Tip-Lys-Val-Cys-Trp-NH 2,

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

AcPhe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2, (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2, (pyro) Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH2, (pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHRl or (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHR¹ · (R¹ is a lower alkyl group), which is not exhaustive.

The microparticles obtained from the abovementioned constituents by the process according to the invention are then used, as already mentioned in the introduction, after suitable sterilization for the preparation of injectable suspensions

The following examples illustrate the invention.

Example 1 g of a 50:50 (mol%) copolymer of D, L-lactic acid and glycolic acid in the form of Kömem with a diameter of 3 to 5 mm were first milled and sieved at a temperature of -5 ° C, wherein microparticles with of an average grain size of 180 μm or less. To this powdered mass was added 0.49 g of finely powdered pamoate of D-Trp 1 -LH-RH (formula of the peptide:

(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2)

The product is composed of microparticles with a grain size of about 10 pm and has an amorphous structure. The resulting mixture was homogenized in a mill at room temperature.

The homogenized mixture was then loaded into a device with a screw connected to a conventional extrusion die. The screw may have a length of about 25 cm and a diameter of about 1.5 cm. It receives a first zone in which the mixture is merely moved, followed by a second zone in which the mixture is compressed and preheated.

As the mixture moves, it is heated from 25 to about 80 ° C with the migration rate adjusted to last about 5 minutes. The actual extrusion is at 98 ° C through an extrusion die having an orifice diameter of about 1.5 mm.

-4AT 397 197 B

The resulting filaments are allowed to cool to room temperature, cut into short pieces and finally ground at -30 ° C. After sieving, the microparticles are collected with an average grain size of 180 ym or less.

The chemical analysis performed on samples of the product after extrusion and milling confirmed the perfect homogeneity of the active substance dispersion in the bulk of the polymer.

The resulting microparticles were sterilized with gamma rays and then suspended in a suitable sterile vehicle. In vivo experiments (determination of blood testosterone level in strains of male rats) confirm the uniform release of the active substance over a period of at least 25 days leads to a decrease in testosterone castration concentrations

Example 2

Using the procedure of Example 1, microparticles were prepared from a 50:50 (mole%) copolymer of DJL-lactic acid and glycolic acid containing a comparable concentration of the pamoate of one of the following decapeptides:

(pyro) Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH2, (pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Leu-Arg Pro-NHRI or (pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHRI (R ^ ethyl).

The activity tests carried out in vivo confirm the uniform release of the active substance over several weeks.

Example 3

13.85 g of a 75:25 (mole%) copolymer of D, L-lactic acid and glycolic acid in the form of grains having a diameter of the order of 3 to 5 mm were first milled and sieved at low temperature to give microparticles with a average grain size of 180 ym or less were obtained.

To this powdered mass was added 1.15 g of the finely powdered pamoate of D-Trp 1 -LH-RH (Formula of the peptide:

(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2). The product consists of microparticles with a grain size of about 10 ym and has an amorphous structure. The resulting mixture was homogenized in a mill at room temperature and finally further treated as described in Example 1.

After cryopulverizing, sieving and sterilizing with gamma-rays, the microparticles were suspended in a suitable sterile diluent. In νίνο tests (determination of blood testosterone level in strains of male rats), a uniform release of the active substance was achieved over a period of at least 40 days confirmed, which led to a decrease in the testosterone castration concentrations.

Example 4

Following the procedure of Example 1, 18 g of the 50:50 (mole%) copolymer of DX-lactic acid and glycolic acid and 2.85 g of the pamoate of a somatostatin analog having the peptide formula:

D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 was used to obtain microparticles of the desired particle size.

The chemical analysis carried out with the product samples after extrusion and milling confirms the perfect homogeneity of the active substance dispersion in the bulk of the copolymer.

In vivo tests also confirmed the controlled release of the active substance (somatostatin analogue) over a period of at least seven days.

Example 5

The procedure of Example 4 was repeated by adding 13.50 g of a 75:25 copolymer of DX-lactic acid / glycolic acid and 1.50 g of the pamoate of the aforementioned somatostatin analog

-5AT 397 197 B was assumed.

The microparticles thus obtained were finally suspended in a suitable sterile diluent after one time of gamma ray sterilization. In νίνο tests (determination of the concentration of somatostatin analogue in the blood serum of rats receiving a single injection at to) show a controlled release of the active substance within a period of at least 15 days.

Example 6

The procedure of Example 4 was used to prepare microparticles from a 50:50 (mole%) copolymer of DX-lactic acid / glycolic acid containing a comparable amount of pamoate of one of the following octapeptides:

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

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

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-Trp-NH2.

The chemical analysis performed on the samples of the product after extrusion and milling confirms the perfect homogeneity of the active substance dispersion in the bulk of the copolymer.

In the above experiments, it was found that the extruded filaments could be used immediately as implants after cutting into short pieces of suitable length and after sterilization. These implants also ensure a delayed and controlled release of the active substance.