NO302856B1 - Process for the preparation of long-acting, biodegradable microparticles - Google Patents

Abstract

Long-acting biodegradable microparticles containing therapeutically active peptides as active ingredient and poly(lactide-co-glycolide) as vehicle, which are obtained by spray drying at temperatures below 60 DEG C with a spray flow above 500 l(STP), are described.

Description

The present invention relates to a method for the production of long-acting biodegradable microparticles based on poly(lactide-co-glycolide) (=PLGA), which contain therapeutically active peptides, respectively. physiologically tolerable salts thereof as active ingredient.

For drugs that are broken down enzymatically in the gastrointestinal tract, such as e.g. proteins or peptides, it is next to a nasal and dermal application, with often only a reduced resorption rate, above all parenteral drug forms of importance. For the long-term treatment of specific diseases, it is desirable to develop parenteral depot drugs that release the drug continuously over several weeks. In the literature, apart from implants (cf. e.g. EP-B-0 058 481), microcapsules/microspheres are also described for this purpose.

To avoid an operative removal of this form of injection, biodegradable polymers are used. Alongside polyamides, polyanhydrides, poly(ortho)esters, polyacetals, polyester is above all also used. For the most part, suitable polymers are described as polyester based on the monomers lactic acid and glycolic acid, or their copolymers.

For the production of microcapsules/microspheres, various methods come into consideration, such as e.g. phase separation (EP-B-0 052 510), solvent extraction (EP-B-0 145 240) or spray drying (EP-A-0 315 875 = ZA patent document 88/8396, CH 666 406, A5, cf. Derwent ref. no. 88-228036/33). Compared to the other methods, spray drying has the advantage that the encapsulation efficiency is usually higher than with the other mentioned methods, and that no further auxiliaries are needed for the pre-encapsulation, which can then lead to possible contamination in the product. The disadvantage is that the spray drying must be carried out at higher temperatures, i.e. usually well above the boiling point of the applicable solvents. If you subject the suspensions, emulsions or solutions of the copolymers of lactic and glycolic acid (PLGA) to normal conditions during spray drying, you do not obtain microparticles, but instead thread-like materials that are not suitable for an injectable depot form (EP-A-0 315 875). The microcapsules which are obtained in CH 666 406 A5 by spray drying at 59° C, do not contain PLGA as biodegradable carrier material, but D, L-oligolacoyl-N-(L)-phenylalanine.

The present invention therefore relates to a method for the production of long-acting biodegradable microparticles, which contain a peptide as active ingredient and (PLGA) as carrier material, characterized by suspending or dissolving the peptide in a solution of the carrier material or emulsifying an aqueous solution of the peptide in a solution of the carrier material and sprays the suspension, solution or emulsion at a temperature below 60°C at a spray flow rate higher than 500 NL/h.

It is possible to spray PLGA polymer solutions and corresponding aqueous suspensions and emulsions into microparticles, when the spray temperature is less than 60°C and the spray flow is above 500 NL/h. This microparticle which still excels compared to microparticles produced by the other methods mentioned, by a lower residual solvent content (organic solvent, water), which is below 1%. The latter is of great importance in the case of injection forms, due to the fact that here greater demands are placed on the purity of the medicine.

The microparticle that is produced contains a peptide as active ingredient and poly(lactide-co-glycolide) (PLGA) as carrier material. They are obtained by spray drying at temperatures below 60°C at a spray flow of over 500 NL/h.

(1 NL = 22.415 litres).

In the above and following embodiments, microparticles also mean microcapsules and microspheres, and also parts where the active ingredient is completely or partially enclosed by polymer, and also parts where the active ingredient is distributed in a PLGA matrix.

Under peptides are understood natural and synthetic peptides as physiologically tolerable salts thereof.

The microparticle produced according to the invention preferably contains water-soluble peptides. The molecular weight of the peptides is preferably above 800. Particularly suitable peptides are LHRH agonists and antagonists, such as Buserelin, HOE 013 (Ac-D-Nal-p-Cl-Phe-D-Trp-Ser-Tyr-D-Ser (α-L-Rha)-Leu-Arg-Pro-Azagly-NH2, cf. EP-A- 0 263 521), nafarelin, triptorelin, leuprorelin and goserelin. Peptides of other types are also suitable, such as e.g. THR, vasopressin, calcitonin, insulin or HOE 427 (=Ebiratide, [4-methionine dioxide, 8-D-lysine, 9-phenylamine]-α-MSH-(4-9)-(8-aminooctyl )-amide- triacetate, cf. EP-A-0 179 332).

The biodegradable carrier material is PLGA. The active substance release from the microparticles is not only influenced by the physico-chemical properties of the active substance, but also by the properties of the polymers or polymer mixtures such as the molecular weight, molar composition, due to the lactic acid and glycolic acid purity in the polymer. The carrier material can e.g. consist of the mixture PLGA 50:50 and PLGA 40:60. The higher the molecular weight, or the inherent viscosity is, the longer the active substance-release-no takes place. The inherent viscosity (20°C, chloroform, 0.1$) is preferably 0.1 to 0.8 dl/g. The molar ratio between lactide and glycolide units is in the range of from 85:15 to 40:60, and preferably poly(d,l-lactide-co-glycolide) 50:50 is used, and which exhibits a viscosity of 0.1 to 0.7 dl/g, especially 0.1 to 0.5 dl/g.

Depending on the properties of the polymer, the active substance is released from the microparticle produced according to the method according to the invention, over a period of preferably 2 weeks to 3 months. The degree of loading likewise has an, if also subordinate, effect on the duration of the active substance release. With the method according to the invention, microparticles are preferably obtained with a peptide load of less than 20%, preferably less than 12%.

The particle size, which affects the release, in the microparticles produced according to the method according to the invention is less than 200 pm, preferably less than 100 pm. The particle size is especially less than 50 pm.

Production of the microparticle takes place by spraying from a solution, emulsion or suspension of the active substance in the polymer solution. The solvent can be added, for example, as chloroform, methylene chloride, DMF, acetone, acetic acid ethyl ester, glacial acetic acid and water or mixtures thereof. Methylene chloride, glacial acetic acid and water are preferably used.

In the method according to the invention, the peptide is preferably dissolved in water, and for dissolving PLGA in e.g. methylene chloride. The resulting emulsion is preferably sprayed. The spraying can also take place after adding e.g. methanol, and here a solution then occurs.

The spray temperature is preferably at 50°C to 30°C, especially at 40° to 30°C. The spray flow must not be too low, as this may otherwise lead to thread formation. It is preferably available at 800 NL/h, as higher spray flow is associated with better drying properties.

The pressure of the spray medium, e.g. air or nitrogen, when using a laboratory spray device (e.g. Biichi Mini Spray Dryer 190) must be in the range 3-8 bar. The pump operation of the spray equipment must be adapted to the situation (3-20 ml/min.) - By correspondingly setting the aspirator's e.g. 18 scale units, it is made possible that all air, resp. nitrogen in the spray medium is removed.

The PLGA polymer should be dissolved in as little solvent as possible, since the polymers with a higher proportion of lactide need a higher amount of solvent. In principle, the concentration of the polymers in the solution, emulsion, suspension to be sprayed is chosen so that spraying is possible under the chosen conditions. Preferably, the PLGA concentration in methylene chloride is less than 15% by weight calculated on methylene chloride.

The invention is now explained using the following examples.

The viscosity stated for PLGA in the examples represents the inherent viscosity of a 0.1% solution in chloroform at 20°C, determined according to the generally accepted methods.

In all the examples, the spray parameters a) aspirator and b) pump capacity are set to 18 (for aspirator) or 6 (for pumping capacity) scale divisions.

EXAMPLE 1: Spray medium - glacial acetic acid

a) 0.064 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is mixed with a solution of

1.936 g PLGA 50:50 (IV = 0.4 dl/g) in 40 g glacial acetic acid and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. One-time temperature 50°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield 1.2 g = 6056 of theory.

Active ingredient content (degree of strain): 356 Residual solvent content: Glacial acetic acid 0.856

b) In a similar manner, microparticles are prepared using 0.192 g of buserelin acetate and 1.808 g of PLGA 50:50

(IV=0.4 dl/g).

Yield 1.2 g = 6056 of theory.

Active ingredient content: 956 Residual solvent content: Glacial acetic acid 0.856

For 20 mg microparticles, the in vitro release rates are determined as follows; 20 mg of microparticles are placed in 2.0 ml of a phosphate buffer solution pH 7.4 and eluted for 24 h at +37° C. After separation of the eluates from the microparticles, the buserelin content in the eluate is determined by means of an HPLC -approach. The retained microparticles are resuspended in 2.0 ml of phosphate buffer and eluted for 24 h at 37 °C. This process is carried out analogously over 56 days.

The amounts of buserelin acetate determined from the eluates are averaged in the following tables:

EXAMPLE 2: Spray medium - chloroform

a) 0.108 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1,892 g PLGA 50:50 (IV = 0.4 dl/g) in 92 g chloroform and sprayed in a laboratory spray dryer.

The spray parameter is set as follows:

1. Inlet temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.9 g = 4556 of theory.

Active substance content: 556.

b) In a similar way, microparticles are produced using 0.152 g of buserelin acetate and in 1.848 g of PLGA 50:50

(IV = 0.4 dl/g).

Yield: 1 g = 5056 of theory.

Active substance content: 756.

For 20 mg microparticles, the in vitro release rates were determined analogously to example 1 over 35 days.

The buserelin acetate amounts determined from the eluates are compiled in the following table:

EXAMPLE 3: Spray medium - methylene chloride/ water/ methanol

a) 0.128 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is mixed with a solution of 1.872

g PLGA 50:50 (IV = 0.4 dl/g) In 46 g methylene chloride. 9 g of methanol are added to the mixture. A clear solution is produced which is sprayed in a laboratory spray dryer.

The spray parameter is set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 1.2 g = 6056 of theory.

Active substance content: 656

Residual solution content: Water 0.956

Methylene chloride 0.0656

Methanol <0.0156

For 20 mg microparticle, the in vitro release rates were determined analogously to Example 1 over 28 days:

The amounts of buserelin acetate determined from the eluates are listed in the following table:

EXAMPLE 4: Spray medium - methylene chloride/ water/ methanol

a) 0.084 g of buserelin acetate is dissolved in 4.0 g of water. The aqueous solution is mixed with a solution of 1.916

g PLGA 50:50 (IV = 0.42 dl/g) in 65 ml methylene chloride. 25 g of methanol are added to the mixture. A clear solution is produced which is sprayed in a laboratory spray dryer.

The spray parameter is set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.8 g = 40 56 of theory.

Active substance content: 456

b) In a similar way, microparticles are produced using 0.168 g of buserelin acetate and 1.832 g of PLGA 50:50

(IV = 0.42 dl/g).

Yield: 0.8 g = 4056 of theory.

Active substance content: 856

For 20 mg of microparticle, the in vitro release rate was determined as follows: 20 mg of microparticle is added to 2.0 ml of a phosphate buffer solution pH 7.4 and eluted for 24 hours at +37°C. After separation of the eluates from the microparticles, the buserelin content in the eluates is determined by means of a radioimmunoassay (RIA). The remaining microparticles are taken up again in 2.0 ml of phosphate buffer and further eluted for 24 hours at 37°C.

This process takes place over 63 days. The amounts of buserelin acetate determined from the eluates are compiled in the following table:

EXAMPLE 5: Spray medium - chloroform/ water/ dimethylformamide

a) 0.13 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.87 g PLGA 50:50 (IV = 0.4 dl/g) in 41.4 g chloroform and 4.6 g dimethylformamide and sprayed in a laboratory spray dryer.

The spray parameter is set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 1.2 g = 6056 of theory.

Active substance content: 656.

For 20 mg microparticles, the in vitro release rate was determined analogously to example 4 during 63 days:

The amounts of buserelin acetate determined from the eluates are given in the following tables:

EXAMPLE 6: Spray medium - methylene chloride

a) 0.085 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.915 g PLGA 50:50 (IV = 0.4 dl/g) in 46 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameter is set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.6 g = 3056 of theory.

Active substance content: 456.

b) In the same way, the microparticles are prepared using 0.128 g of buserelin acetate and 1.872 g of PLGA 50:50 (IV =

0.4 dl/g).

Yield: 0.7 g = 3556 of theory.

Active substance content: 656

c) as well as using 0.170 g buserelin acetate and 1.872 g PLGA 50:50 (IV = 0.4 dl/g).

Yield: 0.4 g = 20% of theory.

Active substance content: 856

For 20 mg of microparticle, the in vitro release rate was determined analogously to Example 4 over 63 days.

The amount of buserelin acetate determined from the eluates is listed in the following table:

EXAMPLE 7: Spray medium - methyl chloride/water

0.032 g of buserelin acetate is dissolved in 1.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer into a solution of 0.968 g PLGA 85:15 in 23 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 40°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.7 g = 7056 of theory.

Active ingredient content: 356

EXAMPLE 8: S<p>ra<y>medium - metvlene chloride/ water

0.064 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.936 g PLGA 50:50 (IV = 0.7 dl/g) in 92 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 40°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.9 g = 4556 of theory.

Active ingredient content: 356

Determination of the active substance release analogously to example 1

EXAMPLE 9: Spray medium - methylene chloride/ water

0.106 g of buserelin acetate is dissolved in 1.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer into a solution of 0.894 g of PLGA 50:50

(IV = 0.1 dl/g) in 23 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 40°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.3 g = 3056 of theory.

Active substance content: 1056

Determination of the active substance release analogously to example 1

EXAMPLE 10: Spray medium - methylene chloride/ water

0.13 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.87 g PLGA 75:20 (IV = 0.5 dl/g) in 46 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 1.2 g = 6056 of theory.

Active substance content: 656

EXAMPLE 11: Spray medium - methylene chloride/ water

0.128 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.872 g PLGA 75:25 (IV = 0.8 dl/g) in 138 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.9 g = 4556 of theory.

Active substance content: 696

EXAMPLE 12: Spray medium - methylene chloride/ water

0.13 g of buserelin acetate is dissolved in 2.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 1.87 g PLGA 47:53 (IV = 0.3 dl/g) in 46 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 1.3 g = 6556 of theory.

Active substance content: 656

EXAMPLE 13: Spray medium - methylene chloride

0.13 g of buserelin acetate is suspended in a solution of 1.87 g of PLGA 50:50 (IV = 0.4 dl/g) and 46 g of methylene chloride using a rotor-stator homogenizer.

The suspension is sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 40°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.5 g = 2556 of theory.

Active ingredient content: 6%

EXAMPLE 14: S<p>ra<y>medium - methylene chloride/ water

a) 0.2 g of Hoe 427 is dissolved in 1.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 3.8 g PLGA 50:50 (IV = 0.4 dl/g) in 92 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 40°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 1.9 g = 4856 of theory.

Active substance content: 556

b) In a similar manner, microparticles are prepared using 0.6 g of Hoe 427 and 3.4 g of PLGA 50:50 (IV = 0.4

dl/g).

Yield 2.8 g=7056 of theory.

Active substance content: 1556

c) as well as using 0.4 g Hoe 427 and 3.6 g PLGA 50:50 (IV = 0.4 dl/g).

Yield: 2 g = 50$ of theory.

Active ingredient content: 10%

EXAMPLE 15: Spray medium - methylene chloride/ water

0.08 g of Hoe 013 is dissolved in 1.0 g of water. The aqueous solution is emulsified using a rotor-stator homogenizer in a solution of 0.92 g PLGA 50:50 (IV = 0.4 dl/g) in 23 g methylene chloride and sprayed in a laboratory spray dryer.

The spray parameters are set as follows:

1. Entry temperature 30°C

2. Spray flow 800 NL/h

The microparticles appear as white, fine powder.

Yield: 0.5 g = 50% of theory.

Active ingredient content: b%

Determination of the active substance release analogously to example 1

Claims (8)

1. Process for the production of long-acting biodegradable microparticles, which contain a peptide as active ingredient and (PLGA) as carrier material, characterized by suspending or dissolving the peptide in a solution of the carrier material or emulsifying an aqueous solution of the peptide in a solution of the carrier material and spraying the suspension, the solution or the emulsion at a temperature below 60°C at a spray flow rate higher than 500 NL/h. 2. Method according to claim 1, characterized in that the peptide is an LHRH agonist or LHRH antagonist. 3. Method according to claim 1, characterized in that the carrier material is PLGA 50:50. 4. Method according to claim 1, characterized in that the inherent viscosity of PLGA is 0.1 to 0.5 dl/g (20°C, chloroform, 0.1%). 5. Method according to claim 1, characterized in that the solvent is chloroform, methylene chloride, glacial acetic acid, methanol, dimethylformamide or water, or a mixture of at least 2 of the mentioned solvents. 6. Method according to claim 1, characterized in that the spray flow amounts to approximately 800 NL/h. 7. Method according to claim 1, characterized in that the peptide is buserelin acetate or HOE 013 8. Method according to claim 1, characterized in that the solvent is methylene chloride, glacial acetic acid or water, or a mixture of 2 of the mentioned solvents.