EP1682089A2

EP1682089A2 - Method for producing multiple emulsions that are stable in storage

Info

Publication number: EP1682089A2
Application number: EP04791035A
Authority: EP
Inventors: Olaf Stange; Martina Mutter; Tanja Oswald; Mark Schmitz
Original assignee: Bayer Technology Services GmbH
Current assignee: Bayer Intellectual Property GmbH
Priority date: 2003-11-05
Filing date: 2004-10-29
Publication date: 2006-07-26
Also published as: WO2005046635A3; WO2005046635A2; RU2367411C2; JP4785746B2; RU2006119296A; AU2004289048A1; BRPI0415717A; US7943154B2; DE10351644A1; JP2007509666A; AU2004289048B2; CN1901883A; US20070253986A1; CN100496462C

Abstract

The invention relates to a method for producing multiple emulsions of the water-oil-water type (W/O/W) that contain one or more active ingredients and are stable in storage. The method comprises the following steps: a) mixing of the active ingredient into an aqueous phase; b) emulsification of the aqueous phase by introducing said phase into an oil phase through a coarse-pored, porous membrane; c) phase inversion of the emulsion obtained in step b) by cooling the mixture at a cooling rate of at least 0.3 K/min and adding an emulsifier to either the aqueous phase in step a) or the oil phase in step b), or to both phases.

Description

Process for the preparation of storage-stable multiple emulsions

The present invention relates to a process for preparing a stable water / oil / water (WOW) type multiple emulsion wherein the outer and inner aqueous phases may contain a pharmaceutically active agent and the oily phase thereof comprises a nonionic surfactant acts as an emulsifier. This multiple emulsion is prepared by introducing the aqueous phase through a porous membrane into the oil phase and then cooling the water-in-oil (W / O) emulsion to form the above W / O / W double emulsion, especially for veterinary purposes is used.

Multiple emulsions are emulsions of emulsions that can be present in two main types, namely W / O / W and O / W / O emulsions. These systems are of great interest in many applications. They allow on the one hand the protection of the active substances in the innermost phase, on the other hand, the introduction of two reacting active substances in two phases of the same formulation. In addition, prolonged efficacy of the drugs in the innermost phase following administration to organisms can be observed.

Today, however, there are only a few multiple emulsions in medicine and cosmetics because their formulation and stability are still a problem and the phenomena of emulsification are still not fully understood. Bases are presented in "Multiple W / O / W Emulsions, theoretical study" Terrisse, L., M. Seiller, et al., (1992) Congr. Int., Technol. Pharm., 6 *

4: 328-35.

In veterinary medicine, vaccines are needed to immunize against one or more pathogens in which the active ingredient is distributed as finely as possible in the vaccine and, in particular, spreads over a prolonged period of time in the animal.

Multiple emulsions are an interesting emulsion system for use in the vaccine formulation. They are emulsions that contain, in the innermost phase, smaller drops of a liquid that corresponds to the continuous outermost phase. One way of formulating such a vaccine is the multiple W / O / W emulsion. In this case, for example, the active ingredient is in the form of a non-soluble protein of a certain particle size in the aqueous innermost phase. The additional oil phase delays absorption into the body, thus prolonging its effectiveness. In addition, the oil acts as an adjuvant, possibly enhancing the antigenicity of the vaccine. The formulation in the form of multiple emulsions is also known from the field of cosmetics. In the published patent application DE 196 30 176 AI, the composition of the ingredients and the preparation of such double emulsions using the phase inversion are described.

The patent specification US-6251 407 Bl describes a composition of oil, emulsifier, aqueous phase and pharmaceutical active substance, which can be used for the production of a vaccine. In it, the individual components can be described in more detail as follows: The oil used is a so-called self-emulsifying oil, which consists of polyglycolized glycerides. The aqueous phase contains an active ingredient which is an antigen.

This double emulsion according to EP-A-489 181 or EP-A-481 982 is prepared by "stirring" the aqueous active ingredient-containing into an oily phase and subsequent phase inversion to form a double emulsion by temperature gradients and subsequent homogenization by stirring.

The disadvantage of the W / O / W emulsion, which results here as in the abovementioned case, is the inhomogeneous, broad droplet distribution and the poor reproducibility of the droplet distribution in the finished emulsion with different stirred tank geometries. Furthermore, this method is possible only in discontinuous operation.

In addition, methods to produce multiple emulsions with membranes are already known. The reference Joscelyne, S.M. and G. Tragardh (2000). "Membrane emulsification - a literature review" J. Membr, 169 (1): 107-107 describes this in detail, in which an aqueous phase, for example, is introduced into one of the phases through a membrane Restrictions result from the limitation of droplet size through the radius of the membrane pore, but it has been found that the diameter of the membrane pore adversely affects the incorporation of an active agent into the emulsion when the drug particle is too large.

The patent application EP 564 738 AI a membrane process for the preparation of emulsions, in which a first emulsion by membrane emulsification, the generation of the double emulsion by stirring, or both are caused by membrane emulsification. The process serves to produce by steps a low-fat spread in the form of a double emulsion, which is characterized by saving stabilizers and gelling agents. The final emulsion droplets have a mean diameter of 10 to 16 μm. All these procedures are tailored to one area of application and therefore can not simply be transferred to other areas of use.

The invention is based on the object to form a stable double emulsion of the type 1) W / O / W, wherein the outer and / or the inner aqueous phase, a pharmaceutical agent, e.g. in the form of an antigen, and whose oily phase contains a surfactant which acts as an emulsifier.

The droplet size of the oil phase should in particular not exceed the mean diameter of 3 .mu.m, that of the inner aqueous phase of 0.3 .mu.m. In addition, the method should in particular have a narrower droplet size distribution and, associated therewith, a higher reproducibility than conventional methods.

In addition, there is the problem that the heterogeneous and partially agglomerated drug particles are not limited by their size, e.g. separated by the membrane used or destroyed by shearing.

It has surprisingly been found, and therein is the solution to the problem that the above-mentioned, storage-stable multiple emulsions of the type W / O / W can be formed by the following process, which is the subject of the invention:

a) stirring the active ingredient into an aqueous phase,

b) emulsifying the aqueous phase by introducing the aqueous phase through a coarsely porous, porous membrane into an oil phase,

c) phase inversion of the emulsion of b), by cooling the mixture at a cooling rate of at least 0.3 K / min, preferably at least 1 K / min, wherein either the aqueous phase in a) or the oil phase in b) or both phases Emulsifier is added.

The formation of the simple emulsion occurs e.g. according to the basically known stirring process. The droplet size (number average) is typically between 10 to 30 microns, the said phase inversion temperature usually 60 to 90 ° C.

Porous inorganic membranes, particularly preferably ceramic membranes, in particular of Al ₂ O ₃ , ZrO 2, TiO 2 and mixtures of these oxides, particularly preferably of Al ₂ O ₃ , are preferably used for the process according to the invention.

The pore size of the membrane is on average preferably 0.2 μm to 5 μm, particularly preferably Suitable ingredients for the continuous phase are, for example, generally oils, for the discontinuous phase, for example, aqueous solutions, in particular with the continuous phase immiscible liquids. The oil phase used is preferably mineral oil, white oil or vegetable oil.

Another essential component is the emulsifier which, depending on the composition, is initially introduced in the aqueous a) and / or oil-containing phase b); in the preferred process it is present as nonionic emulsifier in the oil phase b).

The process is particularly preferably carried out in a temperature range with respect to the emulsification after step a) from 30 ° C to 35 ° C and with respect to the phase inversion a temperature gradient of 3 OK but at least 15K.

It turns out that with the method by an appropriate selection of the starting materials and operating conditions, an extremely narrow droplet size distribution and a mean droplet diameter (oil phase) of 1 .mu.m to 3 .mu.m can be achieved.

The active ingredient introduced in the aqueous phase, that is to say in particular discontinuous phase, can be, for example, a pharmaceutical active substance, preferably for veterinary purposes, in particular an antigen for a vaccine formulation.

Preferably, the active ingredient is selected from the series:

An antigen, such as a virus, a microorganism, more specifically a bacterium or parasite, or a preparation containing a peptide chain. This preparation may be a protein or a glycoprotein, especially a protein or a glycoprotein obtained from a microorganism, a synthetic peptide or a rotein or peptides produced by genetic manipulation.

The above-mentioned virus and / or microorganism may be completely inactivated, live or attenuated.

As a virus, which represents an antigen, may be preferably named: rabies virus, Aujeszky viruses, influenza viruses, the virus of foot-and-mouth disease and HTV viruses.

As a microorganism or a bacterial species which is an antigen, there may be mentioned preferably E.coli and those of the strains Pasteurella, Furonculosis, Vibriosis, Staphylococcus and Streptococcus. As parasites may be mentioned preferably those of the strains Trypanosoma, Plasmodium and Leishmania.

The pressure difference over the membrane (transmembrane pressure) is preferably 0.5 * 10 ⁵ Pa to 25 * 10 ⁵ Pa, but preferably from 0.5 * 10 ⁵ Pa to 5 * 10 ⁵ Pa tinuous depending on the drug concentration in the aqueous phase discontinuously 5 ,

The process can in principle be operated continuously or in batch mode.

Preferably, the process is carried out continuously in all steps.

The overflow of the continuous phase is preferably between 0.5 and 5 m / s, more preferably between 1 and 3 m / s. The disperse phase flux of the discontinuous phase through the membrane is in particular from 50 to 1500 L / (m ² * h), preferably from 800 to 1200 L / (m ² * h).

The discontinuous aqueous phase on which the invention is based preferably consists of an electrolyte, which is preferably a combination of weak acids and weak bases, weak acids and strong bases or strong acids and weak bases.

The electrolytes particularly preferably contain one or more of the following compounds:

Boric acid, phosphoric acid, N-2- (acetamido) -2-aminoethanesulfonic acid, N-2- (acetamido) -2-iminodiacetic acid, alanine, 2-amino-2-methyl-1,3-propanediol, ammonia, N , N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, N, N-bis (2-hydroxyethyl) glycine, 2,2-bis (hydroxyethyl) -iminotris (hydroxymethyl) methane, 2- (cyclohexylamino) ethanesulfonic acid, 3 - [4- (2-hydroxyethyl) 1-piperazinyl] propane sulfonic acid, histidine, imidazole, lactic acid, 2-mohinoethanesulfonic acid, 2-morpholinopro-

Pansulfonic acid, piperazine 1,4-bis (2-ethanesulfonic acid), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid, N- [tris (hydroxymethyl) methyl] glycine, triethanolamine, tris (hydroxymethyl ) - aminomethane, citric acid.

The method can basically be operated sterile.

The method can also be modified such that several different discontinuous aqueous phases with different ingredients are added via several different membranes at different points of the oil phase. This is particularly advantageous if the individual active substances have a mutual incompatibility or reactivity with one another.

An advantageous side effect of the process is the use of the membrane as an emulsifying membrane

^ f \ onf rio * <= »ínn πnr. Λ fiΗ'ff »! τιιr T7-ιl-h * «a + ι. * vn on rt ^ -r onHaron Qo-i + i- * me Λιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιν unwanted agglomerates, impurities or too large particles of active ingredient, which have a larger diameter than the pores of the membrane and can affect the quality of the desired product.

The separation of above-mentioned undesired secondary components can be realized in one process step, as well as in a multi-stage process.

Membrane filtration following membrane emulsification may also serve for product concentration and / or desalination.

The described invention is particularly useful in animal health for the formulation of vaccines as described above, as well as for the formulation of pharmaceutical agents in human medicine, which are characterized by a favorable dosage form and adaptable controlled release properties.

The following examples relate to advantageous erfmdungsgemäße embodiments. The numerical values always mean percentages by weight, based on the total weight of the preparation, unless expressly stated otherwise.

In the figures show:

Fig. 1 Schematic representation of the experimental plant used in the example

Fig. 2 Schpma a multi-stage system for carrying out the method.

Examples

example 1

Material:

A. 50mM HEPES buffer pH 8.32 50.00%

B. Montanide ISA 206 49.95%

C. triethylamine (TEA) 0.05%

The following devices were used:

2 glass jars 1.2 to 2L each

Peristaltic pump 3 Verder SF1500 HPLC pump 4

Membrane module 5

Membrane 6 Inocermic pore size 1.0 μm

3-way valve 7

Hoses, hose connections Heat exchanger 8

Product container 9, glass jar 2L

The following equipment according to the scheme of FIG. 1 was used on a laboratory scale:

The oil phase 1 containing the TEA and the aqueous phase 2 consisting of HEPES buffer are heated to 33 ° C. After reaching this temperature, the two phases are brought together via a ceramic membrane 6, which is located in a module called housing 5, with a pore diameter of 1.0 micron with each other and so long promoted by the pump 3 and 4 in the circulation the entire amount of aqueous phase is combined with the oil phase. After the so-called membrane emulsification, the resulting W / O emulsion is passed through a valve 7 via a heat exchanger 8 and cooled there to 4 ° C, wherein the phase inversion takes place. The cooling rate is 2.5 K / min. The resulting multiple W / O / W emulsion is collected in the product container 9.

It has droplets with a droplet diameter of 2.3 microns.

The complete experimental parameters can be found in Table 1.

Figure 2 shows a modified plant for the introduction of a variety n of active ingredients. In this case, a continuous phase 1 is passed through a plurality of membrane modules 5 via a pump 3 in succession. This phase can be tempered. Once the desired temperature has been attained, different, likewise temperature-controllable, discontinuous phases 2, 7, 9, n with different active substances and electrolytes through the membranes 6, 8, 10, n + 1, which can differ in terms of material and pore size , emulsified. Via the valve 11, the W / O emulsion formed can be passed through a heat exchanger 12 and tempered there again to induce phase inversion. The resulting emulsion can be collected in product container 13.

Example 2

Material:

A. 50mM HEPES buffer pH 8.32 48.50%

B. MKS concentrate (monovalent) 1.50%

C. Montanide ISA 206 49.95%

D. Triethylamine (TEA) 0.05%

The following devices were used:

See Example 1

Membrane 6 Inocermic pore size 1.0 μm

The aqueous phase consisting of HEPES buffer containing the MKS concentrate and the oil phase containing the TEA are heated to 33 ° C. Upon reaching the 33 ° C, the two phases are connected via a ceramic membrane with a pore diameter of 1.0 micron with each other and until the entire amount of aqueous phase is combined with the oil phase in the circulation. The resulting W / O emulsion is cooled by a heat exchanger to 4 ° C, wherein the phase inversion takes place. The cooling rate is 2.5 K / min. The resulting multiple W / O / W emulsion has droplets with a droplet diameter of 2.0 microns. Other experimental parameters correspond to Example 1 and Table 1.

Example 3

Material:

A. 50mM HEPES buffer pH 8.32 45.50%

B. MKS concentrate (trivalent) 4.50%

C. Montanide ISA 206 49.95% The following devices were used:

See Example 1

Membrane 6 Inocermic pore size 3.0 μm

The aqueous phase consisting of HEPES buffer containing the MKS concentrate and the oil phase containing the TEA are heated to 33 ° C. Upon reaching 33 ° C, the two phases are connected via a ceramic membrane with a pore diameter of 3 microns with each other and while still circulated until the entire amount of aqueous phase is combined with the oil phase. The resulting W / O emulsion is cooled by a heat exchanger to 4 ° C, wherein the phase inversion takes place. The cooling rate is 2.3 K / min. The resulting multiple W / O / W emulsion has droplets with a droplet diameter of 2.0 microns.

The complete experimental parameters can be found in Table 1.

Example 4

Material:

A. 50mM HEPES buffer pH 8.32 45.50% B. MKS concentrate (monovalent) 4.50%

C. Montanide ISA 206 49.95%

D. Triethylamine (TEA) 0.05%

The following devices were used:

See Example 1 Membrane 6 Inocermic pore size 3.0 μm

The entire experimental unit was previously steam-sterilized at 121 ° C for 30 min, and the experiment was run under absolute sterile conditions. The further course of the experiment corresponds to Example 3. The cooling rate is 1.4 K / min. The membrane used has a pore diameter of 3 microns. The resulting multiple W / O / W emulsion has droplets with a droplet diameter of 2.0 microns. Subsequent injection into the animal gave an efficacy of 100% based on the efficacy of the vaccine prepared by the conventional method.

The complete experimental parameters can be found in Table 1. Table 1: Experimental parameters of Examples 1 to 4

Claims

claims

1. A process for the preparation of storage-stable, multiple emulsions of the water / oil / water type (W / O / W) containing one or more active substances with the steps a) stirring the active ingredient into an aqueous phase, b) emulsifying the aqueous phase by passing the aqueous phase through a coarsely porous, porous membrane into an oil phase; c) phase inversion of the emulsion from b) by cooling the mixture at a cooling rate of at least 0.3 K / min using either the aqueous phase in a) or the Olphase in b) or both phases, an emulsifier is added.

2. The method according to claim 1, characterized in that as the membrane porous inorganic membram, preferably ceramic membrane, more preferably membranes of alumina, zirconia or titanium oxide, preferably of alumina are used.

3. Process according to Claims 1 to 2, characterized in that the pore size of the membrane used is 0.2 to 5 μm, preferably 0.3 to 3 μm.

4. The method according to any one of claims 1 to 3, characterized in that a substance selected from the series mineral oil, white oil or vegetable oil is used as the oil for the oil phase.

5. The method according to any one of claims 1 to 4, characterized in that a nonionic emulsifier is used as the emulsifier, which is presented in the oil phase.

6. The method according to any one of claims 1 to 5, characterized in that the emulsification cation in step a) at a temperature of 30 to 35 ° C is performed.

7. The method according to any one of claims 1 to 6, characterized in that the phase inversion according to step c) is carried out at a cooling rate of at least 1 K / min.

8. The method according to any one of claims 1 to 7, characterized in that the pressure difference across the membrane is 0.5 * 10 ⁵ Pa to 25 * 10 ⁵ Pa, preferably 0.15 * 10 ⁵ Pa to 5 * 10 ⁵ Pa.

9. The method according to any one of claims 1 to 8, characterized in that the method

10. The method according to any one of claims 1 to 9, characterized in that the active ingredient is a pharmaceutical agent, preferably a pharmaceutical agent for veterinary purposes, more preferably an antigen for a vaccine formulation.

11. The method according to claim 10, characterized in that the active ingredient is selected from the series comprising an antigen, preferably a virus or a microorganism, in particular a bacterium or parasite, or a preparation containing a peptide chain, preferably a protein or a glycoprotein more preferably, a protein or glycoprotein obtained from a microorganism, a synthetic peptide or a protein or peptides prepared by genetic manipulation.

12. Multiple emulsion of the type W / O / W obtainable from a process according to one of claims 1 to 11.

13. Use of the emulsion according to claim 12 as a vaccine for human or veterinary medical purposes.