DE102018010063A1 - Preparation of Vesicular Phospholipid Gels by Screw Extrusion - Google Patents

Abstract

The present invention relates to a process for the preparation of vesicular phospholipid gels (VPG) prepared by means of screw extrusion. In particular, the invention relates to a method for producing VPG comprising at least one pharmaceutical agent, preferably a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus, virus-like particles, or low molecular weight drugs, the preparation comprising the steps of: (i) supplying at least one phospholipid, an aqueous liquid, optionally at least one pharmaceutical agent, optionally at least one further lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes and salts thereof Fatty acids and derivatives thereof in a screw extruder, (ii) homogenizing the aforementioned ingredients in a screw extruder to form a VPG, and (iii) extruding the VPG, which steps may also take place simultaneously. The invention also relates to a process for the preparation of VPGs, which is characterized in that a pharmaceutical active substance is released over a prolonged period.

Description

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of vesicular phospholipid gels (VPG) prepared by means of screw extrusion. More particularly, the invention relates to a method of producing VPG comprising a pharmaceutically active agent, preferably a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus-like particle or low molecular weight drug, the preparation comprising the steps of: (i ) Feeding one or more phospholipids, an aqueous liquid and optionally at least one pharmaceutical agent into a screw extruder, and further optionally at least one other lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, Cholesterol, waxes and salts of the fatty acids and derivatives thereof, (ii) homogenizing the aforementioned ingredients in a screw extruder to form a VPG, and (iii) extruding the VPG, which steps may also take place simultaneously. The invention also relates to a process for the preparation of VPG, which is characterized in that a pharmaceutical active ingredient is released over a prolonged period.

BACKGROUND OF THE INVENTION

Vesicular phospholipid gels (VPG) are semi-solid dosage forms for the prolonged release of proteins, peptides, nucleotides, antibodies, antibody derivatives, viruses, virus-like particles, low molecular weight drugs and other drugs. They consist of up to about 70% phospholipids, the rest make hydrophilic solutions of the drug, usually aqueous buffer solutions, and possibly other lipids. This simple and inexpensive composition, its high biocompatibility, and its extraordinary functionality with release rates of up to several weeks or months, make VPG a valuable depot dosage form.

So far, VPGs have been produced by high-pressure homogenization (HPH) or by means of a Dual Asymmetric Centrifuge (DAC), also known as Speedmixer ( Brandl et al., 1997 and Massing et al., 2008 ). Over the last few years, the DAC has developed as the standard process for the production of VPGs. More recently, as an alternative method for producing VPG, a stirring method has been proposed in which the components are stirred by means of a magnetic stirrer in a glass vessel until a dispersion is formed (Zhong et al., 2013 and Zhang et al., 2015). However, it is questionable whether VPGs of the quality previously achieved with the DAC can be obtained.

All previous manufacturing processes for VPG have various disadvantages such as a long process time (DAC), poor controllability (temperature, shear forces in HPH, DAC), and the fact that extremely high shear forces are exerted on the product (HPH, DAC). The production of VPG can only be carried out in individual batches with DAC processes in succession, since a continuous process with the devices is not feasible. In addition, scaling of batch sizes during production with the DAC is only possible to a very limited extent. With the help of the HPH, VPGs can be produced relatively quickly and in large quantities, but such high shear forces are applied, so that this method is unsuitable for sensitive drugs. For example, proteins aggregate very easily and can no longer be released in native form after preparation with HPH.

Therefore, it would be desirable to provide an improved method for making such VPGs. An improved method for producing VPG would have a shortened process time as well as minimized shear forces and would represent a continuous process, so that the production ideally would not take place in individual batches in succession.

Screw Extrusion, or more precisely twin screw extrusion (TSE), is a simple and continuous manufacturing technology that has become established in the pharmaceutical industry for the manufacture of sophisticated dosage forms. It has long been described extensively as a method (White, 1990 and White, 2002) and characterized in that in a cylindrically or otherwise shaped body ("barrel"), one or more screws are rotating material in one direction through a nozzle discharges. It has gained a reputation for continuous wet granulation and melt-extrusion, as well as for the production of injectable implants (Schulze and Winter, 2009). The fast process with controllable parameters, such as temperature and shear intensity, can be easily adapted to larger or smaller production sizes and continuous production is possible without any problems. Liquid, semi-solid and solid components can be processed simultaneously and a homogeneous mixture can be achieved. The screw extrusion has not yet been used for the production of VPGs.

The screw extrusion is clearly distinguishable from other extrusion processes that are also used in the production of liposomes. Membrane extrusion represents a method in which mostly aqueous liposomal dispersions are forced under pressure through a membrane filter. The liposomes are usually reduced in size and homogenized. For VPG these methods are not suitable. Furthermore, the screw extrusion is clearly distinguishable from extrusion by means of a so-called RAM extruder in which a cylindrical piston in a cylinder presses the contents under high pressure through a nozzle attached to the end of the cylinder.

• • kürzere Prozessdauer
• • verbesserte Temperaturkontrolle
• • geringere Viskosität der VPG als bei DAC
• • möglichst kein Batchverfahren
• • geringere Scherkräfte, damit weniger Stress, Degradation und Aggregation von empfindlichen Wirkstoffen
• • bessere Dispersionsqualität als bei Magnetrührverfahren
• • bessere Lagerstabilität als mit Magnetrührverfahren hergestellte VPG
• • bessere Skalierbarkeit

einfachere Einarbeitung von Wirkstoffen in jedweder Form (gelöst in Lipid, gelöst in wässriger Lösung, emulgiert in einer Emulsion, dispergiert in Liposomen, dispergiert in wässriger Lösung, dispergiert in Lipid, als Reinstoff in fester oder halbfester oder flüssiger Form, z.B. auch als Lyophilisat)wobei die durch das Verfahren hergestellten VPG eine langsame Freisetzung des optionalen pharmazeutischen Wirkstoffs aufweisen.Accordingly, it is an object of the present invention to provide such an improved method for the production of VPG, wherein the VPG optionally comprises at least one pharmaceutical agent in the form of a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus, virus-like particle, or low molecular weight Drug comprises. In summary, based on the technical scope set out above and the resulting need for an improved VPG manufacturing process, the following objectives have been defined:

• • shorter process time
• • improved temperature control
• • lower viscosity of VPG than DAC
• • if possible, no batch process
• • lower shear forces, thus less stress, degradation and aggregation of sensitive drugs
• • better dispersion quality than magnetic stirring
• • better storage stability than VPG produced by magnetic stirring
• • better scalability

simpler incorporation of active substances in any form (dissolved in lipid, dissolved in aqueous solution, emulsified in an emulsion, dispersed in liposomes, dispersed in aqueous solution, dispersed in lipid, as a pure substance in solid or semi-solid or liquid form, eg also as lyophilisate) wherein the VPGs produced by the process have a slow release of the optional pharmaceutical agent.

These and other objects of the present invention, which will become apparent from the description, are achieved by the method of making the VPG and by using the VPG produced in the method according to the independent claims. Preferred embodiments are defined by the dependent claims.

The following are some important references from the state of the art called and commented. However, all proposed systems do not meet the expectations of a relatively short and easily scalable low shear manufacturing process that results in stable VPG with good sustained-release properties of drugs.

US 2005 / 0238721A1 relates to a process for producing a product containing a pharmaceutical ingredient and a polymeric material. The pharmaceutical ingredient may be melt compounded into the polymeric material and extruded through a die to produce a product. This process requires a melt blend of the pharmaceutical ingredient and the polymeric material. The production of other pharmaceutical dosage forms such as VPG with positive long-term release properties of an active ingredient is not mentioned.

EP 2280681A1 describes a method for producing solid oral dosage forms in a finishing line / machine train comprising several parts of devices designed for mixing, extruding, cooling, grinding and finishing. There are no other dosage forms such as VPG with positive long-term release properties of a drug Furthermore, this method is designed to produce granules as solid, oral dosage forms.

EP 1968553A2 relates to a method of producing solid oral dosage forms comprising a sparingly soluble and / or poorly compressible therapeutic compound and a polymer. The process is carried out using an extruder. A volatile plasticizer, z. For example, a liquefied gas, such as supercritical carbon dioxide, is added to facilitate processing of the materials. The volatile plasticizer may serve to lower the viscosity of the mixture to be processed and / or increase the solubility of the therapeutic compound. Polymers and plasticizers are required for this process, and the present process does not disclose anything about the preparation of other pharmaceutical dosage forms such as gels and VPG with positive sustained-release properties of an active ingredient.

EP 1881819A1 describes a method for preparing solid dosage forms containing a difficultly compressible therapeutic compound. The method contemplates the use of an extruder, particularly a twin screw extruder, to melt granulate a therapeutic compound with a granulation aid. In addition, this method provides for the production of granules, as well as the kneading of the mixture is provided in the extruder parallel to heating the mixture. The production of other pharmaceutical forms such as VPG with positive long-term release properties of active ingredient is not mentioned.

In view of the limitations of conventional VPG production methods, there is an obvious need for an alternative method of producing VPG to overcome the disadvantages of traditional manufacturing methods, in terms of process time, process scalability, shear forces, and the properties of the manufactured product VPG, especially with regard to the long-term release properties.

SUMMARY OF THE INVENTION

The aim of the invention is to find a way to produce VPG and VPG with pharmaceutical agents and to avoid the known disadvantages of alternative methods of preparation, such as the disadvantage of the comparatively long process time, the difficult to scale process, the high shear forces and that Batch method is to use, or a high viscosity of the VPG. By way of example, the high viscosity of the prepared VPG and the complicated incorporation of sensitive pharmaceutical active substances, such as, for example, proteinaceous pharmaceutical active substances, may also be mentioned.

Therefore, it is an object of the invention to provide a process for the preparation of VPG which involves low shear production, a shorter batchless process time with improved scalability, and a relatively simple incorporation of pharmaceutically active ingredients.

Another object of the invention is to provide a process for the preparation of VPG which involves improved temperature control of the production as well as a lower viscosity of the VPG than DAC production and a better dispersion quality and thus better stability in magnetic stirring. A further object of the invention is to provide a process for the preparation of VPG wherein the pharmaceutical agent is released in the prepared VPG in a period of more than 5 days, more than 10 days, preferably in more than 20 days, more than 30 days or more, and the initial release of the active ingredient is less than 20% of the total amount of active ingredient within the first 48 hours. Another object is to use the VPG produced by screw extrusion to treat or prevent a disease.

These objects are achieved according to the invention by providing a process for producing VPG by means of screw extrusion.

Surprisingly, by the process for producing VPG by means of screw extrusion, said objects can be achieved. The scalable method of producing the VPG and the optional at least one pharmaceutical agent disclosed herein, and has been found to be particularly favorable due to the ease of scalability, shorter low shear processing time while producing a VPG that provides beneficial long term release properties.

The present invention is based on the unexpected finding that screw extrusion is ideally suited to produce VPG, which VPG may also optionally comprise at least one pharmaceutical agent. The inventive method of preparation is particularly useful for the preparation of VPGs with sensitive substances such as proteins, peptides, polypeptides, nucleotides, antibodies, antibody derivatives, Viruses, virus-like particles, or low molecular weight drugs suitable. Unlike other extruder screw processes known in the art, the inventive method of preparation can and does produce not only VPG but also VPG with sensitive substances such as proteins, peptides, polypeptides, antibody derivatives or antibodies just produce these VPG with advantageous long-term release properties and advantageously low viscosity.

Surprisingly, with the screw extrusion, a mixing and manufacturing process was found which lies exactly between the unfavorable extremes of the DAC, with high, for application unfavorable viscosity, and the stirring process, with poor degree of dispersion and lack of homogeneity, and products which are sufficiently dispersed, and gives ideal release at low viscosity, and moreover is unlimited up-scale bar as it allows continuous process operation. It is particularly surprising that the release at lower viscosity is equal to or even slower than in DAC process, which is particularly advantageous.

In a first aspect, the invention relates to a method of making vesicular phospholipid gel (VPG) wherein the VPG is prepared by screw extrusion.

In certain embodiments of the present invention, the VPG further comprises at least one lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes, salts of the fatty acids and derivatives thereof.

1. (i) Zuführen von mindestes einem Phospholipid und einer wässrigen Flüssigkeit, optional von mindestens einem pharmazeutischen Wirkstoff, bei dem es sich bevorzugt um ein Protein, Peptid, Polypeptid, Nukleotid, einen Antikörper, ein Antikörperderivat, Virus, virus-like particle, oder niedermolekularen Arzneistoff handelt, optional von mindestens einem Lipid ausgewählt aus der Gruppe bestehend aus Fettsäuren, Monoglyceriden, Diglyceriden, Triglyceriden, Sorbitanfettsäureestern, Sphingolipiden, Cholesterol, Wachsen und Salzen der Fettsäuren und Derivaten davon, in einen Schnecken-Extruder,
2. (ii) Homogenisieren des einen oder der mehreren Phospholipide, der wässrigen Flüssigkeit und/oder des optionalen pharmazeutischen Wirkstoffs in dem Schneckenextruder, so dass ein VPG entsteht, und
3. (iii) Extrudieren des VPG,

wobei die Schritte auch gleichzeitig stattfinden können.In preferred embodiments, the method according to the invention comprises the following steps:

1. (i) supplying at least one phospholipid and an aqueous liquid, optionally at least one pharmaceutical agent, which is preferably a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus, virus-like particle, or low molecular weight Drug, optionally of at least one lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes and salts of the fatty acids and derivatives thereof, in a screw extruder,
2. (ii) homogenizing the one or more phospholipids, the aqueous liquid and / or the optional pharmaceutical agent in the screw extruder to form a VPG, and
3. (iii) extruding the VPG,

where the steps can take place simultaneously.

In certain embodiments of the present invention, the at least one phospholipid, the aqueous liquid, the optional at least one pharmaceutical agent, and / or the at least one lipid may be fed separately to the screw extruder, wherein the delivery may occur simultaneously or with a time delay.

In further particular embodiments of the present invention, the at least one phospholipid and the aqueous liquid may be mixed prior to delivery and fed as a mixture to the screw extruder.

In preferred embodiments of the present invention, the screw extrusion is a twin-screw extrusion.

In certain embodiments of the present invention, the temperature during extrusion is between 4 ° C and 60 ° C. In a preferred embodiment, the temperature during extrusion is about 30 ° C.

In a specific embodiment, the rotational speed of the extrusion screw (s) is between 5 min ^-1 and 500 min ^-1 , preferably between 20 min ^-1 and 360 min ^-1 . In one's preferred embodiment, the rotational speed of the extrusion screw (s) 300 min ^-1.

In a preferred embodiment, the temperature during extrusion is between about 4 ° C and about 60 ° C and the rotational speed of the extrusion screw (s) is between about 5 min ^-1 and about 500 min ^-1 , preferably between 20 min ^-1 and 360 min ^-1 . In a particularly preferred embodiment, the temperature during the extrusion is about 30 ° C and the rotational speed of the extrusion screw (s) is about 300 min ^-1 .

In certain embodiments of the present invention, a plurality of extruding steps are performed, ie, the mass is extruded at least once more from one or more phospholipid (s), aqueous liquid, optionally at least one active ingredient, and optionally at least one further lipid after the first homogenizing step in the extruder. Either the extruders are directly connected or the intermediate product is collected and fed to another extruder or the same, previously used extruder.

In certain embodiments of the present invention (i) the extrusion screw (s) has a conveying function and optionally additionally via kneading or mixing segments or differently shaped segments for mixing, transport, temperature control, compression, or discharge, and / or (ii) (iii) the nozzles used for the extrusion have different diameters, lengths and / or shapes, and / or (iv) is the outlet of the extruder to the axis of the extruders -Schnecke (s) arranged at right angles or in a straight line.

In specific embodiments of the present invention, the level of phospholipid (s) in the VPG is about 10-70 wt%, preferably 20-65 wt%, most preferably 30-60 wt%.

In preferred embodiments of the present invention, the at least one pharmaceutical active ingredient is prepared from the prepared VPG for a period of more than at least 5, preferably more than 10 days, more preferably at least 20 days, even more preferably at least 30 days, more preferably at least 45 days and most preferably 55 days and the initial release of the drug is less than 35%, more preferably less than 25%, most preferably less than 20% of the total drug amount within the first 48 hours.

In a further embodiment, the method according to the invention further comprises a step (iv) in which is extruded by a RAM extruder, this step after the steps (i) - (iii) takes place.

In a further embodiment, the process according to the invention comprises a step (v), single or multiple extruding with a screw extruder, wherein optionally the screw extruder is the same as in step (i) - (iii) or another screw extruder, this step being according to the Steps (i) - (iii) and optionally before or after step (iv).

In a further embodiment, in step (v) the same or the same and / or one or more further phospholipids, the same and / or one or more further aqueous liquid (s), the same or the same and / or one or more further pharmaceutical active ingredient (s), and / or the same or the same and / or one or more further lipid (s) as in step (i) are supplied.

Other objects, advantages and features of the present invention will become apparent from the following description.

list of figures

• : Macroscopic appearance of encapsulated methylene blue VPG prepared by magnetic stirring, dual asymmetric centrifugation or twin screw extrusion (from left to right). Samples produced by magnetic stirring show lipid deposits and inhomogeneous appearance, while samples prepared by DAC or extrusion show a homogeneous appearance without lipid deposits.
• :
  • A: Effect of phospholipid content and production process (magnetic stirrer: white bars, ThreeTec extruder: light gray bars, HAAKE extruder: dark gray bars, DAC: black bars) on vesicle size of liposome dispersion prepared from VPGs. The size was determined by means of dynamic light scattering. The VPG was prepared with Lipoid E80 and 20 mM PBS (phosphate buffered saline) pH 7.4 without the inclusion of drugs. (Extrusion parameters: rotational speed 300 min ^-1 , temperature 30 ° C, nozzle diameter 0.5 mm). (N = 3)
  • B: Effect of phospholipid content and manufacturing process (magnetic stirrer: white bars, ThreeTec extruder: light gray bars, HAAKE extruder: dark gray bars, DAC: black bars) on the Polydispersity index (PDI) of the liposomal dispersions prepared from the VPGs. The PDI was determined by means of dynamic light scattering. The VPGs were prepared with Lipoid E80 and 20 mM PBS (phosphate buffered saline) pH 7.4 without the inclusion of drugs. (Extrusion parameters: rotational speed 300 min ^-1 , temperature 30 ° C, nozzle diameter 0.5 mm). (N = 3)
• : Effect of phospholipid content and manufacturing process (magnetic stirrer: white bars, ThreeTec extruder: light gray bars, HAAKE extruder: dark gray bars, DAC: black bars) on the viscosity (at a shear rate of 32.9 s-1) of different prepared VPGs increasing phospholipid content. (Extrusion parameters: rotational speed 300 min ^-1 , temperature 30 ° C, nozzle diameter 0.5 mm). (N = 3)
• : Cumulative in vitro release of FITC-dextran from VPGs with a phospholipid concentration of 45% (m / m) by DAC (black dots, duration 45 min, 3500 rpm), magnetic stirrer (white squares, duration 45 min) or extrusion (Extrusion parameters: rotational speed 300 min ^-1 , temperature 30 ° C, nozzle diameter 0.5 mm, ThreeTec extruder: light gray triangles, HAAKE extruder: dark gray triangles). FITC-dextran 2mg / g. (N = 3)
• : Effect of varying the parameter rotational speed on the product viscosity of the VPG in a single extrusion. Increasing rotational speed leads to smaller vesicle sizes and thus better homogeneity, with constant viscosity.
• : Stability of erythropoietin (EPO) in VPGs after preparation with different manufacturing methods. (Bands 1 and 12: molecular weight markers, 2: native EPO, 3: EPO-VPG (production method DAC), 4: EPO-VPG (production method magnetic stirrer), 5: EPO-VPG (production method ThreeTec extruder), 6: EPO VPG (Production method HAAKE extruder); 7: Native EPO; 8: EPO-VPG (Production method DAC); 9: EPO-VPG (Magnetic stirring production method); 10: EPO-VPG (Production method ThreeTec extruder); 11: EPO-VPG (Production method HAAKE extruders) samples show no aggregation or degradation of the protein regardless of the manufacturing process.
• : Stability of an antibody (type IgG1) in VPGs after preparation with different preparation methods (bands 1 and 10: molecular weight markers, 2: mAb-VPG (preparation method DAC), 3: mAb-VPG (production method magnetic stirrer method), 4: mAb VPG (production method ThreeTec extruder), 5: mAb-VPG (production method Leistritz extruder), 6: antibody stock solution, 7: mAb-VPG (production method DAC), 8: mAb-VPG (production method Leistritz extruder), 9: antibody stock solution The samples show no aggregation or decomposition of the antibody, regardless of the manufacturing process.

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are explained.

The present invention, as hereinafter described by way of explanation, may be suitably carried out in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

The present invention will be described with reference to certain embodiments and with reference to certain figures, but the invention is not limited thereto, but only by the claims.

Where the term "comprising" is used in the present specification and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined below as at least a certain number of embodiments, it should also be understood as a group which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a single noun, e.g. "The" or "one", this includes a plural of this noun, unless expressly stated otherwise. The terms "approximately" or "approximately" in the context of the present invention designate a precision interval which the person skilled in the art understands in order to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ± 10%, and preferably ± 5%.

Concentrations, amounts, solubility, and other numerical data are expressed or presented herein in an interval format. It should be understood that such an interval format is used for purposes of simplicity and brevity and therefore should be interpreted in a flexible manner to encompass not only the numerical values explicitly listed as boundaries of the range, but also all individual numerical values or sub-ranges that are included in this range as if each numeric value and subrange were explicitly specified. By way of illustration, a number range of "about 1 to about 5" should be interpreted to not only include the explicitly stated values of about 1 to about 5, but also to include individual values and ranges within the specified range. Thus, in this numerical range, individual values such as 2, 3, and 4 and subregions such as 1 to 3, 2 to 4, and 3 to 5, etc. are included. The same principle applies to areas that only represent a numeric value. In addition, such a design should apply regardless of the breadth of the range or features described.

As used herein, "preparation", "formulation" and "composition" may be used interchangeably herein and refer to a combination of two or more substances or substances.

Technical terms are used in the sense of common sense or its meaning for the skilled person. When a particular meaning is conveyed to particular terms, the following defines terms in the context of which the terms are used.

In a first aspect, the present invention relates to a method of making a VPG made by screw extrusion.

A "manufacturing process" is a process in which substances are changed in terms of composition, properties and type. In the context of the present invention, a process is a process in which a VPG is produced from different starting materials, the starting materials being changed in terms of their properties. Preferred starting materials are at least one phospholipid and an aqueous liquid. Further preferred starting materials are at least one phospholipid, an aqueous liquid and a pharmaceutical active substance. Further preferred starting materials are at least one phospholipid, at least one further lipid, an aqueous liquid and a pharmaceutical active substance.

For the purposes of the present invention, a "vesicular phospholipid gel" (VPG) is a liposomal formulation which, owing to a high phospholipid content (preferably at least 10% by weight, more preferably 20% by weight, most preferably at least 30% by weight), and thus viscous, ie is no longer pourable and has a high liposome (vesicle) density. A VPG must be distinguished from gels containing liposomes in which the gel properties (viscosity, prevention of free flow) are not caused by a high liposome density, but by a gel-forming component, e.g. Polyacrylamide. However, it should not be excluded that a VPG could be further stiffened by the addition of a gelling agent. VPG contain both aqueous compartments and bilayer membranes that are suitable for the absorption of water-soluble or fat-soluble drugs. Their potential as sustained release depot formulations of low molecular weight drugs has been investigated (Brandl et al., 1995) and various approaches have been described to load VPGs with low molecular weight drugs (Brandl, 2007).

The term "phospholipids" refers to lipid molecules containing one or more phosphate groups, including those derived from either glycerol (phosphoglycerides, glycerophospholipids) or sphingosine (sphingolipids). They include polar lipids and certain phospholipids, which are of great importance for the structure and function of cell membranes and are the most abundant membrane lipids. In certain embodiments, phospholipids are triglyceride derivatives in which a fatty acid has been replaced by a phosphorylated group and one of several nitrogenous molecules. The fatty acid chains are hydrophobic (as in all fats). However, the charges on the phosphorylated and amino groups make this part of the molecule hydrophilic. The result is an amphiphilic molecule.

With respect to the present invention, therefore, a phospholipid is an amphiphilic substance, which generally comprises a hydrophobic group, which in turn comprises a long-chain alkyl group and a hydrophilic group, and a phosphoric acid group in a molecule. Exemplary phospholipids which can be used in the present invention include glycerophospholipids such as phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine and phosphatidylinocitol; Sphingophospholipids such as sphingomyelin (sphingomyelin); natural or synthetic Diphosphatidylphospholipids such as cardiolipin and derivatives thereof; and phospholipids which have been hydrogenated by conventional methods (for example hydrogenated soybean phosphatidylcholine), as well as mixtures of such phospholipids. The above-mentioned phospholipids are sometimes referred to as "phospholipids" hereinafter. The phospholipid component may contain one or more phospholipids and ranges from about 10% to about 70% of the total weight of VPG, preferably 20% to 65%, more preferably between 30% to 60%. In preferred embodiments of the invention, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalins, phosphatidylglycerols and mixtures of several of these phospholipids. In specific embodiments of the invention, a mixture of phosphatidylcholines and / or phosphatidylglycerols can be used as the phospholipid.

Suitable phospholipids for use in the present compositions include phospholipid mixtures, such as those sold under the trade names Lipoid S 20 S, Lipoid S 75, Lipoid S 100, Lipoid S 100-3, Lipoid S 75-3N, Lipoid SL 80, Lipoid E80 and Lipoid SL 80-3 (Lipoid); Phospholipon 85G, Phospholipon 80, Phospholipon 80H, Phospholipon 90G, Phospholipon 90H, Phospholipon 90NG, Phospholipon 100H, Phosal 35B, Phosal 5OG, Phosal 50SA, Phosal 53MCT and Phosal 75SA, (Phospholipon, Cologne, Germany); Alcolec Z-3, (American Lectin Company, Oxford CT); Emulu fluid F30, Emulfluid, Lipotin NE, Lipotin 100, Lipotin SB, Lipotin 100J, Lipotin H, Lipotin NA, Lipotin AH and Lipopur, (Cargill, Degussa Texturant Systems); Terradrill V 408 and Terradrill V 1075 (Cognis); Yellowthin 100, Yellowthin 200, Lecistar Sun 100 and Yellowthin Sun 200 (Star Chemistry); and Lanchem PE-130K (Lambent Technologies, Gurnee, IL).

As used herein, screw extrusion is a process characterized in that in a cylindrically or otherwise shaped body ("barrel"), one or more screws will rotationally carry material in one direction through a nozzle. The screw extruder is a continuous production technology, which apart from controllable parameters, such as temperature and shear intensity, is characterized by the fact that liquid, semi-solid and solid components can be processed simultaneously and a homogeneous mixture is achieved. The goal of the manufacturing process is to produce extrudates (long objects with a solid or semi-solid cross-sectional profile), preferred for the present invention being semi-solid extrudates. At least two major components are required for this purpose: (1) a transport system that can impart a mixing function and (2) a nozzle system that forms the material. The pressure required for extrusion depends on the design of the die, on the rate of extrusion, and especially on the rheological properties of the formulation. The extrusion may be continuous (creation of an infinitely long material) or semi-continuous (generation of many short pieces). With respect to the viscosity adjustment method, the extrusion can be classified into molten systems (hot melt extrusion) and semi-solid systems. Semisolid systems are produced by dispersing a high level of solid material in a liquid phase (Swarbick and Boylan 1992). This technique is often used for the production of granules or pellets, while the (hot) melt extrusion technique is available for the production of other forms of active ingredient (Kissel et al., 2002). Preferred in the present invention is twin-screw extrusion, in which two rotating extrusion screws are arranged in a cylindrical or other shaped body ("barrel"). In certain embodiments, multiple steps of extrusion may take place, i. that the mass of one or more phospholipid (s), aqueous liquid, optionally at least one active ingredient and optionally at least one further lipid after the first homogenization step in the extruder is extruded at least once again. Either the extruders are directly connected or the intermediate product is collected and fed to another extruder or the same, previously used extruder. Preferably, the temperature during extrusion is between 4 ° C and 60 ° C, more preferably between 15 ° C and 40 ° C, most preferably between 25 ° C and 35 ° C.

In one embodiment of the invention, the VPG may comprise one or more additional lipids, including neutral, cationic and anionic lipids. Thus, in other specific embodiments, the pharmaceutical composition further comprises at least one lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes and salts of the fatty acids, and derivatives thereof. These additional lipids account for up to 30%, more preferably up to 10%, most preferably up to 2.5% of the VPG. The additional lipids are to the phospholipids of VPG in a ratio of lipid / phospholipid 1: 1, more preferably 1: 4, most preferably 1:10 or less.

Cationic lipids for use in the present compositions include N, N-dioleyl-N, N-dimethyl ammonium chloride ("DODAC"); N- (2,3-dioleyloxy) propyl-N, N, N -triethylammonium chloride ("DOTMA"); N, N-distearyl-N, N-dimethylammonium bromide ("DDAB"); N- (2,3-dioleoyloxy) propyl) -N, N, N- trimethyl ammonium chloride ("DOTAP"); 3β- (N- (N ', N'-dimethylaminoethane) -carbamoyl) cholesterol ("DC-Chol"), N- (1- (2,3-dioleyloxy) propyl) -N-2- (capricarboxamido) ethyl) -N, N-dimethylammonium trifluoroacetate ("DOSPA"), dioctadecylamidoglycylcarboxypermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"); and N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"). In addition, a number of commercial preparations of cationic lipids can be used, such as LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO / BRL), and TRANSFECTAM (comprising DOGS, in ethanol, from Promega Corp.).

Anionic lipids for use in the present compositions include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, dipalmitoylphosphatidic acid, and other anionic modifying groups attached to neutral lipids.

Any number of neutral lipids can also be used in the present invention, which are either in uncharged or neutral zwitterionic form at physiological pH, including diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

As used herein, screw extrusion is a method characterized in that in a cylindrically or otherwise shaped body ("barrel"), one or more screws, rotating, discharge material in one direction through a nozzle. The screw extrusion is a continuous production technology, which apart from controllable parameters such as temperature and shear intensity is characterized by the fact that liquid, semi-solid and solid components can be processed simultaneously and a homogeneous mixture is achieved. The aim of the manufacturing process is to produce extrudates, long objects with a solid or semi-solid cross-sectional profile, preferred for the present invention are semi-solid extrudates. For this purpose, at least two major components are necessary: (1) a transport system having a mixing function and (2) a nozzle system that forms the material. The pressure required for extrusion depends on the design of the die, on the rate of extrusion, and especially on the rheological properties of the formulation. The extrusion may be continuous (creation of an infinitely long material) or semi-continuous (generation of many short pieces). With respect to the viscosity adjustment method, the extrusion can be classified into molten systems (hot melt extrusion) and semi-solid systems. Semisolid systems are produced by dispersing a high level of solid material in a liquid phase (Swarbick and Boylan 1992). This technique is often used for the production of granules or pellets, while the (hot) melt extrusion technique is available for the preparation of other drug forms (Kissel et al., 2002). Preferred in the present invention is twin screw extrusion in which two rotating extrusion screws are arranged in a cylindrical or other shaped body ("barrel"). In certain embodiments, several steps of extrusion may take place, ie, the mass of one or more phospholipid (s), aqueous liquid, optionally at least one active and optionally at least one further lipid is extruded at least once in the extruder after the first homogenization step. Either the extruders are directly connected or the intermediate product is collected and fed to another extruder or the same, previously used extruder.
In one embodiment, the temperature during extrusion is preferably between 4 ° C and 60 ° C, more preferably between 15 ° C and 40 ° C, most preferably between 25 ° C and 35 ° C.

In one embodiment, the present invention relates to a method comprising the steps of: supplying one or more phospholipids and an aqueous liquid, and optionally at least one pharmaceutical agent, which is preferably a protein, peptide, polypeptide, nucleotide, an antibody, an antibody derivative, virus, virus-like particle, or low molecular weight drug, in a screw extruder, homogenizing the one or more Phospholipids, the aqueous liquid and optionally the pharmaceutical agent in the screw extruder to form a VPG, and extruding the VPG, wherein in the first step also lipids selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol , Waxes and salts of the fatty acids and derivatives thereof can be added and wherein said steps can also take place simultaneously. In certain preferred embodiments, the delivery of one or more phospholipids and an aqueous liquid, and optionally, at least one pharmaceutical agent, which is preferably a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus, virus -like particle, or low molecular weight drug, into a screw extruder, homogenizing the one or more phospholipids, the aqueous liquid, and optionally the pharmaceutical agent in the screw extruder to form the VPG, and extruding the VPG simultaneously.

With respect to the objects of the present invention, the term "aqueous liquid" is used for an aqueous solvent (eg, water or buffer solution). Preferably, the "aqueous liquid" comprises primarily water which may contain dissolved inorganic or organic salts, acids or bases, for example, to set and achieve a specific pH or specific osmotic pressure. However, the aqueous medium may also comprise aqueous solutions of saccharides, amino acids or surfactants or mixtures of water with other solvents such as alcohols, diols or triols. A buffered solution is an aqueous solution comprising a mixture of a weak acid and its conjugate base or a weak base and its conjugated acid. It has the property that the pH of the solution changes very little when a small amount of acid or base is added. Buffered solutions are used to maintain the pH at a nearly constant level in a variety of chemical applications. Suitable buffer systems include e.g. Phosphate, citrate, acetate, succinate, tartrate, maleate, histidine, glycine, TAPS, bicine, tris, tricine, HEPES, TES, and MOPS. These and alternative buffer systems are known to one of ordinary skill in the art. Preferred is a 20 mM phosphate buffered saline (PBS) at pH 7.4.

As used herein, "pharmaceutically active ingredient", "active agent", "bioactive agent", "pharmaceutically active agent" and "pharmaceutically active compound" can be used interchangeably to refer to an active agent, agent, substance or compound that has a having measurable specific or selected physiological activity in a significant or effective amount. It is to be understood that the term "drug" is expressly encompassed by the present definition as many drugs known to have specific physiological activities. These terms are well known in the pharmaceutical and medical fields. Examples of drugs useful in the present invention include, but are not limited to, proteins, peptides, polypeptides, nucleotides, antibodies, antibody derivatives, viruses, virus-like particles, and low molecular weight drugs.

In terms of the objects of the present invention, a "protein" which may be included as a pharmaceutical agent in the present invention comprises a biologically active protein as well as derivatives and mutations thereof and may be naturally occurring, recombinantly engineered or synthesized. In addition, the protein may have a variety of modifications, such as addition, substitution or deletion of an amino acid or domain, or glycosylation and other modifications. The term "proteins" as used herein therefore also includes lipoproteins and glycoproteins. The term "proteins" also includes e.g. Protein aggregates, dimers, trimers, other oligomers.

Examples of "proteins" include human growth hormones, growth hormone releasing hormones, growth hormone releasing peptide, interferons, colony stimulating factors, interleukins, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin , Lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1-antitrypsin, albumin and fragment polypeptides thereof, apolipoprotein-E, erythropoietin, factor VII, factor VIII, factor IX, plasminogen activating factor, urokinase, streptokinase, protein C, C-reactive protein , Renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, bone stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, tissue factor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone R leasing hormone, nerve growth factors such as parathyroid hormone, relaxin, secretin, insulin-like growth factors, adrenocortic hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies to various viruses , Bacteria, toxins, etc. and virus-derived vaccine antigens as well as therapeutic monoclonal or polyclonal antibodies, eg directed against receptors of growth factors. Preference is given to erythropoietin (EPO), G-CSF and INF β-1b.

The terms "polypeptide", "peptide", "protein" and "antibody" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues. As used herein, the term "amino acid" refers to a list of abbreviations, letters, characters or words that represent amino acid residues. Amino acids can be referred to herein either by their well-known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. One convention is that those peptide chains that are short enough to be synthetic from the constituent amino acids to be called "peptide" instead of "proteins". However, with the advent of better synthetic techniques, peptides with hundreds of amino acids can be made, including complete proteins such as ubiquitin. Native chemical ligation has allowed access to even longer proteins, so this convention seems outdated. Another convention sees an informal dividing line at about 50 amino acids in length (some people claim shorter lengths). In accordance with this convention, the term "polypeptide" refers to a single linear chain of amino acids, the term "protein" refers to one or more polypeptides of greater than about 50 amino acids in length, and the term "oligopeptide" or (simply) "peptide" refers to a polypeptide. which is less than 30 to 50 amino acids long.

Accordingly, the terms "protein", "peptide" and "polypeptide" are used interchangeably herein to refer to a polymer or oligomer of contiguous amino acid residues. The term "neuropeptide" is used herein to refer to a peptide that is active in the central nervous system and the term "peptide hormone" is used herein to refer to a peptide that acts as a hormone.

In the context of the present invention, proteins which may be included in the pharmaceutical composition of the present invention include monoclonal and polyclonal antibodies, growth factors such as cytokines and hormones.

"Antibodies", also called immunoglobulins (abbreviated Ig), are gamma globulin proteins that are found in the blood and other body fluids of vertebrates, and that are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. They usually consist of basic structural units - each with two large heavy chains and two small light chains - from which, for example, one-unit monomers, two-unit dimers, or five-unit pentamers are formed. Antibodies are produced by white blood cells called B cells. There are different types of heavy antibody chains, and different types of antibodies, which are grouped into different isotypes depending on the heavy chain they contain. Although the general structure of all antibodies is quite similar, a small region at the tip of the protein is extremely variable, allowing the existence of millions of antibodies with slightly different tip structures. This region is known as a hypervariable region. Each of these variants can bind to a different destination. The enormous diversity of the antibodies allows the immune system to recognize an equally wide variety of antigens. The unique part of the antigen recognized by an antibody is called an epitope. These epitopes bind to their antibodies in a highly specific interaction called induced fit, which allows the antibodies to identify and bind solely to their unique antigen in the midst of millions of different molecules in an organism. Detection of an antigen by an antibody marks it for attack by other parts of the immune system. Antibodies can also neutralize targets directly, for example, by binding to a portion of a pathogen needed to produce an infection.

Targeted monoclonal antibody therapy is often used to treat diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and many forms of cancer, including non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer, and breast cancer. Some immunodeficiencies, such as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or total lack of antibodies. These diseases are often treated by the generation of a transient form of immunity called passive immunity. In preferred embodiments, a (monoclonal) antibody is selected from the group consisting of abciximab, adalimumab, alefacept, alemtuzumab, alirocumab, arcitumomab, atezolizumab avelumab, basiliximab, belimumab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brodalumab, canakinumab, capromab, catumaxomab, certolizumab, certolizumab pegol, cetuximab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, efalizumab, elotuzumab, evolocumab, fanolesomab, gemtuzumab, golimumab, ibritumomab tiuxetan, idarucizumab, imiciromab, infliximab, ipilimumab, ixekizumab, mepolizumab, muromonab- CD3, natalizumab, necitumumab, nivolumab, nofetumomab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, ozogamicin, palivizumab, panitumumab, pembrolizumab, pertuzumab, ranibizumab, Ramucirumab, raxibacumab, reslizumab, rituximab, satumomab, secukinumab, siltuximab, sulesomab, tocilizumab, tositumomab, trastuzumab, trastuzumab emta sine, ustekinumab, vedolizumab, votumumab.

In the context of the present invention, antibodies include monoclonal antibodies, polyclonal antibodies, bi- and multispecific antibodies and chimeric antibodies, as well as parts or fragments thereof. Preferably, fragments or portions of antibodies retain their capacity to specifically recognize and bind antigens. Antibodies may exist as monomers or multimers.

Cytokines are a category of signaling molecules that are commonly used in cellular communication such as hormones and neurotransmitters. The term cytokine includes a large and diverse family of polypeptide regulators produced throughout the body by cells of various embryological origins. Cytokine is a common name; other names include lymphokine (cytokines produced by lymphocytes), monokin (monocyte-derived cytokines), chemokine (cytokines with chemotactic activities), and interleukin (cytokines produced by a leukocyte that act on other leukocytes). Cytokines can act on the cells that secrete them (autocrine effect), on nearby cells (paracrine effect) or in some cases on distant cells (endocrine effect). Cytokines perform a variety of functions, from stimulating immune cells to inhibiting viral replication of infected somatic cells. In the context of the present invention, the cytokine family thus comprises G-CSF, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNFα, TNF-β.

With respect to the present invention, an "antibody derivative" includes fusion proteins in which a portion of the fusion protein is an antibody as defined above, antibody mimetics, e.g. Anticalins, and antibody conjugates, e.g. Antibody-drug conjugates.

In terms of the objects of the present invention, an "antibody-drug conjugate" which may be included as a pharmaceutical agent in the present invention comprises an antibody, an antibody fragment or an antibody-mimetic having covalent linkage (conjugation) with associated with a drug. The antibody, antibody fragment, or antibody mimetic serve to target the conjugated drug to specific targets that are specifically bound and recognized by the antibody, antibody fragment, or antibody mimetic.

In terms of the objects of the present invention, a "nucleotide" which may be included as a pharmaceutical agent in the present invention comprises nucleic acids in both ribonucleic acid (RNA or RNA) and deoxyribonucleic acid (DNA or DNA) strands. Nucleotides within the meaning of the invention are also cDNA, mRNA, siRNA, snRNA, tRNA, rRNA, shRNA, and miRNA.

In terms of the objects of the present invention, a "virus" which may be included as a pharmaceutical agent in the present invention includes infectious viruses, non-infectious viruses, and viral vectors. A virion consists of one or more nucleic acid molecules, which are often surrounded by a protein capsule, the capsid. There may be additional proteins, for example, with enzymatic activities, in the virion. In some viruses, the virion additionally consists of an outer lipid membrane, the virus envelope. Viria can be infectious (viruses) or non-infectious. Viria do not have their own metabolism. Viral vectors are deliberately modified virus particles that are used in genetic engineering to transport genetic material into target cells. These may be cells of a living organism (in vivo) or cells of a cell culture (in vitro). Examples of viruses include e.g. Adeno associated viruses (AAV).

In terms of the objects of the present invention, a "virus-like particle" or "VLP" which may be included as a pharmaceutical agent in the present invention comprises virus particles that are not nucleic acids. As particles of viral origin without viral nucleic acids they can not be propagated in the target cells. Because they contain no functional nucleic acids unlike viral vectors, they are also unable to deliver a transgene. On the other hand, the VLPs of the invention may be e.g. Containing peptides, proteins, antibodies, polypeptides, small molecule drugs, and non-viral non-functional nucleotides.

In terms of the objects of the present invention, a "low molecular weight drug" or "small molecule" which may be included as a pharmaceutically active agent in the present invention comprises drugs whose molecular weight does not exceed about 800 g.mol ^-1 . Due to their small size many low molecular weight drugs are able to penetrate into cells and to develop their effect there. Low molecular weight drugs do not include biologics, ie, no proteins, peptides, antibodies, etc., and are chemically synthesized. Low molecular weight drugs can be used in all areas of the treatment and diagnosis of diseases, as well as their prevention.

A screw extruder is an apparatus characterized in that in a cylindrically or otherwise shaped body ("barrel") there are one or more rotating scrolls which discharge material in one direction through a nozzle. The screw extruder consists of at least one rotating screw inside a stationary cylindrical body ("barrel"). Accordingly, the twin-screw extruder has two rotating screws within a stationary cylindrical one Body. At the end of the barrel is a nozzle, also called outlet, to discharge the VPG produced. The extrusion channel can be divided into three different sections. Within the first zone, the feed zone, the extruder is loaded. After the intake zone follows the compression zone. Within this zone, the pressure increases due to the reduction of the thread pitch while maintaining a constant flight depth or due to the reduction of the flight depth while maintaining the thread pitch. Thus, a compression of the material takes place. Finally, the material enters the discharge zone as a homogeneous mixture suitable for extrusion. In this last zone, the pulsating flow is reduced and a uniform delivery rate through the nozzle is achieved. The nozzles used may have different diameters, lengths and / or shapes. The outlet of the extruder may be arranged perpendicular or straight to the axis of the extruder screw (s). Typical screw extruders are single or twin screw extruders. In a preferred embodiment of the invention, the extruder is equipped with at least one rotating extruder screw. Such extruders include, but are not limited to, twin screw extruders. In further preferred embodiments, the extrusion screws have a conveying function. In further embodiments, in addition to the conveying function, the extrusion screws additionally have kneading and mixing segments or segments designed differently for mixing, transport, temperature control, compression, or discharge. Furthermore, in certain embodiments, the extrusion screws can be operated in the same direction or in opposite directions. The rotational speed of the extrusion screws may be between 5 min ^-1 and 500 min ^-1 , preferably 20 min ^-1 and 360 min ^-1 . The geometry of the extrusion screws may be cylindrically parallel or conical or otherwise shaped.

Screw extruders which have been used in the present process for producing VPG include, among others, the mini-extruder ZE-5 (ThreeTec GmbH, Seon, Switzerland), the MiniLab® Micro Rheology from HAAKE (ThermoFisher Scientific, Karlsruhe, Germany). , the TSE 24 MC (ThermoFisher Scientific, Karlsruhe, Germany), the Process 11 (ThermoFisher Scientific, Karlsruhe, Germany), the EuroLab 16X (ThermoFisher Scientific, Karlsruhe, Germany) L, the Nano 16 extruder (Leistritz Extrusionstechnik GmbH, Nuremberg , Germany), the extruders of the ZSE series (Extrusionstechnik GmbH, Nuremberg, Germany), the extruders of the ZSE Maxx series (Extrusionstechnik GmbH, Nuremberg, Germany), as well as numerous devices of the companies Leistritz, Coperion, Biomation, Xplore, ZSK, Brabender, Thermo, IDE.

Homogenizing is understood to mean the production of a homogeneous, ie uniform mixture of different non-dissolving components of a solution. For the purposes of this invention is meant the preparation of a VPG, wherein the aqueous liquid, the phospholipids, the optional, at least one active ingredient and the optional, at least one further lipid is homogenized.

In one embodiment, the one or more phospholipids, the aqueous fluid, and the at least one optional pharmaceutical agent are separately and simultaneously delivered to the screw extruder. In a further embodiment, the one or more phospholipids, the aqueous liquid and the at least one optional pharmaceutical active ingredient are supplied to the screw extruder separately and with a time offset

In certain embodiments, the one or more phospholipids and the aqueous liquid may be mixed prior to delivery to the screw extruder. In other particular embodiments, the one or more phospholipids as well as at least one other lipid and the aqueous liquid may be mixed prior to delivery to the screw extruder. For mixing the components, various mixing methods are available, such as the magnetic stirring method. For the magnetic stirring method, phospholipid (s) and aqueous liquid are stirred with a magnetic stirrer in a container for a short time at room temperature or slightly elevated temperature. The stirrer preferably covers the entire bottom of the container.

Feeding in the context of the invention means adding the various constituents of a VPG to the screw extruder. The feeding of the various components can be done individually or as a mixture. The feeding is done manually by syringe into the running screw extruder, or automatically, e.g. by a drip device, piston pump or other pump, possibly also by using gravity by the material flows independently into the extruder, by other conveyors such as paddle wheels, slides, presses, screw conveyors, or by pouring the coherent mass into the extruder.

A mixture according to the invention is the substance of (i) aqueous liquid and phospholipid (s) or (ii) aqueous liquid, phospholipid (s) and at least one pharmaceutical agent or (iii) aqueous liquid, phospholipid (s) and at least one another lipid or (iv) aqueous liquid, Phospholipid (s), at least one other lipid and at least one pharmaceutical agent before homogenisation and extrusion. In certain embodiments, the mixtures named from (i) to (iv) may further comprise other substances: in certain embodiments, the mixture according to the invention also comprises at least one adjuvant that modifies the release of the at least one pharmaceutical agent and / or biodegradation of the vesicular phospholipid gel modified and / or the at least one pharmaceutical active substance stabilized during production, storage and release. In addition, the adjuvant could modify the solubility of the at least one pharmaceutical agent. The adjuvant may be, for example, a hydrophilic polymer, a sugar, a polyol, a surfactant, an antioxidant and / or a water-soluble salt, or any other excipient known to accomplish the above-mentioned purposes.

In one embodiment, screw extrusion may be performed as single screw extrusion or as twin screw extrusion. In a particularly preferred embodiment, the screw extrusion is a twin-screw extrusion. Therefore, in certain embodiments, the screw extruder has two extrusion screws.

The temperature during the extrusion and other parameters have an influence on the product properties of the VPG, in particular on the viscosity. In a preferred embodiment, the temperature during extrusion is between 4 ° C and 60 ° C and the rotational speed of the extrusion screw (s) is between 5min ^-1 and 500 min ^-1 , preferably between 20 min ^-1 and 360 min ^-1 . In a particularly preferred embodiment, the temperature during the extrusion is 30 ° C and the rotational speed of the extrusion screw (s) is 300 min ^-1

In certain embodiments, one or more extrusion steps are performed, i. that the mass of one or more phospholipid (s), aqueous liquid, optionally at least one active ingredient and optionally at least one further lipid after the first homogenization step in the extruder is extruded at least once again. The extruders are either directly connected to each other or the intermediate product is collected and fed to another extruder or the same, previously used extruder.

In a further embodiment, in such an at least one further extrusion, the same or the same and / or one or more further phospholipid (s), the same and / or one or more further aqueous liquid (s) ), the same or the same and / or one or more further pharmaceutical active ingredient (s), and / or the same or the same and / or one or more further lipid (s) as supplied in the first extrusion.

In one embodiment (i), the extrusion screw (s) have a conveying function and optionally additionally kneading or mixing segments or other shaped segments for mixing, transport, temperature control, compression or discharge, and / or (ii) the extrusion screw (s) ) and / or (iii) the nozzles used for the extrusion have different diameters, lengths and / or shapes, and / or (iv) the outlet of the extruder is to the axis of the extruder screw (s) arranged at right angles or in a straight line. In a further embodiment, the extrusion screw (s) have a conveying function. In one embodiment, the extrusion screw (s) have a conveying function and in addition via kneading or mixing segments or differently shaped segments for mixing, transport, temperature control, compression, or discharge. The extrusion screws can be operated in the same direction or in opposite directions. In this context, coextensive means that the extrusion screws are operated continuously in the same direction, while in opposite terms in this context means that the extrusion screws are operated in the opposite direction. In one embodiment, the extrusion screws are operated in the same direction. In a further embodiment, the extrusion screws are operated in opposite directions. In one embodiment, the nozzles used for the extrusion have a length between 0.1 mm and 50 mm, preferably a length of 2 mm. In another embodiment, the nozzles used for the extrusion have a length between 10 mm and 100 mm, preferably a length of 20 mm. In another embodiment, the nozzles used for the extrusion have a diameter between 0.1 mm and 5 mm , preferred is a diameter of 2 mm. In another embodiment, the nozzles used for the extrusion have a diameter between 1 mm and 10 mm, preferably a diameter of 5 mm. In one embodiment, the nozzle shapes used for extrusion are round, triangular, quadrangular, pentagonal, or hexagonal or otherwise geometrically shaped. Particularly preferred is a round nozzle shape.
A nozzle whose shape promotes a twist or a lateral movement of the material to be extruded is also possible. In one embodiment, the outlet of the extruder is to the axis of the extruder screw (s) arranged at right angles. In another embodiment, the outlet of the extruder is arranged in a straight line to the axis of the extruder screw (s). Up to 35,000 kg of extrudate per hour can be produced. The geometry of the extrusion screws may be cylindrically parallel or conical or otherwise shaped.

In preferred embodiments of the present invention, the pharmaceutical composition has a phospholipid content of at least 30% by weight (300 mg / g VPG), more preferably at least 40% by weight (400 mg / g VPG), and most preferably at least 45%. -% (450 mg / g VPG); Also included are a phospholipid content of at least 50% by weight (500 mg / g VPG), and a phospholipid content of at least 60% by weight (600 mg / g VPG). Also included are a phospholipid content below 30% by weight (300 mg / g VPG) and above 60% by weight (600 mg / g VPG). In specific embodiments of the invention, the weight ratio between the phospholipid and the at least one pharmaceutical agent is between about 1000: 1 and 5: 1, preferably between 500: 1 and 25: 1, more preferably between 250: 1 and 40: 1, and even more preferably between 150: 1 and 50: 1. In a particularly preferred embodiment of the invention, the weight ratio between the phospholipid and the at least one pharmaceutical agent is about 100: 1.

There are extrusion processes to describe, which are characterized by different temperatures and temperature zones in the extruder. Et al can be heated and / or cooled in the extruder. These temperature-controlled steps can follow each other in any order, length and frequency. It can be extruded by both cooled and heated nozzles.

In one aspect of the invention, the active pharmaceutical ingredient is released from the produced VPG for a period of more than 5 days, more than 10 days, more than 20 days, more than 30 days or more, and the initial release of the active ingredient is less than 35 %, more preferably less than 25%, most preferably less than 20%, even more preferably less than 10% of the total amount of active ingredient within the first 48 hours.

In the context of the present invention, a release means the release of one or more pharmaceutical active ingredients from its formulation, here from the VPG. The initial release is that release that takes place between the time of VPG application and 48 hours after this application. The rate of release of a pharmaceutically active compound from VPG can be determined by a variety of means, such as with flow cells, which are well known and can be readily practiced by those skilled in the art, given the teachings of this invention. According to the present invention, the release rate can be determined in vitro or in vivo.

Accordingly, the VPG encompassed by the pharmaceutical composition exhibits a substantially continuous rate of release of the at least one pharmaceutical agent entrapped in the VPG and greater over a period of at least about 5, preferably 10 days, preferably at least 20 days, more preferably at least 30 days preferably at least 45 days and most preferably 55 days. It is particularly preferred that a release rate be maintained over a period of at least 10 days, preferably at least 20 days, more preferably at least 30 days, even more preferably at least 45 days, and most preferably more than 55 days. The initial release of the active ingredient is less than 35%, preferably less than 25%, even more preferably less than 20%, most preferably less than 10% of the total amount of active ingredient within the first 48 hours.

The pharmaceutical composition of the present invention exhibits a substantially continuous rate of release of the pharmaceutical agent entrapped within the VPG over a period of time. The term "sustained release rate" is used in the context of the invention for a release of the pharmaceutically active substance that is stable and preferably stable in amount. Accordingly, the term "continuous" means release that occurs without interruption and preferably without increase or decrease.

The total amount of active ingredient in the context of the present invention is that amount of a pharmaceutical active ingredient which is in a defined amount of VPG.

In another aspect, the method of the invention further comprises a step (iv) of extruding through a RAM extruder, which step occurs after steps (i) - (iii).

A RAM extruder or piston extruder in the sense of the present invention is an extruder in which a piston is used instead of an extrusion screw. Both RAM extruders with vertically and horizontally arranged pistons can be used. Material to be extruded is gravity fed into a chamber where it is heated to sintering temperature. The hydraulic piston of the RAM extruder then generates pressure on the material to be extruded, whereby the material is forced out of the chamber through an exit, thereby assuming a shape predetermined by the exit.

The VPGs produced by the processes according to the invention have a viscosity between 0.05 Pa * s and 90 Pa * s, preferably between 1 and 40 Pa s, more preferably 1 to 25 Pa s, particularly preferably 1-15 Pa smaller than 10 Pa * s most preferably less than 5 Pa * s.

The VPGs produced by the methods according to the invention can be introduced into the subcutaneous tissue with the aid of commercially available needle and syringe, as well as with the aid of needleless injection. An introduction into other body cavities such as in the periodontal pocket, in the ear, in the inner ear, in the bladder, in the uterus, in the conjunctival sac, in joints or intrathecal is possible.

In a further aspect, the VPG produced according to the invention and comprising at least one pharmaceutical active substance is used for the treatment or prevention of a disease.

A disease or dysfunction within the meaning of the present invention is the presence of a disorder requiring treatment in the sense of medical therapy. An injury falls under the previous definition accordingly. Certain embodiments may be injuries or degenerative diseases of the bones, cartilages, tendons, ligaments, vessels, skin, muscles, or a combination thereof. In other embodiments, diseases or disorders may be AIDS, diabetes, muscular dystrophy or oncological diseases.

A treatment within the meaning of the present invention is the treatment of a disease or dysfunction or injury. One treatment is the therapy with the VPG produced according to the invention, preferably a topical therapy. Also conceivable are other forms of therapy and a mixture thereof, namely a topical therapy (VPG) in the context of a surgical therapy, wherein the VPG is administered. Treatment includes treatment of both humans and animals.

A prevention in the sense of the present invention is the treatment with the VPG produced according to the invention, which can either prevent or delay a disease, dysfunction or injury.

In a further aspect, the VPG produced according to the invention and comprising at least one pharmaceutical active substance is used for cosmetic purposes. The cosmetic application can be done in humans or animals.

In a further aspect, the VPG produced according to the invention and comprising at least one pharmaceutical active substance is used as a dietary supplement or as part of a dietary supplement. The dietary supplement can be intended for both humans and animals.

The production process according to the invention has the advantageous effect of making VPGs scalable and comparatively fast with low shear forces, and it is also possible to include sensitive pharmaceutical agents which have advantageous release properties.

In contrast to the prior art, the method according to the invention can be used for the production of VPG and can be carried out comparatively easily and quickly. Because of these characteristics of the process, sensitive pharmaceutical agents, such as proteins that are relatively unstable drugs, can be included in VPG.

The present invention is further described by the following examples and figures, which are merely for the purpose of illustrating specific embodiments of this invention and are not to be construed as limiting the scope of the invention in any way.

EXAMPLES

Example 1: Preparation of Vesicular Phospholipid Gels by Two Screw Extrusion

As a lipid component, egg lecithin (Lipoid E80, Lipoid GmbH, Germany) was used in a concentration range between 30% (m / m) and 60% (m / m). The components for the preparation of the VPGs (phospholipids and aqueous component) were accurately weighed and premixed for three minutes by slow stirring with a magnetic stirrer. Then, the resulting pre-mixture was supplied to the running extruder. Two different laboratory-scale extruders were used. A mini-extruder ZE-5 (ThreeTec GmbH, Seon, Switzerland) and a MiniLab® Micro Rheology Compounder from HAAKE (ThermoFisher Scientific, Karlsruhe, Germany) was used for production. Various parameters such as the speed of rotation of the screws, the recycling time of the integrated bypass (HAAKE extruder), the nozzle diameter and the nozzle length, as well as temperature and lipid content were varied.

In all cases, VPGs with homogeneous appearance and consistency were obtained. No phase separation was observed, even after prolonged standing (3 months) of VPG at room temperature. Table 1: Macroscopic evaluation of the VPGs after preparation by different methods during storage for 3 months at room temperature. VPGs without visible separation of lipid phase and aqueous phase are referred to as "homogeneous". As "non-homogeneous" are designated VPGs with visible lipid accumulations or visible separation of the aqueous phase from the lipid. Magnetic stirrer DAC extrusion process After production Not homogeneous, small lipid droplets homogeneously homogeneously After a month Not homogeneous, small lipid droplets homogeneously homogeneously After 3 months Not homogeneous, larger lipid accumulations homogeneously homogeneously

Example 2:

Temperature transfer during the manufacturing process

The temperature during production was determined with the aid of a thermometer (Data Logger Thermometer HH147U, OMEGA, Deckenpfronn, Germany) directly after completion of the manufacturing process. For this purpose, the sensor of the thermometer was inserted into the VPG mass immediately after production.

The temperature measurements gave the following temperatures. Table 1: Overview of temperature measurements after production by extrusion. temperature adjustment temperature 10 ° C 13.3 ° C ± 0.2 ° C 20 ° C 20.5 ° C ± 0.2 ° C 30 ° C 29.7 ° C ± 0.2 ° C 60 ° C 54.3 ° C ± 0.7 ° C

For the extruded samples, the measured product temperatures are nearly equal to the set screw and barrel / barrel temperatures. When manufactured using the extruder, the product temperatures are easily controlled by controlling the screw and barrel / barrel temperature. It is easy to produce VPG at temperatures in the range of 13 ° C to 30 ° C, a warming during the process does not take place. It is also possible to produce VPG at elevated, well-defined, constant temperatures.

Example 3:

Quality of the VPGs

VPG was prepared by the method described in Example 1 using the following parameters. A rotational speed of 300 min ^-1 with a temperature of 30 ° C and a nozzle diameter of 0.5 mm was used. VPG was produced with a phospholipid content between 35% and 50%. For control, samples were also prepared using the DAC (3500rpm, 45 min) and magnetic stirring (45 min). The VPG quality after production by extrusion was investigated by using viscosity measurements and by determining the vesicle sizes after dilution to liposome dispersions. Furthermore, the VPGs were assessed macroscopically after preparation.

Macroscopic homogeneity

To assess the macroscopic homogeneity of the prepared VPGs, the gels were evaluated immediately after preparation and photographed. Macroscopic images of the two controls show an inhomogeneous mixture with large lipid droplets after preparation with the magnetic stirrer, while the DAC samples have a very homogeneous appearance. Macroscopic images of the samples prepared by extrusion show a homogeneous mixture. There are no lipid drops visible. (Please refer ).

particle size

To determine the vesicle size after preparation of the VPGs, dilute 10 mg VPG in 1 ml of highly purified water. This dispersion was then diluted a further 1:10 ratio and characterized by Dynamic Light Scattering (DLS). For this purpose, a Zetasizer Nano ZS (Malvern Instruments, UK) was used.
Differences in homogeneity could be detected by DLS measurements. In this case, the homogenous region was sampled in the VPGs prepared with magnetic stirrers. It should be noted that the VPGs prepared with magnetic stirrers in their entirety do NOT have this degree of dispersity, but are to be regarded as inhomogeneous. VPGs made with a magnetic stirrer show high vesicle sizes with high polydispersity after dilution compared to VPGs made with DAC. For the DAC samples, relatively smaller vesicle sizes and lower polydispersity were observed. VPGs prepared by extrusion show vesicle sizes and polydispersity in the range between the other two production methods and are thus much better classified than the samples which were produced by the magnetic stirring process. (Please refer and ,

viscosity

To determine the viscosity, the VPGs were measured with a rotational viscometer (Physica MCR 100, Anton Paar GmbH, Ostfildern, Germany) with a plate-plate geometry (PP-25). 0.3 g VPG were investigated in a shear range from 10 s ^-1 to 100 s ^-1 at 25 ° C. The viscosity was given for comparability at a shear rate of 32.9 s ^-1 .

The results of the viscosity measurements of the extruded VPGs show an increasing viscosity with increasing phospholipid content. The values of the VPGs which were produced by extrusion are between the viscosity of the control groups which were produced with the aid of a magnetic stirrer or a DAC. The injectability of the VPGs is directly correlated with the viscosity. By means of normal needle injection, a correct injection (injection rate 0.1 ml / sec, 27 G cannula) is no longer possible from a viscosity of approx. 0.5 Pa * s. By means of an advantageous needle-free injection, an injection is possible up to a viscosity of about 10 Pa * s, moreover no more.

VPGS prepared by extrusion can thus be advantageously injected against VPGs prepared by the DAC method. For this purpose, the lipid type and lipid levels must be selected so that the maximum possible viscosity for injection is exceeded (See ).

Example 4:

Effect of different extrusion parameters on the viscosity

All VPGs were prepared in a simple extrusion process with a phospholipid content of 45% (m / m). Various extruders were used for the extrusion, as a representative example, the results of the MiniLab® Rheology Compounder from HAAKE are used. Before extrusion, egg lecithin and buffer (20 mM PBS pH 7.4) were premixed by stirring for three minutes.

The rotational speed of the screws was varied between 40 min ^-1 , 100 min ^-1 , 200 min ^-1 and 300 min ^-1 . As the rotational speed increased, smaller vesicles and thus better homogeneity could be achieved without observing an increase in viscosity. (Please refer ).

Furthermore, the effect of different screw and barrel temperatures on the viscosity was determined. For this purpose, the following temperatures were set: 10 ° C, 20 ° C, 30 ° C and 60 ° C. There were no significant differences in viscosity.

Example 5:

In vitro release

An important feature of VPG is its delayed release. It was checked whether this property is influenced by the manufacturing technique.
To investigate the in vitro release, approximately 0.5 g of the differently prepared VPGs (phospholipid content 45% (m / m), loaded with 2 mg / g FITC-dextran, molecular weight 70 kDa) were weighed into specially prepared flow cells made of Teflon. The release cells consist of a bottom donor compartment in which the VPGs reside and an overhead acceptor section through which the release medium (20 mM PBS pH 7.4) is pumped at a constant flow (0.5 ml / hr). For the release experiments, the cells were incubated at 37 ° C in a water bath. The amount of FITC-dextran released (λex = 492 nm, λem = 518 nm) was determined using a fluorescence spectrophotometer (Varian Cary Eclipse, Agilent Technologies, Oberhaching, Germany).

VPG produced by the DAC or magnetic stirrer show prolonged release of FITC-dextran over several weeks. Surprisingly, a slower release rate was found for VPG made by extrusion compared to the two controls. The release is characterized in particular by a long duration and the absence of a significant burst (burst = rapid release at the beginning (within 48 h) of more than 20%). (Please refer ).

Example 6:

Inclusion of proteins and their stability

Ervthropoietin (EPO)

Erythropoietin (EPO) was included by direct inclusion by extrusion in VPGs (consisting of 40% Lipoid E80 and 20 mM PBS pH 7.4 with 3 mg / g EPO). Protein integrity and protein aggregation tendency were assessed by SDS-PAGE. After VPG preparation, the protein-containing VPG was diluted with the appropriate buffer to a protein concentration of 0.05 mg / ml. Tris buffer pH 6.8 with 2% SDS and 2% glycine was added and the samples were denatured at 90 ° C for 10 minutes. A NuPAGE® 4-12% Bis-Tris gel (NOVEX high performance pre-cast gels, Invitrogen, Karlsruhe, Germany) and NuPAGE® MES SDS running buffer (Invitrogen, Karlsruhe, Germany) were used in an XCall II Mini cell system (Novex , San Diego, CA, USA). Each well was loaded with 12 μl sample volume. The molecular weight marker Mark12 ™ used by Invitrogen (Groningen, The Netherlands). The electrophoresis was carried out at a constant voltage of 200 V for about 25 minutes. Subsequently, the gels were immediately stained with SimplyBlue ™ SafeStain (Invitrogen, Karlsruhe, Germany).

anakinra

Anakinra was included by direct inclusion by extrusion in VPGs (consisting of 40% Lipoid E80 and Citrate buffer pH 6.5 with 5 mg / g anakinra). Macroscopically homogeneous VPG was obtained. Protein integrity and protein aggregation tendency were assessed by SDS-PAGE. After VPG preparation, the protein-containing VPG was diluted with the appropriate buffer to a protein concentration of 0.05 mg / ml. Tris buffer pH 6.8 with 2% SDS and 2% glycine was added and the samples were denatured at 90 ° C for 10 minutes. A NuPAGE® 4-12% Bis-Tris gel (NOVEX high performance pre-cast gels, Invitrogen, Karlsruhe, Germany) and NuPAGE® MES SDS running buffer (Invitrogen, Karlsruhe, Germany) were used in an XCall II Mini cell system (Novex , San Diego, CA, USA). Each well was loaded with 12 μl sample volume. The molecular weight marker Mark12 ™ used by Invitrogen (Groningen, The Netherlands). The electrophoresis was carried out at a constant voltage of 200 V for about 25 minutes. Subsequently, the gels were immediately stained with SimplyBlue ™ SafeStain (Invitrogen, Karlsruhe, Germany).

Human serum albumin (HSA)

HSA was included by direct inclusion by extrusion in VPG (consisting of 40% Lipoid E80 and 20 mM PBS pH 7.4 with 5 mg / g HSA). Macroscopically homogeneous VPG was obtained. Protein integrity and protein aggregation tendency were assessed by SDS-PAGE. After VPG preparation, the protein-containing VPG was diluted with the appropriate buffer to a protein concentration of 0.05 mg / ml. Tris buffer pH 6.8 with 2% SDS and 2% glycine was added and the samples were denatured at 90 ° C for 10 minutes. A NuPAGE® 4-12% Bis-Tris gel (NOVEX high performance pre-cast gels, Invitrogen, Karlsruhe, Germany) and NuPAGE® MES SDS running buffer (Invitrogen, Karlsruhe, Germany) were used in an XCall II Mini cell system (Novex , San Diego, CA, USA). Each well was loaded with 12 μl sample volume. The molecular weight marker Mark12 ™ used by Invitrogen (Groningen, The Netherlands). The electrophoresis was carried out at a constant voltage of 200 V for about 25 minutes. Subsequently, the gels were immediately stained with SimplyBlue ™ SafeStain (Invitrogen, Karlsruhe, Germany).

Monoclonal antibody (type IgG1)

A monoclonal antibody was included by direct inclusion by extrusion in VPGs (consisting of 40% Lipoid E80 and 20 mM PBS pH 7.4 with 10 mg / g antibody). Protein integrity and protein aggregation tendency were assessed by SDS-PAGE. After VPG preparation, the protein-containing VPG was diluted with the appropriate buffer to a protein concentration of 0.05 mg / ml. Tris buffer pH 6.8 with 2% SDS and 2% glycine was added and the samples were denatured at 90 ° C for 10 minutes. A NuPAGE® 3-8% Tris-Acetate gel (NOVEX high performance pre-cast gels, Invitrogen, Karlsruhe, Germany) and NuPAGE® Tris-Acetate SDS running buffer (Invitrogen, Karlsruhe, Germany) were loaded into an XCall II Mini cell system ( Novex, San Diego, CA, USA). Each well was loaded with 12 μl sample volume. Used as the molecular weight marker HiMark12 ™ Pre-stained Protein Standard by Invitrogen (Groningen, The Netherlands). The electrophoresis was carried out at a constant voltage of 200 V for about 25 minutes. Subsequently, the gels were immediately stained with SilverXPress® Silver Staining Kit (Invitrogen, Karlsruhe, Germany).

Beriglobin

Beriglobin was included by direct inclusion by extrusion in VPGs (consisting of 40% Lipoid E80 and 20 mM PBS pH 7.4 with 5 mg / g beriglobin). Macroscopically homogeneous VPG was obtained. Protein integrity and protein aggregation tendency were assessed by SDS-PAGE. After VPG preparation, the protein-containing VPG was diluted with the appropriate buffer to a protein concentration of 0.05 mg / ml. Tris buffer pH 6.8 with 2% SDS and 2% glycine was added and the samples were denatured at 90 ° C for 10 minutes. A NuPAGE® 3-8% Tris-Acetate gel (NOVEX high performance pre-cast gels, Invitrogen, Karlsruhe, Germany) and NuPAGE® Tris-Acetate SDS running buffer (Invitrogen, Karlsruhe, Germany) were loaded into an XCall II Mini cell system ( Novex, San Diego, CA, USA). Each well was loaded with 12 μl sample volume. Used as the molecular weight marker HiMark12 ™ Pre-stained Protein Standard by Invitrogen (Groningen, The Netherlands). The electrophoresis was carried out at a constant voltage of 200 V for about 25 minutes. Subsequently, the gels were immediately stained with SilverXPress® Silver Staining Kit (Invitrogen, Karlsruhe, Germany).

Depending on the sensitivity of the proteins to shear stress, all candidates showed comparable or even slightly improved stability compared to the DAC and the magnetic stirrer. (Please refer for EPO, for mAb).

Example 7:

Storage stability of the VPGs

VPGs prepared by magnetic stirring, VPGs prepared by DAC and VPGs prepared by TSE were stored at room temperature for 3 months. VPGs with a phospholipid content of 40% (m / m) consisting of egg lecithin (Lipoid E80, Lipoid GmbH, Germany) and 20 mM PBS pH 7.4 were used for this study. It was found that the VPGs produced by means of magnetic stirring did not show any homogeneous dispersion after three months. Clear lipid deposits are visible here. VPGs made by DAC or TSE were both macroscopically and microscopically stable.

In vitro release assay with VPG loaded with monoclonal antibody showed that the released protein was monomeric, i. not aggregated, was.

REFERENCES

• Brandl M, Drechsler M, Bachmann D, Bauer KH. Morphology of semisolid aqueous phosphatidylcholine dispersions, a freeze fracture electron microscopy study, Chem. Phys. Lipids. 87 (1997) 65-72. doi: 10.1016 / S0009-3084 (97) 00028-5 ,
• Brandl M. Vesicular phospholipid gels, in: Liposomes - Methods Protoc., 2003: pp. 205-212 ,
• Brandl M. Vesicular Phospholipid Gels: A Technology Platform, J. Liposome Res. 17 (2007) 15-26 ,
• Brandl M. Proc. International Symposium on Controlled Release of Bioactive Materials 1995, 22, 472-473
• Kissel T., Li Y. and Unger F. ABA-triblock copolymers from biodegradable polyester A-blocks and hydrophilic poly (ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins. Advanced Drug Delivery Reviews (2002): 54 (1): 99-134 ,
• Massing U, Cicko S, Ziroli V. Dual asymmetric centrifugation (DAC) -A novel technique for liposome preparation, J. Control. Release. 125 (2008) 16-24. doi: 10.1016 / j.jconrel.2007.09.010 ,
• Schulze S, Winter G. Lipid extrudates as novel sustained release systems for pharmaceutical proteins, J. Control. Release. 134 (2009) 177-185. doi: 10.1016 / j.jconrel.2008.11.026 ,
• Swarbick, J and Boylan JC. Extrusion and extruder. In Encyclopedia of Pharmaceutical Technology, Marcel Dekker, Inc., New York, 1992, pp. 395-441
• White JL. Twin screw extrusion - Technology and Principles, 2nd Ed., Hanser Publishers, Munich, 1990 ,
• White JL, Potente H. Screw Extrusion - science and technology, Hanser Publishers Munich, 2002 ,
• Zhong Y, Chen L, Zhang Y, Li W, Sun X, Gong T, Zhang Z. Vesicular phospholipid gels using low concentrations of phospholipids for the sustained release of thymopentin: pharmacokinetics and pharmacodynamics, pharmacy. 68 (2013) 811-815 ,
• Zhang Y, Zhong Y, Hu M, Xiang N, Fu Y, Gong T, Zhang Z. In vitro and in vivo sustained release of exenatide from vesicular phospholipid gels for type II diabetes, Drug Dev. Ind. Pharm. 9045 (2015) 1-8 ,

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 2005/0238721 A1 [0011]
• EP 2280681 A1 [0012]
• EP 1968553 A2 [0013]
• EP 1881819 A1 [0014]

Cited non-patent literature

• Brandl et al., 1997 and Massing et al., 2008 [0003]
• Brandl M, Drechsler M, Bachmann D, Bauer KH. Morphology of semisolid aqueous phosphatidylcholine dispersions, a freeze fracture electron microscopy study, Chem. Phys. Lipids. 87 (1997) 65-72. doi: 10.1016 / S0009-3084 (97) 00028-5 [0133]
• Brandl M. Vesicular phospholipid gels, in: Liposomes - Methods Protoc., 2003: pp. 205-212 [0133]
• Brandl M. Vesicular Phospholipid Gels: A Technology Platform, J. Liposome Res. 17 (2007) 15-26 [0133]
• Brandl M. Proc. International Symposium on Controlled Release of Bioactive Materials 1995, 22, 472-473 [0133]
• Kissel T., Li Y. and Unger F. ABA-triblock copolymers from biodegradable polyester A-blocks and hydrophilic poly (ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins. Advanced Drug Delivery Reviews (2002); 54 (1): 99-134 [0133]
• Massing U, Cicko S, Ziroli V. Dual asymmetric centrifugation (DAC) -A novel technique for liposome preparation, J. Control. Release. 125 (2008) 16-24. doi: 10.1016 / j.jconrel.2007.09.010 [0133]
• Schulze S, Winter G. Lipid extrudates as novel sustained release systems for pharmaceutical proteins, J. Control. Release. 134 (2009) 177-185. doi: 10.1016 / j.jconrel.2008.11.026 [0133]
• Swarbick, J and Boylan JC. Extrusion and extruder. In Encyclopedia of Pharmaceutical Technology, Marcel Dekker, Inc., New York, 1992, pp. 395-441 [0133]
• White JL. Twin screw extrusion - Technology and Principles, 2nd Ed., Hanser Publishers, Munich, 1990 [0133]
• White JL, Potente H. Screw Extrusion - science and technology, Hanser Publishers Munich, 2002 [0133]
• Zhong Y, Chen L, Zhang Y, Li W, Sun X, Gong T, Zhang Z. Vesicular phospholipid gels using low concentrations of phospholipids for the sustained release of thymopentin: pharmacokinetics and pharmacodynamics, pharmacy. 68 (2013) 811-815 [0133]
• Zhang Y, Zhong Y, Hu M, Xiang N, Fu Y, Gong T, Zhang Z. In vitro and in vivo sustained release of exenatide from vesicular phospholipid gels for type II diabetes, Drug Dev. Ind. Pharm. 9045 (2015) 1-8 [0133]

Claims (14)

Process for the preparation of vesicular phospholipid gel (VPG), wherein the VPG is prepared by screw extrusion. Process for the preparation of VPG according to Claim 1 wherein the VPG further comprises at least one lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes and salts of the fatty acids and derivatives thereof. Method according to Claim 1 or 2 composition comprising the steps of: (i) supplying at least one phospholipid and an aqueous liquid, optionally at least one pharmaceutical agent, which is preferably a protein, peptide, polypeptide, nucleotide, antibody, antibody derivative, virus, virus -like particle, or a low molecular weight drug, optionally of at least one lipid selected from the group consisting of fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, sphingolipids, cholesterol, waxes and salts of the fatty acids and derivatives in a screw extruder, (ii ) Homogenizing the one or more phospholipids, the aqueous liquid and / or the optional pharmaceutical agent in the screw extruder to form the VPG, and (iii) extruding the VPG, which steps may also take place simultaneously. Method according to Claim 3 wherein the at least one phospholipid, the aqueous liquid, the optional, at least one pharmaceutical agent, and / or the optional, at least one lipid are fed separately to the screw extruder, wherein the supply can be carried out simultaneously or with a time delay. Method according to Claim 3 wherein the at least one phospholipid and the aqueous liquid are mixed prior to feeding and fed as a mixture to the screw extruder. A method according to any one of the preceding claims, wherein the screw extrusion is a twin-screw extrusion. Method according to one of Claims 3 to 6 , wherein the temperature during the extrusion is between 4 ° C and 60 ° C and the rotational speed of the extrusion screw (s) between 5min ^-1 and 500 min ^-1 , preferably between 20 min ^-1 and 360 min ^-1 . Method according to one of Claims 3 to 7 in which several steps of extrusion are performed. Method according to one of Claims 3 to 8th in which (i) the extrusion screw (s) have a conveying function and optionally additionally kneading or mixing segments or differently shaped segments for mixing, transport, temperature control, compression or discharge, and / or (ii) the extrusion screw (s) are the same or (iii) the nozzles used for the extrusion have different diameters, lengths and / or shapes, and / or (iv) the outlet of the extruder to the axis of the extruder screw (s) is rectilinear or rectilinear is arranged. A process according to any one of the preceding claims, wherein the proportion of phospholipid (s) in the VPG is 30-60% by weight. Method according to one Claims 3 to 10 wherein the at least one pharmaceutically active agent is released from the produced VPG over a period of more than 5 days and the initial release of the active ingredient is less than 20% of the total amount of active ingredient within the first 48 hours. Method according to one of Claims 3 to 11 , further comprising a step (iv) extruding through a RAM extruder, said step taking place after steps (i) - (iii). Method according to one of Claims 3 to 12 , further comprising a step (v) single or multiple extruding with a screw extruder optionally with the screw extruder being the same as in step (i) - (iii) or another screw extruder, this step after steps (i) - (iii) and optionally before or after step (iv) takes place. Method according to Claim 13 in step (v), the one or more other and / or one or more other phospholipids, the same and / or one or more further aqueous liquids, or the the same and / or one or more additional active pharmaceutical ingredients, and / or the same or both and / or one or more other lipid (s) as in step (i).

Images