EP2760437A1 - Système de pulvérisation permettant de produire une matrice formée in situ - Google Patents

Système de pulvérisation permettant de produire une matrice formée in situ

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Publication number
EP2760437A1
EP2760437A1 EP12762603.4A EP12762603A EP2760437A1 EP 2760437 A1 EP2760437 A1 EP 2760437A1 EP 12762603 A EP12762603 A EP 12762603A EP 2760437 A1 EP2760437 A1 EP 2760437A1
Authority
EP
European Patent Office
Prior art keywords
polymer
spray system
solvent
plga
matrix
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12762603.4A
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German (de)
English (en)
Inventor
Rudolph CARSTEN
Senta ÜZGÜN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ethris GmbH
Original Assignee
Ethris GmbH
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Filing date
Publication date
Application filed by Ethris GmbH filed Critical Ethris GmbH
Publication of EP2760437A1 publication Critical patent/EP2760437A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7015—Drug-containing film-forming compositions, e.g. spray-on
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • the invention relates to a spray or application system for use in preventing adhesions, in particular surgical adhesions.
  • the peritoneum as a serous skin, coats the abdominal cavity and consists of a visceral and a parietal leaf with a serous cleft space filled with 5 to 20 ml of fluid, allowing free movement of the organs. Histologically, the peritoneum consists of a single-layered squamous epithelium, also called the mesothelial layer, and a narrow layer of subserous connective tissue.
  • a breach of the mesothelial layer leads to the formation of solid adhesions between the two leaves and the surrounding tissue within a few days.
  • the mesothelial layer can be damaged by the use of swabs, the drying out of the surface during surgery, or by contact with talcum from appropriately powdered gloves. Even with minimally invasive surgery, such as laparoscopy, the activation process is set in motion.
  • FIG. 1 summarizes the pathogenesis of peritoneal adhesions with therapeutic options. Traumatization of the peritoneal tissue is believed to produce an inflammatory response with exudation of inflammatory cells and soluble fibrin monomers. Within about 3 hours, this results in the formation of the first fibrous structures, which, with sufficient fibrinolytic activity by the serine protease Plasmin can be resolved within the first few days. If this does not happen, it leads to the formation of collagen-rich connective tissue, the permanent adhesion, which then causes the difficulties.
  • neutrophilic leukocytes While neutrophilic leukocytes are involved in the inflammatory process in the first two days after the injury, macrophages and mesothelial cells play a crucial role in the development of permanent adhesion. Both cell types are able to release plasminogen, a precursor of plasmin, into the bloodstream. In the blood capillaries, plasminogen is converted to plasmin by the serine protease plasminogen activator (Tissue / urokinase plasminogen activator, t-PA / u-PA). These proteases are also secreted by the mesothelial layer.
  • TNF tumor necrosis factor
  • TGF ⁇ tumor necrosis factor
  • interleukins interleukins
  • PAI-1 plasminogen activator inhibitor type 1
  • t-PA / PAI-1 balance is the key site for the development of peritoneal adhesions.
  • fibrinolytics have been considered to be promising for therapy, but none of these approaches has prevailed because of the associated disadvantages in the clinic. It turned out indicates that currently available fibrinolytic agents have inadequate antiadhesive activity, which may be due, inter alia, to their short plasma half-life. Due to the therefore required high dosage severe side effects occur, which prevent the use.
  • Known thrombolytic agents are streptokinase, urokinase and t-PA protein alteplase recombinantly produced (available as Actilyse ®) and its modified form reteplase (commercially available form Rapilysin ®). Alteplase has a plasma half-life of only 3 to 6 minutes, which could be increased to 13 to 16 minutes for the modified form of reteplase. For this reason, multiple applications and infusion pumps are necessary to achieve continuous levels of effect that produce high side effects.
  • resorbable tissues are known for this purpose, such as, for example, oxidized cellulose fibers, a combination of hyaluronic acid and carboxymethyl cellulose or PEG gels.
  • an adhesion barrier comprising a plurality of particles of a polymer combination of a biodegradable polymer and at least one water-soluble polymer deposited in the form of a film on a fabric to prevent adhesion.
  • the water-soluble polymer is intended to attract water from the fabric after application, to swell and thereby to allow the formation of film and provide water, so that the particles gradually degrade and release the active ingredient optionally contained.
  • the active ingredient in these particles, e.g. contain an anti-inflammatory agent.
  • the properties of the films obtained with this formulation depend on the amount of water available at the applied site and are not set to be replicable.
  • WO 2004/01 1054 describes a polymeric matrix composition having a polymer matrix of various types of low to high molecular weight polymers, which contains a solvent which is hardly miscible with water, which is intended to improve the plasticity of the polymer.
  • the proposed composition is a complex system of different types of polymers and therefore expensive to manufacture and use.
  • DE 100 01 863 describes implants that are formed at the intended location by mixing a carrier material and a solvent for this purpose shortly before use, so that at least part of the carrier material dissolves, in order then to form liquid-crystalline phases in the body.
  • the carrier material is provided in powder form and is used e.g. obtained by spray drying.
  • an active ingredient it must be ensured that sufficient mixing takes place in order to distribute the active ingredient evenly in the matrix produced.
  • a product based on Atrigel ® technology is commercially available in the form of a hormone preparation for the treatment of advanced, hormone-dependent prostate cancer.
  • a further object of the present invention is to provide an application system which can be sprayed directly onto the intended site, which is suitable for taking up and releasing in a controlled manner active ingredients, in particular hydrophilic active ingredients such as nucleic acids, proteins and peptides, which can produce a stable film at the site of application whose release properties can be adjusted and optimized.
  • active ingredients in particular hydrophilic active ingredients such as nucleic acids, proteins and peptides, which can produce a stable film at the site of application whose release properties can be adjusted and optimized.
  • an application system should be provided which is physiologically compatible. is not inhibiting the activity of proteins, peptides and nucleic acids and thus allows the release of active products.
  • the above objects are achieved with a sprayable application system as defined in claim 1.
  • the sprayable application system also referred to below as a spray system, comprises at least one lipophilic component which is formed at least from a polymer dissolved in a solvent, and an aqueous component and optionally at least one active substance. It may include other ingredients.
  • a carrier material is provided which is easy to use, which is stable, which can be applied at the desired site and to the desired extent and provide an active ingredient for the desired duration and at a desired rate.
  • a spray system consisting of two components, one component having at least one polymer dissolved in one solvent and the other component having at least one aqueous solvent by mixing the components directly before or during application and by spraying are applied, wherein from the components of the invention in situ, a matrix is formed, which decomposes after a predetermined period of time and selectively releases the active ingredient optionally contained in this time.
  • the film formed by the system according to the invention has a high quality and remains for a predetermined time at the applied location and exerts its effect there. Only by combining the features according to the invention is a carrier material having the desired properties obtained.
  • Important features of the present invention are the nature of the polymer and the solvent, as well as the form of application, i. contacting the two components directly before or during spraying or during spraying.
  • the lipophilic component which contains at least one glycolic acid and lactic acid-based polymer and at least one biocompatible solvent for the polymer, the solvent having a predetermined logP value, as explained in more detail below.
  • This lipophilic component is then mixed with at least one hydrophilic component which is at least containing at least one aqueous solvent, mixed immediately before or during spraying, which by precipitation of the polymer produces a matrix which forms a physical barrier at the sprayed location which can effectively prevent surgical adhesions.
  • the lipophilic component and / or the hydrophilic component contains at least one active substance which is enclosed in the film during spraying and film formation and released therefrom in a controlled manner, which additionally blocks the surgical adhesions in a physiological or biological manner.
  • the particular advantage of the present application system is that, by selecting the specially applied ingredients, upon spraying by precipitation of the polymer from the solvent, a polymer matrix is formed which, if the composition contains an active agent, also includes it.
  • precipitation of the polymer is meant that the solubility limit of the polymer is exceeded and the polymer is no longer or no longer completely dissolved in the solvent.
  • the precipitation and flame formation rate and thus the porosity of the resulting matrix can be predetermined, which leads to controlled release properties. Due to the variability of these polymers, there are many ways to set the optimal properties, but the optimal solution for the individual case is not always easy to find. In the following, therefore, parameters are described which allow the selection of an optimal system.
  • Critical to the system according to the invention are in particular the polymer used, the solvent used to dissolve the polymer, the proportion of polymer, solvent and aqueous component and optionally the proportion of active ingredient and its form.
  • the material used to form the matrix should be sterilizable and must allow controlled release of an entrapped drug for a period of time during which adhesions and scarring may form. This is a period in the range of at least two weeks and up to six weeks and preferably from two to four weeks. In addition, the material must have such a quality that it retains its strength long enough to fulfill the desired purpose of preventing adhesions.
  • the selection of the appropriate polymer is made by the inherent viscosity (natural logarithm of the relative viscosity relative to the concentration C of the solute). In a manner known per se, for PLGA polymers, the inherent viscosity is measured at 0.1% in CHCl 3 at 25 ° C.
  • the polymers used for the system according to the invention are those having an inherent viscosity in the range from 0.1 to 0.8, in particular 0.15 to 0.7 prefers. If the value is below 0.1, the polymers are often too small to have a sufficiently long effect. If the value becomes too large, sufficient film quality can not be ensured, and the startup time for the release may be too long. In order to achieve optimum properties, it is also possible to use mixtures of polymers of different molar mass.
  • the molecular weight of the PLGA polymers can also be determined by conventional methods, e.g. with gel permeation chromatography. It has been found that PLGA polymers with a molecular weight in the range of 10 to 63 kDa are well suited.
  • the system of the invention must be sprayable, which implies that it must be soluble or suspendable in a biocompatible solvent.
  • a measure of the quality and mechanical strength of the film formed from the application system according to the invention is the matrix or film quality, which can be determined by the method described in the examples.
  • FIG. 2 shows the results of tests on the film quality of some combinations of polymers and solvents described in the examples.
  • the quality is essentially determined by the choice of the solvent and the molecular weight of the polymer. For their determination, the percentage of the polymer in the supernatant (loss) and in the precipitate (matrix grade) based on the total amount of polymer used is determined.
  • the matrix quality of the resulting layer or film is critical to the system of the present invention because the system is expected to perform its function for at least two and up to six weeks.
  • the matrix grade should therefore be in a range of 80 to 100%, preferably 90 to 100% and most preferably 95 to 100%, the value being determined by the method described in the example at room temperature, i. about 25 ° C, is determined.
  • the matrix quality depends inter alia on the molecular weight of the polymers. It has been found that by using higher molecular weight polymers, a larger amount of polymer could be incorporated into the matrix, while with lower molecular weight polymers, polymer (for film formation) was lost. Thus, it has been found that for a PLGA polymer having a ratio of lactide to glycolide of 75:25 and an inherent viscosity of 0.5 to 0.7, ie, a relatively high molecular weight and using a polymer having esterified end groups, almost 100% of the amount of polymer used formed the matrix.
  • one of the essential components of the system is the polymer used.
  • poly (lactide-co-glycolide), usually poly (D, L-lactides-co-glycolide), hereinafter also referred to as PLGA polymers used Both D, L-lactide based polymers and those based on the enantiomerically pure L-lactide based polymers can be used.
  • Milk and / or glycolic acid based polymers have been known for some time, even for controlled release systems.
  • PLGA polymers are processed into microparticles or implants, which can then be used in different ways.
  • PLGA polymers are biocompatible and biodegradable and their properties can be tailored to suit their purpose.
  • glycolic acid and lactic acid based polymers dissolved in a solvent are used whose release kinetics are adjusted by molecular weight, molecular weight distribution and end groups.
  • the resulting matrix can be selected by appropriate selection of the polymer and the solvent whether the resulting matrix should effect a diffusion-controlled, erosion-controlled or both diffusion- and erosion-controlled release.
  • a rapid formation of the high-quality matrix contributes significantly to a linear release kinetics without initial loss of active ingredient.
  • PLGA polymers are better suited for the system according to the invention than pure polylactide (PLA) or pure polyglycolide (PGA).
  • PLA polylactide
  • PGA pure polyglycolide
  • ratio of lactide to glycolide units always refers to the molar ratio of the units in a polymer
  • Mixtures of different PLGA polymers can also be used Mixtures of all types of PLGA polymers can be used, for example mixtures of polymers in which molar ratio of lactide to glycolide units and / or molecular weight or inherent viscosity and / or type of lactide units (D / L or L) and / or end groups
  • D / L or L type of lactide units
  • end groups The most suitable mixture for the particular purpose can be found by routine experimentation.
  • the degradation rates of PLGA polymers depend on the proportion of PGA or PLA, with PLGA copolymers generally having shorter degradation rates than PLA polymers or PGA polymers. For this reason, PLGA polymers are preferred.
  • the shortest degradation times are achieved with polymers in which the ratio of lactide to glycolide is 50:50.
  • Increasing the PGA content in the polymer or using the pure stereoene enantiomer L-lactic acid over the monomer D- / L-lactic acid also increases the degradation times of the polymers by increasing the crystallinity of the polymer as water diffuses more easily into amorphous regions and thus these regions be broken down faster than crystalline. Thus, during degradation, the crystallinity of the polymer steadily increases.
  • the crystallinity and the degradation time can thus be predetermined. Furthermore, the degradation rates can be accelerated by shorter polymer chains and free end groups. Free end groups, ie, free hydroxy groups and free carboxy groups increase the hydrophilicity of the polymer, thereby increasing the rate of diffusion of the water and its level in the polymer matrix. In addition, by lowering the pH inside the matrix, free carboxyl groups catalyze the hydrolysis of the polymers. Therefore, according to the invention, preference is given to using polymers which have free end groups.
  • PLGA polymers having free end groups which have a ratio of lactide to glycolide units of 40:60 to 60:40, more preferably about 50:50 and / or which have an inherent viscosity of less than 0.6.
  • PLGA polymers having esterified end groups those having a ratio of lactide to glycolide units of 75:25 are preferred.
  • Suitable PLGA polymers are commercially available, for example Resomer polymers (available from Evonik Industries AG, Essen, Germany), especially those of Resomer ® H-Series or Resomer ® S series. Particularly suitable polymers are, for example, Resomer ® 502H, 503H and 504H or Resomer ® RG755S. Table 1 below lists some properties for preferred polymers: Table 1: Properties of the Resomer ® RG polymers used
  • the release data are from the publication by Eliaz and colleagues [44 [Eliaz, 2000 # 257].
  • the release data refers for Resomer ® RG 755 S and 503 H to the release of bovine serum albumin [44] and for Resomer ® RG 502 H and 504 H to the release of thymus DNA [45] of an injectable implant (10% to 20% PLGA (m / v) in tetraglycol).
  • the degradation of the polymer matrix occurs via ester hydrolysis to the biocompatible monomers lactic acid and glycolic acid, which are then metabolized to C0 2 and water through the Krebs cycle.
  • the degradation behavior of PLGA implants is due to bulk erosion, which is characterized by the fact that water diffuses faster into the polymer matrix than the degradation of the polymer takes place. Accordingly, there is a homogeneous mass loss over the entire cross section of the polymer matrix.
  • the degradation process can generally be divided into three sections:
  • Hydration The polymer absorbs water and swells, already splitting a small proportion of ester bonds. However, there is still no mass loss.
  • the degradation times are crucial for the release of encapsulated macromolecules and nanoscale carrier materials, since these are mainly released by matrix erosion because of their size and thus a targeted adjustment of the release rates is made possible by the degradation rates.
  • the degradation times of the PLGA polymers can be controlled by their composition and the molecular weight of the polymers, wherein the inherent viscosity is usually given as a measure of the molecular weight for the commercially available PLGA polymers.
  • the viscoelastic properties of the system also play a role, as shown in FIG. 3 and in the examples.
  • the carrier system formed from the administration system according to the invention is loaded with active ingredient, it is additionally necessary that the active ingredient with the desired release kinetics be released.
  • the matrix quality of the film obtained from the application system according to the invention depends on the polymer used, the solvent used for its solution and its water solubility. It has been found that the solvent used for the dissolution of the PLGA polymer has a considerable influence on the quality of the matrix produced therewith.
  • the matrix produced by combining the PLGA dissolved in the solvent with the aqueous phase thus depends on the nature and amount of the solvent, in particular its hydrophilicity.
  • the selection of the solvent also depends on the type of polymer used.
  • the more lipophilic the polymer the more lipophilic the solvent must be.
  • the lipophilicity of the polymer depends, inter alia, on the end groups, since a PLGA with free acid groups is more hydrophilic than a PLGA with esterified end groups.
  • the solvent must sufficiently dissolve the selected polymer so that the polymer can be sprayed; on the other hand, the solubility of the solvent in water must be sufficiently high so that precipitation can be rapid after spraying the two components.
  • One parameter useful in selecting the appropriate solvent is the logP value. Since, as stated above, the matrix quality changes as a function of the molar mass of the polymer by the solvent, a further essential feature of the invention is the solvent.
  • An important parameter for the selection of the solvent is the miscibility with water. The higher the miscibility with water, the faster the matrix formation, however, the porosity is higher, the lower the miscibility with water, the slower the matrix formation, the higher but also the quality.
  • the water miscibility of a solvent can be assessed by the logP value.
  • the logP value denotes the octanol / water partition coefficient, i. the ratio of the concentration of the solvent in a two-phase system of 1-octanol and water.
  • the definition of the logP value is:
  • logP iog-
  • - logc ⁇ - log cj.
  • the calculation or determination of the logP value is known per se.
  • One algorithm suitable for determining the logP value is XlogP3 as described in Cheng et al. (Cheng T., Zhaoy, Lix, Lin F., Xu Y., Zhang X. et al., Computation of Octanol-Water Partition Coefficients by Guiding to Additive Model with Knowledge. J. Chem. Inf. Model ; 47: 2140-2148).
  • the logP value calculated in this way gives positive values for lipophilic substances and negative values for hydrophilic substances.
  • suitable solvents for the system according to the invention are those whose lipophilicity is not too high, so that preference is given to those solvents which have a negative or at least very small positive XlogP3 value.
  • a more lipophilic solvent has a poorer water miscibility and therefore leads to a high matrix quality.
  • solubility of the polymer in the solvent also plays a role. The better the polymer dissolved in the solvent, the more water is required later to precipitate the polymer out of the film and form a film. On the other hand, the solubility must be such that a sufficient amount of polymer can be dissolved in the solvent. It has been found that a solvent which has a solubility for the PLGA to be used at room temperature of at least 5% (mass / volume) (m / v), preferably from 5 to 60%, and in particular a solubility of 10 to 30%, suitable for forming a high quality film.
  • the solubility of a solvent for a polymer decreases with increasing molecular weight of the polymer.
  • a very lipophilic polymer is used with a highly water-soluble solvent, the polymer is only slightly dissolved, while a non-lipophilic polymer, e.g. one with free acid groups and lower molecular weight in a hydrophilic solvent can be solved well.
  • the more water is required for the precipitation the better solved is the polymer.
  • a well-suited solvent thus combines good water miscibility with such polymer solubility that, for the desired amount of polymer, the solubility in the solvent at the application temperature, i. is between 30 and 40 ° C, near the saturation limit, wherein the solubility at room temperature must be sufficient in any case, that a stable solution is formed.
  • Tetraglycol, glycerol formal and dimethyl isosorbitol have proven particularly suitable.
  • the solvent tetrahydrofurfuryl alcohol-poly- ethylene glycol, termed tetraglycol or glycofurol, is a solvent that has long been used for paren- teralia concentrations up to 50% are used and at this dilution the solvent shows only low toxicity.
  • Glycerol formal is an odorless, low toxicity solvent consisting of a mixture of 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol, which is an excellent solvent for many pharmaceuticals and cosmetics. It is mainly used in veterinary medicine as a solvent for injections. Is commercially available for example as glycerol Ivumec ® and PTH ®. Ivumec TM is approved at 0.27% for subcutaneous application in pigs and is usually used at 0.1 ml / kg.
  • DMI Dimethyl isosorbide
  • Triacetin 18.75 0.2 35% parenteral k. A.
  • the layer thickness of the matrix formed from the spray system according to the invention plays a role in the rate of diffusion of the water.
  • surface erosion could additionally be detected.
  • the layer thickness of the films for the longer-chain polymer increased analogously to the observed release kinetics of DMI via glycerol formal to tetra glycol.
  • Another very important feature of the solvent to be used for the application system according to the invention is biocompatibility or tissue compatibility.
  • the tissue compatibility is determined by the influence of the solvent on the metabolic cell stability over a period of 11 hours. A method of determination is described in the examples.
  • the observed LD 5 o value is the measure of the toxicity.
  • the LD 50 must be at least 1, preferably at least 10 mg / ml in order to consider a solvent for the present application system.
  • the above-mentioned particularly preferred solvents fulfill this requirement.
  • Glycerol formal which has an LD 50 value of about 1 g / ml at an incubation time of less than 6 hours, has proven particularly suitable in this context. Glycerolformal is therefore a particularly preferred solvent for the inventive system.
  • Figure 5 shows LD 5 o values for preferred solvents depending on the incubation time.
  • the application system according to the invention consists only of a lipophilic component with polymer and solvent, as described above, and water as the second component. With these ingredients, if they meet the above conditions, mixing and spraying can be used to generate a film in situ which can effectively prevent surgical adhesions.
  • the spray system according to the invention contains the lipophilic component and the aqueous component separated from one another until they are sprayed on. Only at or just before spraying or during spraying may the Components are mixed. It has been found that even the addition of comparatively small amounts of water leads to a precipitation of polymer. If this precipitation took place too early, the film formation could be disturbed and, if necessary, the spraying device could be blocked by deposition of polymer. Preference should therefore be given to mixing directly during the spraying, for example by the respectively intended amounts of the two components are fed into a mixing chamber and sprayed directly therefrom during mixing from this. Mixing and spraying should thus preferably take place essentially simultaneously.
  • an application system which additionally contains an active ingredient.
  • active ingredients all substances useful for the intended application site are considered.
  • the application system according to the invention is particularly suitable for the release of nucleic acids, proteins and peptides. Thus, both proteins and peptides can be released directly as well as the nucleic acids encoding them or even a mixture thereof. It has been found that the application system according to the invention and the film formed therefrom release the nucleic acids in such a form that their subsequent expression is possible. Since the system according to the invention is intended for the prevention of adhesions, fibrinolytic proteins and peptides or the corresponding nucleic acids coding for them are preferably used as active ingredients.
  • the active ingredient may be dissolved or dispersed in one of the two components. It has been found that an excessively high proportion of aqueous phase can influence the quality of the film formed. Therefore, if it is desired to add an active ingredient whose solubility in water is not sufficiently high to produce highly concentrated solutions, it is preferable to use the active ingredient in already precipitated form, e.g. to be added in dried form. Particularly suitable are lyophilisates or polyplexes in finely divided solid form, which can be dispersed in the lipophilic component. This has the further advantage that the active ingredient in solid form is more stable for storage.
  • tissue-specific plasminogen activators and their inhibitors in particular play a role in the formation of adhesions.
  • a "gene-activated" film formed in situ is applied locally by spraying for the treatment of peritoneal adhesions, since, as described above, after an operation in the abdominal cavity in a time window of 2-3 weeks permanent adhesions can occur and there If it is assumed that the trigger for this is an imbalance between the tissue-specific plasminogen activator (tPA) and its inhibitor (PAI-1), this imbalance is modified according to the invention by providing tPA and / or inhibiting PAI-1 the film formed in situ, the tPA and / or PAI-1 inhibitor and / or this coding nucleic acid contains acids.
  • the spraying system according to the invention particularly preferably contains both at least one tissue-specific plasminogen activator or a nucleic acid encoding it and at least one plasminogen activator inhibitor inhibitor or a nucleic acid encoding same.
  • the spraying system according to the invention can therefore contain either at least one tissue-specific plasminogen activator or at least one PAI-1 inhibitor or a combination of both or in each case the corresponding nucleic acids.
  • the desired effect can be set very variably with the system provided according to the invention.
  • the nucleic acid may be RNA, DNA, mRNA, siRNA, miRNA, piRNA, shRNA, antisense nucleic acid, aptamer, ribozyme, catalytic DNA, and / or a mixture thereof.
  • DNA includes all suitable forms of DNA such as cDNA, ssDNA, dsDNA, etc.
  • RNA includes all suitable forms of RNA such as mRNA, siRNA, miRNA, piRNA, shRNA, etc.
  • the nucleic acid may be linear or circular, it may be single-stranded or double-stranded.
  • nucleic acid is also understood to mean a mixture of nucleic acids, each of which can code for identical or different proteins or peptides, All forms of nucleic acids which encode the desired protein or peptide and can express it at the desired site are suitable.
  • nucleic acid can originate from any source, eg from a biological or synthetic source, from a gene library or collection, it can be genomic or subgenomic DNA
  • the nucleic acid may contain the elements necessary for its amplification and expression, such as promoters, enhancers, signal sequences, ribosome binding sites, tails, etc.
  • the nucleic acid may be a DNA or RNA and may have one or more genes or fragments.
  • the nucleic acid may be an autonomously replicating or integrating sequence, it may be as a plasmid, vector or other form well known to those skilled in the art. It may be linear or circular and single or double stranded. Any nucleic acid active in a cell is suitable here. Since "naked" nucleic acids are not very stable and are rapidly inactivated and degraded in the cell, it is preferred to coat the nucleic acid with a layer, so-called polyplexes being a particularly preferred embodiment.
  • polyplexes are nucleic acid molecules surrounded by a polymer shell.
  • a cationic polymer is used as the shell material. It has been found that cationically charged particles can be more easily taken up by the cell than neutral or anionically charged particles, but they also tend to promote nonspecific deposits.
  • cationic shell materials are preferred, since nucleic acids can be enveloped and protected very easily with cationic substances. Corresponding methods are well known to the person skilled in the art.
  • the shell material may be a naturally occurring, synthetic or cationically derivatized natural substance, eg a lipid or a polymer or oligomer.
  • a natural oligomer is spermine.
  • synthetic polymers are nitrogen-containing biodegradable polymers, in particular those with protonatable nitrogen atoms.
  • Particularly suitable are polyethyleneimines, in particular branched polyethyleneimines, which are commercially available.
  • a branched polyethyleneimine having an average molecular weight of 25 kDa which is commercially available, is suitable. It has been found that this polymer is very compatible with the other constituents of the spray system according to the invention.
  • lipids in particular cationic or neutral lipids, as natural, optionally derivatized, layer-forming shell material. Lipids are available in many variants and can be used for example for the formation of liposomes.
  • the ratio of shell material to nucleic acid should be adjusted in a manner known per se so that the nucleic acid is sufficiently protected but can be expressed upon release. If too little shell material is present, the nucleic acid will not be sufficiently protected. If the proportion of shell material is too high, on the one hand problems with the compatibility can occur and on the other hand an excessive amount of shell material can lead to the fact that the nucleic acid can no longer be released and / or can no longer be expressed. In both cases, the efficiency of the transfer suffers. The expert can find the most suitable ratio in a few routine tests. Particularly suitable is a ratio of shell material to nucleic acid in the range of 10: 1 to 1: 4, by weight, proved.
  • the proportion of the polymer can also be given by the molar ratio of polymer nitrogen to the proportion of DNA phosphate (N / P);
  • N / P the molar ratio of polymer nitrogen to the proportion of DNA phosphate
  • the N / P ratio is in a range of 1 to 10, more preferably 4 to 8.
  • the polyplex molecules are designed so that the nucleic acid is protected during storage, transport and application, and then the nucleic acid is released and expressed at the target site.
  • Suitable polymers have been widely described in the literature, and those skilled in the art can choose from a variety of materials the most appropriate one.
  • Non-viral gene transfer systems represent a safe alternative to viral systems in terms of immunogenicity and mutagenesis potential.
  • Non-viral gene therapy approaches have been described. The application of naked nucleic acid in combination with physical methods such as electroporation, as well as the use of nanoscale complexes with synthetic carrier systems such as cationic polymers, which are also referred to as polyplexes.
  • the spray system of the present invention provides a new promising approach for achieving long-lasting gene expression.
  • a film in the form of a gene-activated depot system whose local application can lead to a constant nucleic acid level over a defined period of time in the area of application, advantageous properties are achieved. Dosage frequency and dosage amount can thereby be reduced, unwanted side effects such as transfection of other tissues, so-called off-target effects, prevented, unphysiological protein levels and the burden of the patient with nucleic acid and support material can be avoided and patient acceptance can be improved.
  • a spray system which contains as active ingredients a combination of PA and PAI-1 inhibitor or nucleic acids encoding them, wherein the ratio of PA and PAI-1 inhibitor in the range of 5: 1 to 1: 5, using the appropriate nucleic acids, the ratio can be adjusted so that at the target site after expression, a ratio of PA: PAI-1 inhibitor from 5: 1 to 1: 5 results. It has been found that when such a combination is applied, the formation of surgical adhesions can be suppressed particularly effectively.
  • the spray system according to the invention is characterized in that, when the two components are mixed, the polymer is precipitated very rapidly to form a film, with any active ingredients present in one or both of the components simultaneously being integrated into the film.
  • the two components which are stored in separate containers before use, are sprayed so that they are sprayed or mixed directly before or sprayed during mixing.
  • the two components of the spray system according to the invention are thus mixed for application. This is preferably done by the two separate components are performed for spraying in a mixing chamber and sprayed directly from it.
  • a device is used for spraying, as is known per se, in which, when the spray valve is actuated from two storage chambers, one dose in each case is conducted into a mixing chamber and sprayed together therefrom.
  • Spray applicators suitable for mixing / spraying two components previously separated are known in the art.
  • a known and suitable for the application according to the present invention device is shown in Figure 8 and is available as a spray kit from Baxter.
  • the dose to be dispensed between the two components can be adjusted. It depends on the type of use, the type of components, if necessary, the active ingredient.
  • the two components should be applied at a ratio (based on the volume of solutions / liquids) of 10:90 to 90:10, preferably 25:75 to 75:25 and more preferably in a ratio of 40:60 to 60:40 be mixed.
  • the amount of components dispensed for the film to be formed depends on the desired size and thickness of the film. It can be adjusted in a conventional manner. For administration in the abdomen an amount of 0.5 to 5 ml, preferably 0.7 to 3 ml of each component has been found suitable.
  • lipophilic component 10% (m / v) PLGA solution (Resomer ® RG H series) in glycerol formal, tetraethylene glycol or DMI,
  • hydrophilic component water for injections
  • Active ingredient component pDNA / l-PEI polyplexes, dissolved as lyophilisate in the hydrophilic phase (embedded variant A) or dispersed in the lipophilic PLGA solution by means of a homogenizer (embedded variant B).
  • the spray system according to the invention is provided for therapeutic use. Areas of application for matrix systems produced thereby are the prevention of postoperative adhesions, in which permanent adhesions can occur after abdominal surgery, triggered by an imbalance between the tissue-specific plasminogen activator and its inhibitor [56, 64, 65]. Critical is a time window of 2 weeks, which includes an acute phase of 2-5 days after surgery. Depot systems which contain a plasmid coding for tPA as the active component are particularly suitable. In addition to the pharmacologically active component of the polymer film additionally provides an anti-adhesive barrier against adhesions. It is also possible, for example, the spraying of the spray system according to the invention via endoscope, z. B. in an endoscopic procedure in the abdominal cavity.
  • FIG. 1 shows a schematic representation of the pathogenesis of surgical adhesions
  • FIG. 3 shows diagrams illustrating the results of examinations of the viscoelastic films: A) storage modulus (G '), B) loss modulus (G ") of Resomer ® RG H- series with different solvents in comparison at a frequency of 1Hz.
  • Figure 5 shows LD 5 o values of the solvents tested compared: LD 50 of the solvents tested as a function of incubation time on mesothelial cells. In each case, the metabolic cell viability was determined by ATPlite assay
  • FIG. 6 shows diagrams for release kinetics of different formulations in situ formed films:
  • A Resomer ® RG 502 H, polyplexes in a hydrophilic phase
  • B Resomer ® RG 502 H, polyplexes in a lipophilic phase
  • C Resomer ® RG 504 H, Polyplexes in hydrophilic ler phase
  • D Resomer RG 504 H, polyplexes in the lipophilic phase.
  • Figure 7 shows the transfection efficiency of lyophilized 1-PEI / pDNA polyplexes on lung cell lines using different cryoprotectants.
  • FIG. 8 shows a test setup for the production of in situ formed films
  • FIG. 10 shows results for in vitro application of in situ formed films on mesothelial cells.
  • A matrix release 504 H films based and
  • B Fluorescence image of the embedded plasmid DNA after staining with Propidium- iodide from Resomer ® RG.
  • FIG. 11 shows cotransfection of plasmid DNA / siRNA on mesothelial cells: a) PAI-1 and tPA detection in the Western Blot after 48 h, b) tPA / PAI-1 ratio as a function of time.
  • the polyplexes consisting of pCMV-tPA-IRES-Luc (ptPA) or a wild-type plasmid (pUC) and various siRNAs (PAI-1, EGFP) were treated with 1-PEI at an N / P ratio of 10 (relative to the pDNA amount) in HBS.
  • ptPA pCMV-tPA-IRES-Luc
  • pUC wild-type plasmid
  • PAI-1, EGFP various siRNAs
  • Figure 13 shows a dilution series of 1-PEI / pDNA polyplexes in PBS
  • Figure 14 shows a standard curve of the human tPA antigen assay
  • Figure 15 shows the transfection efficiency of powdered polyplexes using different cryoprotectants: transfection efficiency of lyophilized 1-PEI / pDNA polyplexes on lung cell lines under (A) using different cryoprotectants (10% (m / v) sucrose or mannose, 4% (m / v) dextran 5000, B) after homogenization of lyophilized polyplexes using 10% (m / v) sucrose as cryoprotectant.
  • the plasmid pCMVLuc which is available as described in [19], contains the luciferase gene (Luc) of the fire fly Photinus pyralis under the control of the CMV promoter, a promoter from cytomegalovirus.
  • the pMetLuc construct also encodes the luciferase gene of the rower crawler Metridia longa, a secreted luciferase enzyme, under the control of the CMV promoter [20].
  • the construct pCMV-tPA-IRES-Luc was cloned and is shown schematically in FIG. It contains, in addition to the sequences for the luciferase enzyme (Luc) and the tissue-specific plasminogen activator (tPA), a CMV promoter (CMV-IE, cytomegalovirus immediate-early).
  • the cloning of the pCMV-tPA-IRES-Luc plasmid was carried out using the pl-RES-Luc vector [21].
  • a tissue-specific plasminogen activator (tPA) -coding sequence was cloned into under the control of the CMV promoter using the restriction endonucleases Mlul and Fsel (New England Biolabs Inc., USA).
  • the sequence (insert) from the plasmid pCMV-tPA was amplified by means of polymerase chain reaction (PCR) [22].
  • the pIRES-Luc vector contained an internal ribosomal entry site (IRES), which allowed translation of both transcripts independently of one another.
  • control plasmid used was pUC21 vector (Invitrogen, Germany), which contains no expression cassette but only the bacterial backbone.
  • siRNA was screened against plasminogen activator inhibitor 1 (PAI-1, 5 '-GGAACAAGGAUGAGAUCAG [4, 23] -3') and as control a siRNA against EGFP
  • Linear polyethylenimine with a molecular weight of 22 kDa was synthesized according to a protocol by Plank and colleagues [24].
  • Linear PEI was obtained analogously to the procedure by acid hydrolysis of the propionic acid poly (2-ethyl-2-oxazoline) 50 Da, wherein the liberated propionic acid as an azeotrope mixture was continuously withdrawn from the synthesis approach, so that the reaction could proceed almost completely.
  • the free base was then precipitated by means of sodium hydroxide solution at pH 12, washed and lyophilized.
  • the lyophilized 1-PEI was stored at 4 ° C and dissolved in distilled water as needed, adjusted to pH 7.4, dialyzed (ZelluTrans dialysis membranes T2, MWCO 8-10 kDa) and sterile filtered.
  • Quantification of the PEI solution was carried out photometrically by means of the copper sulfate method at 285 nm on a spectrophotometer (Ultrospec 3100 Pro) [25].
  • a 1-PEI charge of known concentration was used.
  • the purity of the synthesis product was checked by 'H-NMR spectroscopy (Bruker 250 MHz, Düsseldorf).
  • the measurement of the molecular weight was carried out by gel permeation chromatography with a multi-angle laser scattering detector (GPC-MALLS) and gave a molecular weight of 20-22 kDa.
  • Pleural mesothelial cells human, short Met5A, from ATCC, Germany (CRL-9444) were used.
  • the cell line was cultured in a 1: 2 mixture of Ml 99 (Gibco-BRL, UK) and MCDB 105 (Sigma-Aldrich, Germany) at 37 ° C, 5% CO 2 and 100% humidity.
  • Ml 99 Gibco-BRL, UK
  • MCDB 105 Sigma-Aldrich, Germany
  • fetal calf serum PPA Laboratories, Austria
  • an epidermal growth factor (5 ng / ml, Sigma-Aldrich, Germany)
  • hydrocortisone 400 ng / ml, Sigma-Aldrich, Germany
  • the complexes were formed spontaneously by electrostatic bonding forces, the properties of the polyplexes formed thereby being essentially dependent on the ionic strength of the medium, the polymers used and the N / P ratio. This gives the molar molar ratio of protonated nitrogen atom (N) of the polymer structure to the negatively charged phosphate atom (P) in the nucleic acid.
  • N protonated nitrogen atom
  • P negatively charged phosphate atom
  • nucleic acid solution equal volumes of the lower charge density solution, the nucleic acid solution, were pipetted to the higher charge density solution, the polymer solution, and mixed by pipetting up and down (5-8 times). The solution was then incubated at RT for 20 min before further experiments were performed.
  • the medium used was water for injection (siRNA, plasmid DNA lyophilisate) and HBS pH 7.4 (plasmid DNA liquid).
  • the polyplexes were prepared using pCMVLuc and 1-PEI at a N / P ratio of 10 as described above in water for injections. To test different cryoprotective substances, the polyplexes were incubated after incubation with a 20% (m / v) sucrose solution, a 20% (m / v) mannose solution or a 4% (m / v) dextran 5,000 solution Diluted 1: 2, mixed and aliquoted. These could then be flash frozen in nitrogen and lyophilized for about 24 hours at maximum power in the lyophilizer. The lyophilizates were resuspended to a final concentration of 0.02 ⁇ g / ⁇ l (same starting concentration) in the respective medium and transfected onto BEAS-2B cells in 96-well plates analogously as described below.
  • Sucrose was added in powder form after an incubation time of 10 min and the complexes were incubated for a further 10 min, the particle size being controlled by PCS before and after addition of sucrose.
  • the powder was in a mortar homogenized and pestle and subsequently by means of homogenizer, a cylindrical glass vessel with glass pestle (debris Labortechnik, Germany), or by means of Ultra-Turrax ® (stage 3, 14 sec, Ika Labortechnik, Germany) PLGA solution to be suspended.
  • the powder was resuspended directly or previously homogenized by mortar in water for injection.
  • the lyophilizates were resuspended to a final concentration of 0.02 ⁇ g ⁇ l in the respective medium.
  • the determination of the hydrodynamic diameter of the polyplexes was carried out by means of photon correlation spectroscopy in a semimicrocuvial cuvette with 600 ⁇ Polyplex solution in bidistilled water (0.02 ⁇ g / ⁇ l pDNA), the zeta potential by means of electrophoretic light scattering in a macro cuvette with 1.6 ml Polyplex solution (0.02 or 0, / ⁇ 1 pDNA).
  • the following settings were used: 5 measurements (size measurement), 5 runs of 10 cycles per sample (zeta potential); Viscosity of water (0.89 cP) or HBS (1.14 cP); Ref index 1.33; Dielectric constant 78.5; Temperature 25 ° C.
  • the zeta potential was calculated according to Smoluchowski.
  • the evaluation of the size was based on a standard curve.
  • the device was pulsed with polystyrene latex particles 92 nm in size (Duke Scientific Cooperation, CA, USA) and zeta potential reference Bl-LC-ZRZ with a +50 mV charge (Laboratory chemistry, Vienna, Austria) at regular intervals Checked intervals.
  • Agarose gel electrophoresis can be used to determine the degree of complexation of the nucleic acid (plasmid DNA, mRNA) in polyplexes.
  • polyplexes were prepared as described above, mixed with 6-fold concentrated loading buffer and 100 ng each pDNA on a 0.8% agarose gel spiked with ethidium bromide (10 ug / 100 ⁇ ) applied. For reference, a corresponding size marker was applied. Electrophoresis was carried out at 125 V for approximately 1.5 h in 1x TAE buffer. Subsequently, the bands of the nucleic acid were detected under UV light (360 nm) and recorded by gel camera.
  • the supernatant was removed and dried in a vacuum system (Speed Vac, Dieter Piatkowski, Germany) to constant weight.
  • a freeze dryer Liscovac GT 2, LH Leybold, Germany
  • the matrix in a freeze dryer was also dried to constant weight, which then determines the percentage of the polymer in the supernatant (loss) and in the precipitate (matrix grade) based on the total amount of polymer used could be.
  • cytotoxicity of the solvents was determined by means of an ATP-based test assay (ATPlite, Perkin Elmer).
  • ATPlite ATPlite, Perkin Elmer
  • cells were seeded in a 96-well plate 24 hours before the experiment, the medium was removed immediately before the experiment, the cells were washed once with PBS and 50 .mu.l serum-containing medium with antibiotic addition (Penicillin / Streptomycin 0.1% (v / v); gentamycin 0.5% (v / v), Gibco-BRL, UK).
  • each 50 ⁇ different concentrations of solvent (16-500 ug / ul) diluted in water for injection, added and incubated for different lengths of time at 37 ° C, 5% C0 2 and 100% humidity (15, 30, 60, 221 , 360 and 640 min). After the incubation period, the medium was aspirated, the cells were washed once with PBS, 50 .mu.l PBS per well presented and determined cell viability according to the manufacturer. The measurement of luminescence was carried out in a plate reader (Wallac Victor2 / 1420 Mulitlabel Counter, PerkinElmer Inc., USA), using the luminescence of untreated cells (50 ⁇ M water for injection) as a reference value with a viability of 100%. For each time point, the concentration of the solvent against the measured cell viability (mean ⁇ standard deviation from n 4 batches) was plotted and a non-linear standard function was adjusted:
  • Both response variables can be mathematically transformed into the memory module G "and loss modulus G " ', the memory module characterizing the stored and thus recyclable portion of the introduced kinetic or deformation energy (elastic component) and the loss modulus, a measure of the heat released per oscillation Energy and thus lost share represents (frictional share).
  • the quantification of the released plasmid DNA from the matrix formed in situ was carried out photometrically.
  • the samples were shaken out before the measurement with chloroform (1 ml, 400 g, RT, 10 min) in order to separate PLGA degradation products which would disturb a photometric quantification [27].
  • the samples were then measured photometrically at 260 nm (Nanodrop-1000, PEQLAB Biotech, Germany).
  • 1-PEI / pDNA polyplexes pDNA concentration 100 ⁇ g / ml
  • 1-PEI / pDNA polyplexes pDNA concentration 100 ⁇ g / ml
  • a standard series was determined by serial dilution with PBS on 5 individual days at 260 nm, on the basis of which the concentration of released complexed plasmid DNA could be calculated.
  • the results are shown in FIG.
  • unloaded films were examined (background basic correction), small deviations in the volumes were taken into account by weighing the samples over the density of water.
  • L-PEI / plasmid DNA polyplexes (N / P ratio 10, 100 ⁇ g pDNA / batch) were formulated as described above, lyophilized with 10% sucrose, and homogenized using a mortar and pestle, which was dosed by weight and applied either in a sterile filtered PLGA solution or resuspended in the water phase (water for injection) could be resuspended. Water for injections without additives was used as a negative control.
  • plasmid DNA pMetLuc and pCMV-tPA-IRES-Luc were used in equal proportion.
  • Met5A cells were seeded on hanging inserts (1 ⁇ M PET Millicell) with a polyethylene terephthalate (PET) membrane, which allowed light microscopic control of the cells.
  • PET polyethylene terephthalate
  • Each 1.5 ml cell culture medium was presented, the inserts 2 minutes in equilibrated therein and then seeded 250,000 cells per well in 1, 5 ml of medium on the membrane.
  • the medium was removed, washed once with PBS, and the samples were sprayed on the cells as described above. Samples were taken daily at the beginning, and every two to three days later, with the medium being changed completely, the samples immediately placed on ice and stored at -80 ° C. until the analytical determination. Determination of transfection efficiency by luciferase activity measurement
  • the luciferase activity 24 h after transfection was measured by washing the cells once with PBS, per well with 100 ⁇ lx cell lysis buffer (25 mM Tris / HCl pH 7.8, 0.01% Triton-X 100) and after an incubation time of 10 min at RT were shaken for 60 sec.
  • luciferin substrate 100 ⁇ luciferin substrate (470 ⁇ D-luciferin, 270 ⁇ coenzyme, 33.3 mM DTT, 530 ⁇ ATP, 1.7 mg (MgC0 3 ) 4 Mg 2 ⁇ 5 H 2 O) were then automatically added to an aliquot of 50 ⁇ , measured 2.67 mM MgS0 4, 0.1 mM EDTA 0.1 mM, 20 mM tricine) was added and the light emission over a period of 5 sec in a plate reader (Wallac Victor 2/1420 Mulitlabel Counter, PerkinElmer Inc., USA) become. Before addition of the substrate, the background was also determined over the period of 5 sec.
  • the luciferase activity measured as emitted photons (Relative Light Units, RLU) was integrated after background correction over a period of 10 seconds and related to the total protein amount of the cell mass.
  • the total protein was previously determined by means of a standard protein assay (Biorad method).
  • the total tissue plasminogen concentration in selected samples was determined by ELISA (Human tPA Total Antigen Assays, Alternative Research, Dunn Laboratory GmbH, Germany) in the supernatant of the cells, with the assay used in addition to free and thus active tPA also the latent and bound to the inhibitor form was detected. Since these were supernatants from the cell culture, the standard was diluted analogously to the samples in cell culture medium of the cells used without FCS. The positive control (bolus dose) was diluted as follows: 1:50 (48 h, 9 d), 1:10 (16, 23 and 29 d).
  • the samples from the inner compartment were filled in (30 ⁇ sample ad 100 ⁇ ), whereas the samples from the outer compartment were analyzed undiluted.
  • the assay was performed according to the manufacturer's instructions and the absorbance at 450 nm over a period of 0.1 sec in a plate reader (Wallac Victor 2/1420 Mulitlabel Counter, PerkinElmer Inc., USA). The standard curve is shown in FIG. The negative controls were used as described above.
  • the medium was removed and the plasmid DNA remaining in the matrix was stained with propidium iodide.
  • the matrix was incubated with propidium iodide in a 1:10 dilution in PBS for 10 min at RT, washed again with PBS before taking up and taken pictures with an epifluorescence microscope (Axiovert 135, Carl Zeiss, Jena, 10x objective).
  • the excitation of propidium iodide was at 470 ⁇ 20 nm, whereas the emission was detected at 540 ⁇ 25 nm.
  • the software Axiovision LE 4.5 was used and analyzed with an Alexa 560nm filter at Brightfield.
  • the transfection was carried out as above in 24-well plates with some special features. In each case 750 ng of plasmid DNA and 30 pmol siRNA complexed with 1-PEI at an N / P ratio of 10 (based on the amount of plasmid DNA) were used. The change of the medium took place after 6 h. The proteins, the tissue-specific plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1), were analyzed after transfection by Western blot.
  • PAI-1 tissue-specific plasminogen activator and plasminogen activator inhibitor type 1
  • the separation of the proteins was carried out according to their molecular weight by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To destroy secondary and tertiary structures of the proteins, the batches were assayed (3.75 ⁇ sample, 15 ⁇ 4x application buffer (130 mM Tris / HCl pH 7.4, 20% glycine, 10% SDS, 0.06% bromophenol blue, 4% DTT). ad 60 ⁇ water for injection) previously denatured for 5 min at 95 ° C.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the labeled proteins were detected by ECL chemiluminescence (Amersham Bioscience, USA) on a film (Amersham Hyperfilm ECL, GE Healthcare, German) and analyzed for quantification using Image J Basics Version 1.38. Normalization was via the actin band of the untreated cells.
  • tetraglycol tetrahydrofurfuryl alcohol-polyethyleneglycol
  • Tetraglycol has been used since the 1960s as a solvent for parenteral (iV, im) in concentrations up to 50% (v / v) and shows low toxicity at this dilution [30].
  • Glycerolformal is an odorless, also low toxicity, solvent consisting of a mixture of 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol [30] and is an excellent solvent for many pharmaceuticals and cosmetics.
  • solvent consisting of a mixture of 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol [30] and is an excellent solvent for many pharmaceuticals and cosmetics.
  • Ivomec TM 0.27% is approved for subcutaneous administration in pigs and is used at 0.1 ml kg [31].
  • DMI dimetylisosorbide
  • ethyl lactate is used as a parenterally administrable vehicle for steroid formulations and, despite the GRAS number, is considered to be relatively toxic with narcotic and slightly hemolytic activity.
  • DMI is used topically as a penetration enhancer with low haemolytic activity [30, 34].
  • Matschke and colleagues showed that glycerol esters with good compatibility are suitable solvents for PLGA / PLA polymers [33].
  • triacetin a low-toxicity short-chain triglyceride, has been tested [35, 36] and previously described as an alternative to NMP and DMSO for in situ formed depot drug forms [29, 37-39].
  • the amount of polymer in the supernatant (loss) and in the precipitate (implant) was quantified in spray tests by weighing back the dried matrix. If one now plots the distribution coefficient P of the solvents against the matrix quality, as shown in FIG. 4B, the film quality is linearly dependent on the water-miscibility of the solvent. The graph clearly shows that the matrix formation could be improved with increasing water-miscibility of the solvents, with about 80% of the amount of polymer used being incorporated into the matrix with a P value ⁇ 0.25.
  • FIG. 5 shows the LD 50 values calculated from the experiments for all solvents tested as a function of the incubation time.
  • the compatibility of the solvents decreased in the following order; Glycerol formal>>DMI> tetraglycol.
  • Glycerol formal showed the lowest toxicity of the tested solvents with an LD 5 o value of approximately 1 g / ml with an incubation time of less than 6 h. In comparison to DMI and tetraglycol, this meant a 220-fold or 400-fold better compatibility after the end of the experiment.
  • biodegradable copolymers of lactic acid and glycolic acid poly (D, L-lactic-co-glycolic acid) (PLGA) polymers
  • PLGA poly (D, L-lactic-co-glycolic acid)
  • Boehringer Ingelheim were investigated (trade name Resomer RG ® ), which are already approved by the FDA for parenteral administration ,
  • Figure 2 shows the results for polymers having a composition of (a) PLA / PGA 50:50 with free acid groups (H series) and (b) PLA / PGA 75:25 with esterified end groups (S series). The latter are more lipophilic than the H series due to the higher proportion of lactic acid and the esterified end groups.
  • the graphs illustrate that with higher molecular weights of the polymers in both series, a larger amount of polymer could be incorporated into the matrix.
  • This effect was significant for DMI in the H-series, and for both solvents in the S-series, 755 S almost 100% of the amount of polymer used in the matrix trained using glycerol formal and Resomer ® RG (97.6 ⁇ 0.6% ). Comparing the polymer series in combination with the solvents tested, glycerol formal with the S-Series showed a 20% lower loss of polymer used compared to the H series and compared to DMI in both series of polymers.
  • DMI dissipation factor
  • plasmid DNA was developed as a model active substance. Theoretically, the plasmid DNA could be incorporated into the matrix both in "naked” and complexed form. However, since "naked" plasmid DNA transfected cells only very inefficiently, the plasmid DNA was complexed prior to embedding in the film with 1-PEI (N / P ratio 10) and incorporated as nanoscale polyplexes in the matrix. As a result, it was additionally able to be protected against a drop in pH within the matrix, which occurs during the degradation of the polymer structure due to the release of polymer monomers within the matrix and is generally a problem for sensitive macromolecules [47].
  • the incorporation of the polyplexes into the matrix could be carried out dissolved in the aqueous phase [44] or dispersed in the PLGA solution [28, 45].
  • a direct addition of small quantities of water about 5%
  • the use of highly concentrated plasmid DNA solutions was required, which, however, have a low stability and tend to aggregate formation. It was therefore advantageous to disperse the polyplexes as lyophilisate analogously to protein formulations [28, 45] in the PLGA solution or to resuspend before use in the aqueous phase.
  • the formulations were structured as follows:
  • the lipophilic phase (1 ml): 10% (m / v) PLGA polymer in glycerol, tetraethylene glycol, or DMI (Resomer ® RG 502 H, 504 H)
  • Hydrophilic phase (1 ml): water for injections 3) Active Ingredient Component: lyophilized polyplexes resuspended in the lipophilic or hydrophilic phase
  • Freeze-drying is one of the standard methods in the pharmaceutical industry for stabilizing formulations during storage. By including the molecules in an adjuvant matrix, formulations can be stabilized during the drying process, it being possible to use different protectors depending on the drying phase.
  • cryoprotectants prevent crystallization of the solution during freezing. The system solidifies as a supercooled melt without complete phase separation (solidified liquid, glass).
  • lyoprotectants provide protection in the further course of drying by replacing the bonds of the active ingredient to the water by forming hydrogen bonds.
  • Polyplexes can also be lyophilized with the addition of cryoprotectants and lyoprotectors, so that aggregation can be prevented after resuspension [48, 49].
  • polyplexes can be stored better [48] and the solution can be concentrated to a plasmid DNA concentration of 1 mg / ml [50].
  • Sugars such as sucrose or trehalose act as lyo- and cryoprotectants and have been shown to stabilize polyplexes [48, 49].
  • Water-soluble substances such as these can also accelerate the release of macromolecular drugs from in situ formed PLGA based films. During the matrix formation, water-filled pores are formed by the dissolution of these substances, through which the active substance can subsequently diffuse out of the matrix. A similar effect was described for high loading of the matrix [27, 33].
  • UT Ultra-Turrax
  • H glass homogenizer
  • the release of active substances from implants can in principle be effected by i) diffusion of the active substance from the polymer matrix (diffusion-controlled) or ii) by erosion of the matrix (erosion-controlled) [51, 52].
  • diffusion-controlled diffusion-controlled
  • erosion-controlled erosion-controlled
  • initial release of the active agent may occur until complete precipitation of the polymer.
  • the release kinetics of films formed in situ was investigated as a function of the molar mass of the polymer, of the solvent used and of the embedded variant. The following combinations were tested:
  • Lipophilic phase (1 ml): Resomer ® RG 502 H or 504 H to 10% (m / v) in glycerol formal, tetraethylene glycol or DMI
  • Active component lyophilized polyplexes (1-PEI / pDNA) in homogenized form
  • the polyplexes were homogenized in a mortar and either resuspended in the hydrophilic phase (embedding variant A) or dispersed in the lipophilic PLGA solution by means of a homogenizer (embedding variant B).
  • the Frei GmbHspro-glare are shown for Resomer ® RG 502 H and 504 H using different solvent for both variants embedding in FIG. 6
  • phase 1 concave release profile
  • phase 2 linear kinetics
  • glycerol formal showed a low initial, followed by a slow diffusion-controlled release. It was only with the beginning of erosion of the matrix erosion that an accelerated release of the polyplexes was observed, which began 15 or 26 days, depending on the chain length of the polymers used.
  • films based on tetraglycol showed a moderate initial release of 32% (Resomer RG 502 H) and 50% (Resomer RG 504 H) a moderate to no release in the observed time frame. Only in the case of the longer-chain polymer was it due to the erosion of the matrix to a low release after 26 days (Fig. 6C).
  • DMI showed the fastest release for all combinations of long or short chain polymer and the different embedding variants.
  • Continuous release up to 100% drug release was achieved with Resomer ® RG 504 H by incorporation of the drug component into the hydrophilic phase.
  • Polyplexes incorporated in tetraglycol-based films showed no diffusion-controlled release. Over the observed period, an additional 14% of the pDNA amount could be released after initial release, whereby the initial release varied between 0 and 48%. This was relatively high in the case of embedding variant A, whereas no initial release was observed when the polyplexes were dispersed in the lipophilic phase.
  • Films based on glycerol formal showed a low initial release irrespective of the embedding method, although the polyplexes could not be released until after 23 days by erosion of the matrix even when these films were used.
  • the polyplexes were dissolved in the hydrophilic phase.
  • the formulations were first tested in vitro using the active component 1, which was complexed with 1-PEI and lyophilized with the addition of 10% sucrose.
  • the pMetLuc plasmid which codes for a luciferase enzyme secreted by the cell, was additionally used in a 1: 1 mixture.
  • mesothelial cells were cultured on inserts and the polymer film was subsequently sprayed onto the cell layer.
  • the use of the inserts made it possible to divide the wells into a two-chamber system with outer and inner compartments comparable to the on-site anatomy in the peritoneum, between which a constant mass transfer was possible, so that the cells could be supplied with medium from the apical and basolateral side.
  • An optical control of the cell morphology was carried out by light microscopy, but was complicated by the sprayed on film.
  • the expression of the reporter gene luciferase could be examined by using the inserts in both compartments over a period of 30 days.
  • FIG. 10 Shown in FIG. 10 is the upper compartment.
  • the films formed in situ showed a lesser amount of luciferase by a factor of 10 3 to 10 4 .
  • the course of gene expression was as described above.
  • films based on DMI had shown an initial release of 56% of the amount of pDNA used and subsequently a further 38% to day 26 release. Following this release profile, an increased gene expression was observed for the DMI-based film after only 2 days, which dropped to basal levels after a further 7 days and increased again to moderate levels by day 23.
  • the tPA expression from the matrix consisting of glycerol and Resomer ® RG 504 H is shown in comparison to the single dose and at a film without active ingredient component (inactive film) in Figure 10A.
  • the active substance component Without the depot system, very high protein levels were obtained over a period of 29 days, analogous to luciferase expression, whereby a 100 to 40-fold increase in the tPA concentration compared to the basal values could be achieved.
  • Similar concentrations were achieved after intraperitoneal administration of recombinant tPA (alteplase) in plasma [82]. The values that could be achieved using the matrix formulation therefore appear much closer to the physiological conditions.
  • the tPA / PAI-1 ratio when co-applied to the untreated cells increased to 8-fold, with the application of pDNA alone or in combination with a non-functional siRNA (EGFP siRNA) only an increase of the 4 - or 5 times could be achieved.
  • EGFP siRNA non-functional siRNA
  • pUC placebo plasmid
  • Glycerol formal showed the best tolerability on mesothelial cells in the cell viability test, with LD 50 values being 200 and 400 times higher, respectively, than DMI and tetraglycol. These were below 6 g incubation time at about 1 g / ml, ie with an application of about 780 ⁇ pure Glycerolformal die off 50% of the mesothelial cells.
  • Literature data indicate similar LD50 values after iv administration in rodents for DMI and glycerol formal, whereas tetraglycol is more toxic by a factor of 2-3.
  • an LD50 of 4 to 4.8 g / kg body weight (mouse) with iv administration of a 50% strength by weight refer Glycerolformallö- solution.
  • the EMA describes this in a final report by the Committee for Veterinary Products [85].
  • the acute toxicity after intravenous administration of DMI hardly differs from glycerol formal.
  • an LD 50 of 5.4 g / kg body weight (rat) with application of 40% DMI in isotonic saline solution (v / v) and an LD 50 of 6.9 g / kg body weight (mouse) with application of a 20% Solution seems even better tolerated DMI after intravenous administration.
  • Tetraglycol has been used as a solvent for parenterals (iv, im) since the 1960's in concentrations up to 50% (v / v) and is considered to be non-irritating at this dilution.
  • the LD50 after intravenous administration without dilution is 3.8 g kg body weight (mouse) lower than described for the other two solvents [86].
  • Jain RA The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices. Biomaterials. 2000; 21: 2475-2490.
  • Diamond MP Reduction of de novo postsurgical adhesions by intraoperative precoating with Sepracoat (HAL-C) Solution: a prospective, randomized, blinded, placebo-controlled multicenter study. The Sepracoat Adhesion Study Group. Fertil Sterile. 1998; 69: 1067-1074.
  • Gago LA Saed GM
  • Chauhan S Elhammady EF
  • Diamond MP Diamond MP.
  • Sepraufm modified hyaluronic acid and carboxymethylcellulose acts as a physical barrier. Fertil Sterile. 2003; 80: 612-616.

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Abstract

L'invention concerne un système de pulvérisation permettant de produire une matrice se formant in situ. Ledit système contient au moins un composant lipophile contenant au moins un polymère à base d'acide glycolique et d'acide lactique et au moins un solvant biocompatible présentant une valeur XlogP3 inférieure à 0,2, et au moins un composant hydrophile. Les deux composants ou plus sont présents séparément les uns des autres avant l'utilisation et ne sont mélangés que pour la pulvérisation ou au moment de la pulvérisation. Les composants forment un film au moment de la pulvérisation sur un tissu du corps humain.
EP12762603.4A 2011-09-28 2012-09-25 Système de pulvérisation permettant de produire une matrice formée in situ Withdrawn EP2760437A1 (fr)

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DE102011114986A DE102011114986A1 (de) 2011-09-28 2011-09-28 Sprühsystem
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US20160129044A1 (en) * 2013-06-05 2016-05-12 Fundacion Pública Andaluza Progreso Y Salud Use of mesothelial cells in tissue bioengineering and artificial tissues
CA2916800C (fr) 2013-06-28 2022-10-25 Ethris Gmbh Compositions comprenant un composant comportant des fractions oligo(alkylene amine) caracteristiques
JP2017507946A (ja) 2014-02-26 2017-03-23 エスリス ゲーエムベーハーｅｔｈｒｉｓ ＧｍｂＨ Ｒｎａを胃腸内投与するための組成物
US9655842B1 (en) 2015-12-04 2017-05-23 Covidien Lp Injectable non-aqueous compositions and methods of treating vascular disease
US11013501B2 (en) 2017-12-08 2021-05-25 Davol, Inc. Method of protecting the peritoneum against tearing and other injury before an active surgical intervention at or near the peritoneum
EP3628309A1 (fr) * 2018-09-28 2020-04-01 Universität Heidelberg Procédé de fabrication de formes posologiques orales

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GB8815435D0 (en) * 1988-06-29 1988-08-03 Smith & Nephew Non autoadherent dressings
US4938763B1 (en) * 1988-10-03 1995-07-04 Atrix Lab Inc Biodegradable in-situ forming implants and method of producing the same
TW247878B (fr) * 1991-07-02 1995-05-21 Takeda Pharm Industry Co Ltd
EP0560014A1 (fr) * 1992-03-12 1993-09-15 Atrix Laboratories, Inc. Pansement sous forme de film biodégradable et méthode pour sa fabrication
ES2359973T3 (es) * 1998-03-19 2011-05-30 MERCK SHARP & DOHME CORP. Composiciones poliméricas líquidas para la liberación controlada de sustancias bioactivas.
WO1999047073A1 (fr) * 1998-03-19 1999-09-23 Merck & Co., Inc. Compositions polymeres liquides pour la liberation controlee de substances bioactives
US6103266A (en) * 1998-04-22 2000-08-15 Tapolsky; Gilles H. Pharmaceutical gel preparation applicable to mucosal surfaces and body tissues
JP4540762B2 (ja) 1999-01-18 2010-09-08 本田技研工業株式会社 排気２次空気弁を備えた鞍乗型車両
EP2030611A1 (fr) 2002-07-31 2009-03-04 Alza Corporation Compositions de dépôt de polymère multimode injectable et utilisations associées
US20060003008A1 (en) 2003-12-30 2006-01-05 Gibson John W Polymeric devices for controlled release of active agents
DK1888031T3 (da) * 2005-06-06 2013-02-18 Camurus Ab GLP-1-analogformuleringer
WO2007120818A2 (fr) * 2006-04-12 2007-10-25 Massachusetts Institute Of Technology Compositions et méthodes permettant d'inhiber les adhérences
WO2010142660A2 (fr) * 2009-06-09 2010-12-16 Novartis Ag Système d'administration de médicaments
US9649331B2 (en) 2009-08-27 2017-05-16 Ara Medical Llc Sprayable polymers as adhesion barriers

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US20140243395A1 (en) 2014-08-28
ZA201401557B (en) 2015-09-30
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WO2013045455A1 (fr) 2013-04-04
DE102011114986A1 (de) 2013-03-28

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