Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
  • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
  • A61L26/0023—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
  • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
  • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
  • A61L15/28—Polysaccharides or their derivatives

Definitions

• Wound dressing compositions especially for delivery of protease inhibitors
• the present invention relates to dry and hydrated, i.e. wet wound dressings and delivery systems also suitable for active ingredients, their use for the treatment of wounds and skin diseases, preferably chronic wounds, and methods of preparing them. More particularly, the present invention relates to a xerogel or film comprising a mixture of a cellulose ether and a gellan gum. Such systems can be used directly as wound dressings or alternatively as dry, storage stable delivery systems for active ingredients, preferably proteins, in the field of cosmetics and medicine. Before use and/or during application in a moist environment, especially wounds, the dry composition is rehydrated, thus serving as a hydrogel loaded with active ingredients which are released at a controlled rate.
• Such a system can be used for moist wound healing or dermal delivery of therapeutic substances and for other medical or cosmetic purposes.
• the wound dressing according to the invention comprises a cellulose ether, a gellan gum and alpha- 1-antichymotrypsin (ACT).
• ACT alpha- 1-antichymotrypsin
• the present invention can be used for delivery of active ingredients, especially alpha- 1- antichymotrypsin, into wounds.
• active ingredients especially alpha- 1- antichymotrypsin
• the mechanisms of wound healing in general and characteristics of different wound healing phases are well known. Since 1962 moist wound healing has become a widely accepted treatment.
• a problem which has often been encountered is that the topical delivery system for active ingredient does not adequately stabilize or release the active ingredient and/or that it is not adequate regarding patient compliance.
• proteins as active ingredients which are typically labile, the above prerequisites are difficult to meet.
• hydrogels are preferred over solutions with low viscosity as they keep the wound moist, do not evaporate fast and therefore have to be applied only once daily.
• the solution Eurokinin, for example, has to be applied continuously onto a compress on the wound.
• Regranex is a hydrogel which shows good wound healing and handling properties, but inadequate storage stabilities.
• Gels can provide a controlled delivery system for protein on a wound site. Controlled release refers to a drug release sufficient to maintain a therapeutic level over an extended period of time. This is an important advantage because it permits less frequent application of the formulation to the wound and thereby permits less disturbance of the wound.
• a variety of gels and ointments for application to wounds are described in the prior art for a variety of purposes.
• the gel may for example be used to clean wounds, to promote healing of the wound or to prevent infection.
• the gel may include an active ingredient which is administered to the patient by topical application of the gel.
• Intrasite® produced by Smith & Nephew Ltd. This gel contains hydrated carboxymethyl cellulose-Na as a main ingredient and is packaged and applied to wounds in gel form as a primary treatment in order to debride the wound.
• Gels have the further advantage of having a high water content, easy application to a wound, and easy removal from the wound after application by washing.
• the gel may also assist in preventing the wound from drying out, thereby promoting the healing process.
• gels are mobile they offer the advantage of intimate contact with the often irregular surface of a wound, something that is often not achieved with a more rigid or liquid wound dressing.
• the advantage of good contact is, however, tempered by the conflicting needs of making the gel sufficiently mobile that it can be applied to the wound but not so mobile that it runs out of the wound under the influence of gravity. Gels currently in use suffer from the disadvantage that they can run out of the wound.
• the drying procedure can be carried out by various methods. Freeze drying and warm air drying are preferred drying procedures for the present invention. Particularly preferred is freeze-drying. Drying methods using organic solvents are less preferred due to the unwanted interactions of labile active ingredients with organic solvents and the problems of residual solvent contents in the product that at least affords additional testing routines adding to production costs.
• a dry storage system can either be hydrated before use with a suitable aqueous solution and/or during use in contact with aqueous body fluids, especially when applied topically to a wound.
• Preferred dry storage systems for hydrogels are films and xerogels, especially xerogels. The xerogel or film take up water upon hydration, swell and eventually form a hydrogel.
• a desirable, optimal active ingredient product would have a dry, storage stable form and could be hydrated to a hydrogel before use.
• Such storage stable forms need not to be stored at very low temperatures and thus allow also easy and cheap transportation.
• Such products may also be stored by the patient himself without complications. Thereby, high costs which occur when treatment has to be effected at hospitals are avoided. It would be useful to have a dry, ready to use, single dose product covering a defined contact area, which can also be cut reproducibly into pieces in order to achieve defined doses.
• the patient to be treated could adapt the medicament or cosmetic composition according to his needs, for example to immobilize the gel by hydration to prevent flowing out of the wound or to reconstitute it to a mobile gel that can insert in even cleft regions of a wound without gaps.
• an active medicament unstable to heat with a biodegradable protein carrier such as collagen, atelocollagen, or gelatin to form a carrier matrix having sustained release properties.
• the resultant mixture is then dried, and the dried material is formed into an appropriate shape, as described in US 4,774,091.
• active ingredients for this purpose are given as t-Pa, prostaglandines, prostacyclines biohormones, e.g. hGH, bGH, GRF, somatomedins, and calcitonin, interferons, interleukins, tumor necrosis factor and other cytokines such as macrophage activating factor, migration inhibitory factor, and colony stimulating factor.
• JP 2002143290 describes a freeze dried matrix made from a gel formed by a mixture of PLGA-copolymer and bovine atelocollagen I. Additionally, the matrix is cross-linked by glutaraldehyde vapour.
• EP 083491A2 discloses a polysaccharide sponge made by solvent drying.
• US 5,189,148 describes a stabilized FGF composition and production thereof.
• US 2003/0105007 discloses a solid formulation for growth factors, e.g. PDGF in fibronectin.
• WO 91/19480 describes a freeze-dried hydrogel preparation containing a wound healing medicament which is compressed after freeze drying.
• the polymer in this invention is Hydroxyethylcellulose (HEG), EP 0308238Al describes stable lyophilised formulations containing growth factors.
• US 5,714,458 discloses a stable lyophilised formulation of FGF in a xerogel.
• DE 19503338 Al describes a film for the release of collagenase. The film can consist of various polymers. Gellan gum is not mentioned.
• WO03/034993 and Bl 102118 describe collagen sponges as wound/tissue healing material.
• CA2246895 describes bioabsorbable solid materials including films and sponges.
• Cellulose derivatives have been used to formulate therapeutic proteins or polypeptides for topical use. See, e.g. EP 267,015, EP 308,238, and EP 312 208, which disclose formulations of a polypeptide growth factor having mitogenic activity, such as TGF beta, in a polysaccharide such as methylcellulose.
• EP 261,599 discloses human topical applications such as TGF beta.
• EP 193,917 discloses a slow release composition of a carbohydrate polymer such as cellulose and a protein such as growth factor.
• GB 2,160,528 describes a formulation of a bioactive protein and a polysaccharide.
• US 4,609,640 describes a therapeutic agent and a polymer selected from polysaccharides, cellulose, starches, dextroses.
• DE4328329 claims a freeze dried biomatrix consisting of natural polysaccharides and modified polysaccharides.
• GB 2357765 describes an alginate foam crosslinked by di- and bivalent cations.
• the above-described wound dressing materials provide important advantages. The materials are of natural, biological origin (albeit chemically modified), and consequently tend to have low antigenicity. Furthermore, some of these materials can have positive therapeutic effects on wound healing. In principle, they are suitable for the delivery of protein active ingredients.
• a drawback of some collagen- and gelatine-based wound dressing materials is that the collagen breaks down too fast in vivo, due to the action of collagenase enzymes in the wound. This can be countered to some extent by cross-linking the collagen/gelatine with a covalent cross-linking agent such as glutaraldehyde or dicyclohexylcarbodiimide. Residual contents of these agents, however, can cause unwanted interactions with loaded protein active ingredients and the wound milieu itself. Besides, by cross-linking a strong irreversible immobilization is effected.
• the immobile sponge in the first period after application may not cover cleft wound grounds and in the later stages the sponge is liquefied by enzymatic degradation what as well does not provide good contact to the wound ground.
• collagen as a component of wound dressings is prone to denaturation when it is sterilized by gamma-irradiation.
• Collagen and gelatine also are extracted from natural animal sources. So 3 the problems of contamination with pathogens for animal or human, like the bovine spongiform encephalopathy (BSE) and others are relevant.
• BSE bovine spongiform encephalopathy
• it can be antigenic to certain patients unless stringent measures are taken to purify the collagen, which add to its cost.
• Non cross-linkable materials like non-ionic cellulose ethers suffer from the rheological disadvantage that they can run out of the wound and so lose close contact with the wound surface.
• Materials that are covalently cross-linked are always endangered of having unwanted effects caused by residual contents of cross-linking agents antagonising wound healing.
• Di- and bivalent cations used e.g. for crosslinking alginate also can have antagonising effects on the wound healing process.
• solutions of these cations in the needed high concentrations are not used in clinical practice. This complicates the possibility that the cross-linking is carried out by the applicant when it seems appropriate for the individual wound condition.
• alginates are not suitable for steam sterilization.
• Biodegradable polymers like PLGA, collagen, gelatine, and others in some cases are known to interact with labile drugs due to their fragmentation. So, fragments of PLGA, i.e. lactic acid and glycolic acid, can change pH and the osmotic pressure within the matrix to awkward proportions.
• Sodium salts of polymers like carboxymethyl cellulose sodium or carboxymethyl starch sodium are unfavourable for lyophilisation because their combination with phosphate buffer, which is essential for many protein formulations, results in a pH-shift into the sour region, which is counterproductive for protein stability.
• wound dressing materials suitable for delivery of active ingredients if necessary, to mammalian wounds, and especially to human chronic wounds, such as venous ulcers, decubitus ulcers and diabetic ulcers.
• the wound dressing compositions of the invention are surprisingly suitable for stabilizing and releasing proteins, in particular alpha- 1- antichymotrypsin.
• the present invention relates a wound dressing composition
• a wound dressing composition comprising a cellulose ether and a gellan gum.
• the cellulose ether is hydroxyethyl cellulose (HEC).
• a wound dressing composition made out of gellan gum and HEC is especially advantageous since it is very soft and comfortable.
• HEC is advantageous over e.g. the use of carboxymethyl cellulose (CMC), since CMC matrices generally are harder in texture due to higher density, more brittle and show reduced swelling in comparison to HEC.
• CMC is usually applied in the form of sodium CMC.
• the ionic groups of sodium CMC may interact with active, therapeutic ingredients, which may result in a destabilization or at least partial inactivation of the ingredient, especially if it is a protein.
• the gellan gum is a deacetylated gellan gum.
• the gellan gum is available under the trade name Kelcogel F.
• the composition comprises hydroxyethyl cellulose (HEC) and deacetylated gellan gum.
• HEC hydroxyethyl cellulose
• deacetylated gellan gum deacetylated gellan gum
• the cellulose ether and the gellan gum are homogeneously mixed.
• the composition is in dry form, particularly a xerogel or film.
• the wound healing composition is a hydrogel.
• the hydrogel is obtained by hydration of a dry storage form of the gel, in particular by hydration of a xerogel or film.
• the composition may contain, if desired, one or more additional excipients like sugars, sugar alcohols, surfactants, amino acids, antioxidants, polyethylene glycols.
• the excipients comprise at least one non-ionic surface active component preferably selected from Poloxamer® 188, Tween 80 and Tween 20.
• the excipients comprise at least one buffer agent, preferably selected from phosphate and Tris.
• the excipients comprise at least one amino acid, preferably arginine.
• the excipients comprise at least one polyethylene glycol, preferably selected from PEG 400 suitable as plastiziser and PEG 2000 suitable as pore former.
• the excipients comprise at least one polyvinyl pyrrolidone, preferably Kollidon 17PF, which is suitable as strengthener or pore former or stabilizer.
• the wound dressing compositions according to the present invention preferably are a homogeneous mixture of gellan gum and hydroxyethyl cellulose.
• the wound dressing composition comprising the homogeneous mixture of gellan gum and hydroxyethyl cellulose is in a suitable vehicle, such as a solvent, in an even more preferred embodiment the solvent is aqueous and the composition is a hydrogel. In another embodiment the hydrogel is formed by hydration of a film, xerogel or sponge.
• the wound dressing composition comprising the homogeneous mixture of gellan gum and hydroxyethyl cellulose is dry.
• the dry composition is a film or a sponge or xerogel.
• Hydroxyethyl cellulose can be obtained by the process described in US 4,084,060.
• HEC is a non-ionic water-soluble cellulose ether, formed by reaction of cellulose with ethylene oxide. It is widely used in pharmaceutical compositions and very well known in the art. It can be dispersed in cold or hot liquids, but is insoluble in most organic solvents. This material offers numerous advantages including the features that it is biocompatible, biostable, non-immunogenic and readily commercially available. It can be obtained in various degrees of molecular weight at high levels of purity.
• the hydroxyethyl cellulose has a molecular weight of at least 500 kDa. Gels of hydroxyethyl cellulose can be steam sterilized.
• Gellan gum is a microbial polysaccharide derived from Pseudomonas elodea. It consists of a tetrasaccharide unit, D-D-glucose, D-D-glucuronic acid, D-D-glucose and 0-L-rhamnose.
• Gellan is produced with two acyl substituents present on the 3-linked glucose, namely, L-glyceryl, positioned at 0(2) and an acetyl substituent at O(6).
• the native polysaccharide is partially esterified; the 1,3-D-Glc residue can be linked to L-glycerate at C-2 and/or to acetate at C- 6, and there is 1 mol of glycerate per repeating unit and 0.5 mol of acetate per repeating unit.
• Gellan gum has been suggested for use in wound healing as solution/gel in US 6,596,704 and fibers in EP 0454373 as well as in WO 95/05204.
• Gellan gum has the CAS No. 71010-52- 1.
• Well known Gellan gum products are available under the tradenames Gelrite® and Kelcogel®.
• "Deacetylated Gellan Gum" according to the present invention is to be understood as gellan Gum obtainable by partial or complete deacetylation of native Gellan Gum.
• the deacetylated Gellan Gum contains less than 10%, more preferably less than 25%, in particular less than 50%, most preferably less than 15% of the acetyl groups present in native Gellan Gum.
• Native Gellan Gum can be obtained by aerobic fermentation using Sphingornon ⁇ s elode ⁇ .
• Deacylated Gellan gum is readily soluble in water and is characterized by high viscosity at low concentration.
• the gum also has a high rheological yield point. Changes of pH in the range 3-11 do not substantially affect the viscosity of the gel.
• the viscosity of such gel is also stable in the range of 20 0 C - 70 0 C. Above this temperature it reversibly liquefies; i.e. the thermoreversibility is formed after heating and cooling. Following heating in the presence of various cations deacetylated gellan gum produces firm, non-elastic or brittle gels. Cations especially useful in the formation of gels with are those of sodium, potassium, magnesium, calcium. Gellan gum is used as a thickening, suspending and stabilizing agent in aqueous systems in various fields including nutrition.
• both gelling agents can be sterilized by steam sterilization, which strongly simplifies production processes.
• the swelling and thermoreversible behaviour of gellan gum also are activated by steam sterilization.
• they can be lyophilised and they also form stable films when air dried.
• a very low content of each gelling agent is needed for forming gels with high viscosities.
• the pore sizes of freeze dried hydrogels of the invention i.e.
• both HEC and gellan gum are commercially available with a range of defined and controllable properties offering the possibility to adjust and control the properties of the wound healing compositions of the present invention as needed to an exceptional degree.
• the mechanical texture, viscosity, the rate of hydration, porosity and density of the materials can be adjusted in a wide range as it seems appropriate.
• hydroxyethyl cellulose has the disadvantage that it is a non-crosslinked gelling agent.
• Such HEC-gels not containing gellan gum show a pseudoplastic rheology. That means there is no rheologic yield point and it is therefore free-flowing under the influence of gravity.
• the free-flowing HEC gels without gellan gum will flow out of the wound site and so lose contact with the wound ground resulting in a loss of their therapeutic effects.
• deacetylated gellan gum in the absence of HEC has the disadvantage that it forms brittle gels in presence of cations that would not be suitable for administration to a highly sensitive region such as a wound. Moreover, it lacks of the flexibility and flow tendency to be able to be applied without gaps to uneven and cleft wound surfaces.
• This, however, is essential for a wound dressing because the suitability of such wound dressing is greatly dependent on the interaction between the hydrogel wound dressing and the wound fluid: the release from the wound dressing can only take place by diffusion of the active substances at the interface between hydrogel wound dressing and wound fluid, For accurate, controlled, and reproducible release of drugs, especially in case of highly potent substances at low doses, e.g. proteins, a close contact between wound dressing and the wound is a prerequisite.
• the mixture of a HEC and a gellan gum has all the advantages of the single components alone and overcomes disadvantages of the single components, which makes the mixture exceptionally suitable for its use as wound healing composition.
• the rheology of the wound healing composition comprising a mixture of a gellan gum and HEC in the gel state can be adjusted as needed offering a broad range of advantageous wound dressing compositions:
• Hydration of a xerogel/film comprising gellan gum and HEC, and optionally at least one therapeutic wound healing agent with water The resulting gel behaves more like a HEC gel than a gellan gum gel. It still has a good flowability and is very smooth.
• a xerogel/film comprising Gellan gum and HEC, and optionally at least one therapeutic wound healing agent with electrolyte solutions, e.g. isotonic NaCl, Ringer, etc:
• the resulting gel is immobilized and provides a higher yield point and a highly pronounced plastic rheology. Moreover, it still is flexible enough to adjust to any surface morphology. Moreover, the soft pressure of a bandage is enough for the gel pad to overcome the yield point and start to creep and to deform to the given shape of the wound site.
• the film/xerogel can be cut into parts.
• the part(s) for the deep cave of the wound ground can be hydrated in water. They will start to flow and creep into the deepest wholes of the wound like a normal free flowing hydrogel.
• the second part(s) will be hydrated in electrolyte solutions and get immobilized, They are placed over the first non-immobilized part(s) of the gel into the wound and serve as a clot. So the wound is completely filled without gaps, but the gel is still immobile and will stay in the wound accurately as long as desired.
• the weight ratio of HEC to gellan gum is in the range from 1:5 to 50:1, preferably from 1:1 to 10:1 in the wound dressing compositions of the invention.
• the hydroxyethyl cellulose and gellan gum together make up at least 25% by weight of the material on a dry weight basis, more preferably at least 30% by weight of the material on a dry weight basis, even more preferably at least 40% by weight of the material on a dry weight basis, particularly at least 50% by weight of the material on a dry weight basis.
• the content of gelling agent mixture; i.e the sum of HEC and gellan gum is preferably between 1% and 10 %, preferably between 2 and 4%.
• the material essentially consists of the hydroxyethyl cellulose and gellan gum.
• the composition is in form of a hydrogel or a dried or lyophilised form thereof, i.e. a film, xerogel or sponge. Such compositions may be used directly for topical applications in the field of cosmetics or medicine.
• composition according to the present invention may be in any convenient form, such as powder microspheres, flakes, a mat, a sponge, a xerogel or a film.
• the composition according to the present invention is in the form of a semisolid or gel ointment for topical application.
• the wound dressing compositions of the present invention are in form of a hydrogel.
• wound dressing compositions of the present invention are in form of a freeze-dried or solvent-dried xerogel or sponge.
• composition according to the present invention is in the form of a flexible film, which may be continuous or interrupted (e.g. perforated).
• the flexible film preferably comprises a plasticiser to render it flexible, such as PEG 400, glycerol, or propylenglycol.
• the invention in another embodiment relates to a wound dressing package comprising a sterile wound dressing composition of the invention, packaged in a microorganism- impermeable container.
• a wound dressing package comprising a sterile wound dressing composition of the invention, packaged in a microorganism- impermeable container.
• Such packages may usually represent single or multi unit dosage forms of the present compositions.
• These packages can be easily used by the patient himself, especially if the package of the invention contains a wound dressing composition in dry from, especially in form of a xerogel, sponge of film.
• Such packages are especially preferred embodiments of the invention,
• the wound dressing compositions are located on an inert support, preferably selected from adhesive strip, adhesive wrap, bandage, gauze bandage or compress system.
• the present invention also relates to wound dressing systems comprising an inert support and a wound dressing composition of the invention.
• wound dressing . systems can also be packaged in a microorganism- impermeable container or foil and thus represent wound dressing packages with high patient compliance.
• the wound dressing compositions, systems and packages of the present invention may be used for the treatment of wounds, especially badly healing wounds like chronic wounds, in particular for the treatment of diabetic, venous, decubitus or neuropathic ulcers or infected wounds.
• compositions may also be used for cosmetic or medical treatment of skin diseases and skin conditions or diseases of a mucosa or eye in general, especially when an active ingredient is part of the compositions and the compositions therefore serve as topical delivery system for the active ingredient.
• the compositions suitable for cosmetic and/or medical use of the invention can be used, for example for treating and/or preventing ophthalmologic, nasal diseases, wounds or skin disorders, e.g.
• dermatoses selected from psoriasis, dermatoses, eczema, urticaria, lupus erythematosus, vitiligo, pigmentation disorders, wrinkling, aged skin, ichthyoses, hyperkeratoses, contact dermatitis, hand eczema or atopic dermatitis. It may also be used, depending on the active ingredient, for transdermal delivery of active ingredients.
• the invention therefore relates to a wound dressing composition according to the present invention comprising HEC and Gellan gum, wherein the composition further comprises one or more active ingredients.
• the invention relates to a a wound dressing composition according to the present invention comprising HEC and Gellan gum, wherein the composition further comprises one or more active ingredients for use as a medicament.
• the invention relates to the use of a wound dressing composition according to the present invention comprising HEC and Gellan gum for the preparation of a medicament for the treatment and/or prevention of ophthalmologic, nasal diseases, wounds and skin disorders.
• the present invention also relates to a wound dressing composition of the present invention, wherein the composition further comprises one or more active ingredients which are active in the treatment and/or prevention of ophthalmologic, nasal diseases or skin disorders, e.g. selected from psoriasis, dermatoses, eczema, urticaria, lupus erythematosus, vitiligo, pigmentation disorders, wrinkling, aged skin, ichthyoses, hyperkeratoses, contact dermatitis, hand eczema or atopic dermatitis.
• active ingredients which are active in the treatment and/or prevention of ophthalmologic, nasal diseases or skin disorders, e.g. selected from psoriasis, dermatoses, eczema, urticaria, lupus erythematosus, vitiligo, pigmentation disorders, wrinkling, aged skin, ichthyoses, hyperkeratoses, contact dermatiti
• Such active ingredients comprise steroids, like hydrocortisone and betamethasone, Calcineurin inhibitors like tacrolimus, Pimecrolimus and Cyclosporin A, clobetasol, vitamin A derivatives like retinoic acid and esters and amides thereof, Tazarotene, vitamin D derivatives like calcitriol and calcipotriol, to enumerate a few.
• the invention thus relates to the use of a wound dressing composition of the present invention comprising at least one active ingredient for the preparation of the medicament for the treatment of mucosal diseases, ophthalmologic diseases, wounds and skin diseases,
• the wound healing compositions can be used directly for the treatment of wounds, without any further active ingredient due to the beneficial effects of the components HEC and gellan gum on wound healing alone as well as their optimal rheologic and moist properties when mixed to homogeneity.
• the invention therefore relates to a wound dressing composition of the present invention for use as a medicament.
• the invention thus relates to the use of a wound dressing composition of the present invention for the preparation of the medicament for the treatment of wounds, especially badly healing wounds like chronic wounds, in particular for the treatment of diabetic, venous or neuropathic ulcers or infected wounds.
• wound dressing composition suitable for cosmetic and/or medical use can be applied directly, independent of whether it is in a "wet" state, especially as a hydrogel or in a dry storage form, especially as a xerogel or film.
• Dry wound dressing compositions of the present invention can alternatively be reconstituted with water or other adequate solutions before application. This is necessary especially when compositions are to be applied to dry skin surfaces or if specific, especially rheologic, characteristics of the rehydrated hydrogel are needed. Thus, depending on the viscosity to be achieved, the hydration medium can be selected as needed.
• a dry wound dressing composition comprising a mixture of hydroxyethyl cellulose and gellan gum, and where necessary, one or more active ingredients, in an amount that the mixture will form a topical gel upon hydration of the mixture;
• the present invention relates to a method for treating wounds comprising the steps of:
• a dry wound dressing composition comprising a mixture of hydroxyethyl cellulose and gellan gum, and where necessary, one or more active ingredients, in an amount that the mixture will form a topical gel upon hydration of the mixture;
• step 4 of the preceding method i.e. covering the wound for example with a plaster, compress or bandage, may be performed after step 3 of the present method.
• a dry wound dressing composition comprising a mixture of hydroxyethyl cellulose and gellan gum, and where necessary, one or more active ingredients, in an amount that the mixture will form a topical gel upon hydration of the mixture;
• the wound dressing compositions comprising a gellan gum and HEC, especially in form of a hydrogel, are per se beneficial for wound healing.
• active substances beneficial for wound healing in the following called wound healing therapeutic substances, may be added in order to further improve the beneficial effect of the compositions on the wound healing process.
• the HEC/gellan gum wound dressing compositions of the invention have also an excellent ability to stabilize and deliver proteins in a controlled way, in particular when the protein is a protease inhibitor, most particularly when the protease inhibitor is alpha- 1-antichymotrypsin. It was found that the wound dressing compositions of the present invention are suitable for stabilizing ACT in a storage form allowing easy storage, and which release ACT at a controlled rate (see example 7). Thus the present invention also relates to wound dressing compositions of the present invention, wherein the compositions further comprise one or more wound healing therapeutic substances.
• Such wound healing therapeutic substance maybe present in a concentration of about up to about 10% by weight, preferably from about 0.001 to about 5% by weight, typically from about 0.1 to about 2% by weight of one or more wound healing therapeutic agents, such as non-steroidal anti-inflammatory drugs, e.g. acetaminophen, steroids, like hydrocortisone or betamethosone, local anaesthetics, antimicrobial agents, growth factors (e.g. fibroblast growth factors or platelet derived growth factor), or protease inhibitors, in particular alpha- 1-antichymotrypsin (ACT) or alpha- 1 -antitrypsin (AAT).
• the antimicrobial agent may, for example, comprise an antiseptic, an antibiotic, or mixtures thereof.
• Preferred antibiotics include cephalosporins (cephalexin, cefoxytin, and others), penicillins (amoxycillin, ampicillin, phenoxymethylpenicillin, and others), tetracyclines (minocycline, doxycycline, and others), aminoglycosides (gentamicin, neomycin, and others), antifungals (isoconazole, clotrimazole, amphotericin, and others), sulphadiazine, chloramphenicol, erythromycin, vancomycin, trimethoprim, and others.
• Preferred antiseptics include silver, including colloidal silver, silver salts including one or more silver salts of one or more of the anionic polymers making up the material, silver sulfadiazine, chlorhexidine, povidone iodine, triclosan, sucralfate, quarternary ammonium salts and mixtures thereof.
• concentrations refer to the concentration in the wet, hydrogel state of the wound dressing compositions of the invention.
• the wound healing therapeutic substance is a protein.
• the wound healing therapeutic substance is a protease inhibitor belonging to the Serpin family.
• Preferred serpins are ACT, alpha-1-antitrypsin, antithrombin III, alpha-2-antiplasmin, Cl Inhibitor (ClINH), pancreatic trypsin inhibitor, plasminogen activator inhibitor- 1 (PAI-I), Plasminogen activator inhibitor type-2 (PAI-2), Heparin Cofactor II, active protein C inhibitor, PN-I, Maspin, SERPINB12, Protease inhibitor 14, SERPINB3 and -4, SERPINBl.
• the protease is selected from ACT and alpha-1-antitrypsin. Ih the most preferred embodiment of the invention the wound healing therapeutic substance is ACT.
• a wound healing composition in a wet, hydrogel state comprising a gellan gum, HEC and a wound healing therapeutic substance, particularly ACT
• contains the wound healing therapeutic substance in a concentration of about ACT is between 10 ⁇ g/ml and 10 mg/ml in the wet, hydrogel state, preferably between 100 ⁇ g/mi and 10 mg/ml.
• a dry wound healing composition comprising a gellan gum, HEC and a wound healing therapeutic substance, particularly ACT, contains the wound healing therapeutic substance in a concentration of about between 0,1 ⁇ g/cm 2 and 1000 ⁇ g/cm 2 of wound dressing surface.
• the present invention makes available a method of treatment of a chronic wound in a mammal, such as a neuropathic ulcer decubitus ulcer, a venous ulcer or a diabetic ulcer or an infected wound.
• the method comprises applying a wound dressing according to the invention comprising a wound healing therapeutic substance to the wound.
• the wound dressing according to the invention is applied to the chronic wound for a period of at least 1 hour, preferably at least 24 hours, more preferably at least 48 hours, and most preferably at least 72 hours.
• the treatment may be extended for several days, weeks or months, with dressing changes as appropriate, if necessary for chronic wounds.
• sterile wound healing compositions can be performed as follows comprising the steps of:
• HEC and gellan gum as dry powders are levigated in a mortar and thereby mixed.
• the mixture is dispersed in a suitable solvent, preferably an aqueous solution and is homogenised by stirring.
• the dispersion is then autoclaved, whereby sterility and the gelling notably of the gellan gum component, which is temperature dependent, is achieved at the same time.
• the preparation is poured at a temperature between 70°C to 9O 0 C into a suitable dish or may get cast out with a scraper on a suitable base. Solvent is removed from the dispersion to leave a solid material comprising a homogeneous mixture of HEC and gellan gum.
• the optional, additional components in the materials according to the present invention are preferably included in the dispersion prior to autoclavation.
• an additional working step may be added: After the removal of solvent the solid is again hydrated by the addition of a suitable solution of the protein or other sensitive substance with suitable excipients. Then an additional step of removing the solvent is performed.
• the solvent can be removed from the dispersion by evaporation, for example by evaporation from the dispersion in a tray resulting in a dry film.
• the solvent preferably water
• the solvent is removed by freeze-drying (lyophilising) or solvent-drying to obtain the material in the form of a xerogel or sponge.
• the method of lyophilisation is carried out including an annealing step before primary drying, see example 9.
• the present invention also relates to a method for freeze-drying of an dispersion of gellan gum and HEC, characterized in that an annealing step is performed before the primary drying. Further preferred embodiments of the method of the invention can be taken from the attached claims.
• the invention further relates to a dry wound dressing composition, obtainable by the method of the invention.
• the invention further relates to the use of a gellan gum and HEC for the preparation of a wound dressing composition for topical administration. All embodiments discussed above for the wound dressing composition of the invention also apply to this use of the invention.
• the use of the invention further comprises the use of an active ingredient. All embodiments discussed above with respect to the active ingredients of the wound dressing composition of the invention also apply to the use of the invention.
• Xerogel according to the present invention is to be understood as porous, sponge-like matrix obtainable from a hydrogel e.g. by freeze-drying comprising at least one gelating substance wherein the matrix has the potential to swell and form hydrogels when in contact with aqueous solutions.
• “Film” according to the present invention is to be understood as polymer-based foil of flat- shaped form of uniform thickness and consistency obtainable from a hydrogel by drying, e.g. evaporative drying or by casting from organic solutions. The matrix has the potential to swell and form hydrogels when in contact with aqueous solutions.
• “Dry” according to the present invention is to be understood as containing a very low content of water, preferably less than 5% (w/w) moisture, more preferably less than 2% (w/w) moisture, especially preferably less than 1% (w/w) moisture. Moisture can be determined by coulometric Karl-Fischer titration, for example using KF 373 (Metrohm GmbH & Co, Filderstadt, Germany).
• Active ingredient is to be understood as any substance which causes a biological effect, either directly or when released from its pro-drug form in vivo and which is thus beneficial for the medical treatment or prevention of diseases and /or disorders or for cosmetic treatment of conditions of the body.
• wound is to be understood as any injury of the skin resulting in a partial or complete destruction of the skin barrier function by partial or complete local destruction of the skin integrity. Such wounds may result from mechanical impact as slash wounds or stab wounds, or maceration and/or ulceration as observed in decubitus ulcers or diabetic ulcers, or burn wounds resulting from high temperature or chemicals. Preferred wounds according to the present invention are chronic wounds which heal slowly or badly or not all under standard treatment, especially diabetic ulcer, venous ulcers, neuropathic, ulcers and decubitus ulcers or infected wounds.
• Alpha- 1-antichymotrypsin or "ACT” according to the present invention is to be understood as an alpha- 1-antichymotrypsin protein showing at least approximately 70%, in particular at least approx. 80%, especially at least approx. 90%, more preferred at least 95%, even more preferred at least 98% sequence identity to human wildtype alpha- 1- antichymotrypsin and having at least 1%, more preferably at least 10%, even more preferably at least 50% inhibitory activity versus Cathepsin G compared to mature wildtype alpha- 1-antichymotrypsin isolated from human blood serum.
• the inhibitory action can be determined by assays well known in the art.
• An ACT protein according to the present invention may be isolated from a mammalian organism, preferably from human serum or may be produced recombinantly, for example by use of viral, bacterial, fungal or mammalian expression systems. Such ACT may be glycosylated, partially glycosylated or unglycosylated. hi a preferred embodiment, ACT is mature human wildtype ACT. hi another embodiment, the ACT lacks all or part of the N-terminal signal sequence, more particularly lacks the signal sequence.
• Sequence identity is understood as degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of, for example, BlastP 2.0.1 and in the case of nucleic acids by means of, for example, BLASTN 2.014, wherein the filter is set off and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).
• BLASTN 2.014 BLOSUM is 62
• Ih humans one ACT gene encoding ACT is known, with polymorphism having been described, particularly in the signal peptide sequence (Rubin, 1989, database entry).
• the sequence of human wildtype ACT polypeptide sequence with signal peptide is shown in SEQ ID No. 1.
• ACT polypeptides which exhibit ACT activity comparable to ACT polypeptides according to SEQ ID No. 2 and 3 are polypeptides according to SEQ ID No. 4 and SEQ ID No. 5 which can be e.g.
• N-terminal Methionine obtained by recombinant expression in yeast and which are characterized by an N-terminal Methionine followed by the sequence of the mature ACT polypeptides according to SEQ ID No. 7 and SEQ ID No. 10.
• N-terminal extensions for example a Methionine extension which may be introduced in order to facilitate expression and/or purification do not affect ACT activity.
• polypeptides according to any of SEQ ID No. 1 to 5 and functional variants thereof are preferred ACT polypeptides according to the invention. Particularly preferred is the use of mature ACT polypeptides lacking the signal peptide according to SEQ ID No. 2 to 3 and functional variants thereof, especially functional variants according to SEQ ID No 4 to 5.
• ACT polypeptides which can be used in accordance with the invention, which variants possess protease inhibitor specificity with regard to cathepsin G which is similar to that of the mature wildtype ACT polypeptide.
• functional variants of ACT polypeptides according to SEQ ID No. 1 to 5 possess at least approximately 70%, in particular at least approx. 80%, especially at least approx. 90%, more preferred at least 95%, even more preferred at least 98% sequence identity with one of the sequences SEQ ID No. 1, SEQ ID No.: 3, SEQ ID No.: 4, or SEQ ID No, 5 and have the Cathepsin G protease inhibitory activity similar to that of the mature wildtype ACT polypeptide which can be determined as described in Example 7.
• Similar inhibitory activity is to be understood as activity which is at least 1%, preferably at least 10%, even more preferably at least 50% of the activity of a mature human wildtype ACT polypeptide.
• Functional variants of the polypeptides can also be parts of the polypeptides used in accordance with the invention which, when compared with the wildtype ACT polypeptides, do exhibit similar protease inhibitor activity.
• the first amino acid i.e. methionine
• an N-terminal Methionine may be added to an ACT polypeptide or functional variants thereof, e.g.
• a methionine may be added to the N-terminus of a mature ACT polypeptide without there being any significant change in the function of the polypeptide.
• N- and/or C- terminal and/or internal deletions of the polypeptide in the range of approx. 1-60, preferably of approx. 1-30, in particular of approx. 1-15, especially of approx. 1-5, amino acids are also included provided the protease inhibitor specificity remains essentially unaltered as compared with that of the respective wildtype polypeptide.
• deletions which affect the signal peptide, or parts thereof, at the N terminus of an ACT polypeptide e.g.
• an ACT polypeptide having the last 4 amino acids of the signal peptides followed by the sequence of the mature ACT polypeptide according to SEQ ID No. 3 retains full ACT activity (US 5,367,064).
• ACT polypeptides having an additional N-terminal Methionine are also preferred ACT polypeptides according to the invention, especially ACT polypeptides according to SEQ ID No. 4 and SEQ ID No. 5.
• Figure 1 shows the results of example 7.
• Figure 2 shows a xerogel pad after freeze drying without annealing step
• Picture 3 shows a xerogel pad after freeze drying with annealing step
• Fig. 2-1 Amino acid sequence of human ACT with numbering.
• Fig. 2-3 deacetylated gellan gum.
• Fig. 2-4 Warm air drier for vials and eppendorf caps.
• Fig. 3-1 pH stability profile of ACT in 5OmM potassium phosphate buffer after a 5 days storage at 40 0 C measured by activity assay.
• Fig. 3-2 pH dependent stability of ACT in 5OmM potassium phosphate buffer after a 5 days storage at 40 0 C measured by SDS-PAGE: lane 1: pH 2.2 with 0.5% 10OkDa aggregate, 3.2% 4OkDa, 0.7% 2OkDa fragments; lane 2: pH 4.5 with 9.7% 10OkDa aggregate, 3.8% 4OkDa, 0.9% 2OkDa fragments; lane 3: pH 6.5 with 8.5% 10OkDa aggregate; lane 4: pH 7.4 with 4.7% 10OkDa aggregate; lane 5: pH 8.0 with 2.5% 10OkDa aggregate; lane 6: pH 11.0 with 1.7% 10OkDa aggregate; lane 7: marker; control without degradation is visualised in Fig. 3-4.
• Fig. 3-3 pH stability profile of ACT in 5OmM potassium phosphate buffer after 5 F/T cycles in liquid nitrogen and 25°C, resp. measured by activity assay.
• Fig. 3-4 SDS-PAGE gel for evaluation of freeze/thaw stress dependent stability of ACT in 5OmM potassium phosphate buffer pH 7.2 after 5 F/T cycles: lane 1 F/T stressed sample with 9.8% 10OkDa aggregate; lane 2 unstressed control sample without fragment and aggregate formation; lane 3 marker.
• Fig. 3-5 Recovered relative activities of ACT after 9 days at 4O 0 C storage in 25mM phosphate buffer and varying citrate contents (o) and after 5 F/T cycles in 5mM phosphate buffer and varying citrate contents (A) measured by activity assay.
• Fig. 3-6 Recovered relative activity of ACT in 5OmM potassium phosphate buffer pH 7.2 with different salt additives before (above) and after (below) a 6 days storage at 40°C measured by activity assay.
• Fig. 3-7 Response surface calculated from recovered relative activities of ACT in samples with potassium phosphate buffer pH 7.2 and salt (KCl) contents according to a simplex- lattice design after temperature treatment - 40°C for 9 days.
• Fig. 3-8 Recovered relative activities of ACT after 5 F/T cycles in varying potassium phosphate buffer contents pH 7.2 measured by activity assay.
• Fig. 3-9 Recovered relative activity of ACT in dependence of the concentration of surfactants in ACT samples in 5OmM potassium phosphate buffer after a 9 days storage at 40°C measured by activity assay; D: Poloxamer ® 188, 0: Tween ® 80, ⁇ : Solutol ® HS15.
• Fig. 3-10 Recovered relative activity of ACT in dependence of the concentration of surfactants in ACT samples in 1OmM phosphate buffer pH 7.2 after 15 F/T cycles measured by activity assay; above: Tween ® 80; below: Poloxamer ® 188.
• Fig. 3-12 Recovered relative activity of ACT in 5OmM potassium phosphate buffer pH 7.2 with cyclodextrin additives (2%) before (above) and after (below) a 9 days storage at 40 0 C measured by activity assay.
• Fig. 3-13 Recovered relative activity of ACT in dependence of cyclodextrin addition (2%) in ACT samples in 1OmM potassium phosphate buffer pH 7.2 after 15 F/T cycles measured by activity assay.
• Fig. 3-14 Recovered relative activity of ACT in dependence of HP- ⁇ -cyclodextrin concentration in ACT samples in 1OmM potassium phosphate buffer pH 7.2 after 15 F/T cycles measured by activity assay; data points correspond to molecular ratios of ACT : HP- ⁇ -CD being 1:0, 1:1, 1:5, 1:10, 1:20, 1:50, 1:100.
• Fig. 3-15 HP- ⁇ -CD concentration dependent stability of ACT in 1OmM potassium phosphate buffer pH 7.2 after 15 F/T cycles measured by SDS-PAGE: Concentration is given in molecular ratio ACT : HP- ⁇ -CD; lane 1: 1:1, 11% aggregate; lane 2: 1:5, 8.5% aggregate; lane 3: 1:10, 5.8% aggregate; lane 4: 1:50, 3.2% aggregate; lane 5: 1:100, 1.5% aggregate; lane 6: untreated control; lane 7: marker.
• Fig. 3-16 Recovered relative activity of ACT in 5OmM potassium phosphate buffer pH 7.2 with different amino acids before (above) and after (below) a 9 days storage at 40 0 C measured by activity assay.
• Fig. 3-17 SDS-PAGE gel on influence of amino acids on stability of ACT in 5OmM potassium phosphate buffer pH 7.2 after thermal treatment (9 days, 40 0 C): lane 1: methionine 9.6% dimer, 1.2% fragment; lane 2: arginine only monomer; lane 3: phenylalanine 4.5% dimer, 3.6% fragment; lane 4: lysine 6.7% fragment; lane 5: cysteine 21.6% dimer; lane 6: glycine 1.4% dimer; lane 7: alanine 3.6% dimer; lane 8: marker.
• Fig. 3-18 Recovered relative activity of ACT in 5OmM phosphate buffer pH 7.2 with different arginine contents after a 9 days storage at 40°C measured by activity assay.
• Fig. 3-19 Recovered relative activity of ACT in 5OmM potassium phosphate buffer pH 7.2 with preservatives before and after a 9 days storage at 40 °C measured by activity assay.
• Fig. 3-21 Ln k values of carboxymethyl cellulose sodium 10.000 (D), hydroxyethyl cellulose 100.000 ( ⁇ ), and hydroxyethyl cellulose 10.000 (A) gels in dependence of the gelling agent content measured after steam sterilisation.
• Fig. 3-23 Flow curves in amplitude sweep of 2.0% hydroxyethyl cellulose / 1.0% gellan gum; in 5OmM potassium phosphate buffer pH 7.2 with storage modulus G' (0), loss modulus G" ( ⁇ ), loss factor tan ⁇ P h 0S phate buffer, yield point ⁇ Y phosphate buffer; in water with storage modulus G' (D), loss modulus G" ( ⁇ ), loss factor tan ⁇ wa ter •
• Fig. 3-24 Recovered relative activity of ACT in 5OmM potassium phosphate buffer pH 7.2 and 0.1% Poloxamer ® 188 with various polymers at 0.5% content after a 9 days storage at 40°C measured by activity assay; polymers are HEC 10.000, CMC Na 10.000, HPC 100.000, HPMC 15.000, gellan gum LTlOO, PVP 17, PEG 2000, PVA 100.000.
• Fig. 3-25 Recovered relative activity of ACT in 1OmM potassium phosphate buffer pH 7.2 and 0.1% Poloxamer ® 188 with various polymers at 0.5% content after 15 freeze/thaw cycles in liquid nitrogen measured by activity assay; control is formulated in buffer; polymers are HEC 10.00O 5 CMC Na 10.000, HPC 100.000, HPMC 15.000, gellan gum LTlOO, PVP 17, PEG 2000, PVA 100.000.
• Fig. 3-27 Recovered relative activities of a hydrogel formulation consisting of 60 ⁇ g/ml ACT, 1OmM arginine, 0.1% Tween ® 80, 0.05% PVP 17, 2.5% hydroxyethyl cellulose in a 5OmM potassium phosphate buffer pH 7.2 at 6 °C (D) and 40°C ( ⁇ ) in linear scale measured by activity assay.
• Fig. 3-28 Recovered relative activities of a hydrogel formulation consisting of 60 ⁇ g/ml ACT, 1OmM arginine, 0.1% Tween ® 80, 0.05% polyvinyl pyrrolidone 17, 2.5% hydroxyethyl cellulose in a 5OmM potassium phosphate buffer at 6°C (G) and 40°C ( ⁇ ) in logarithmic concentration scale with fitted logarithmic functions resulting in evens in the one-sided logarithmic scale as predicted by first-order time law; measured by activity assay.
• Fig. 3-29 Recovered relative activities of a hydrogel formulation consisting of 60 ⁇ g/ml ACT, 1OmM arginine, 0.1% Poloxamer ® 188, 1.5% polyvinyl pyrrolidone 17, 1.0% PEG 400, 1.0% gellan gum, 2.0% hydroxyethyl cellulose in a 5OmM potassium phosphate buffer at 6°C measured by activity assay; relative activity is in logarithmic scale; fitted logarithmic function and 95% confidence intervals are shown; 10% loss line marks the lower specification limit.
• Fig. 3-33 Xerogel pads consisting of hydroxyethyl cellulose 100.000 made from 2.5% hydrogels by conventional freeze drying process without annealing; left: top, right: bottom Fig. 3-34 Xerogel pads consisting of hydroxyethyl cellulose 100.000 made from 2.5% hydrogels by the freeze drying process with annealing step during freezing; left: top, right: bottom.
• Fig. 3-35 Temperature/pressure - time diagram of freeze drying process with annealing step during freezing; — plate temperature, — product temperature, — cabin pressure.
• Fig. 3-36 X-ray patterns of xerogels consisting of hydroxyethyl cellulose 100.000 made from 2.5% hydrogels without (above) and with (below) annealing step during lyophilisation; curves are shifted on the y-axis for better demonstration.
• Fig. 3-37 Swelling of xerogels after 3 minutes exposure to water; matrices are made from different hydroxyethyl cellulose qualities in different concentrations but comparable viscosity in the hydrated state.
• Fig. 3-38 Force diagram of texture analysis of xerogels made form 5% hydroxyethyl cellulose 4000; xerogel is compressed to 50% of height followed by release to beginning; integrated areas are deformation and restoring energy.
• Fig. 3-39 Swelling behaviour of xerogels made from hydrogels containing 2.5% hydroxyethyl cellulose 100.000 and various phosphate buffer ( ⁇ ) and TrisHCl buffer (D) contents.
• Fig. 3-40 Swelling behaviour of xerogels made from hydrogels containing 2.5% hydroxyethyl cellulose 100.000 and various Tween ® 80 ( ⁇ ) and Poloxamer ® 188 (D) contents.
• Fig. 3-41 Swelling behaviour of xerogels made from hydrogels containing 2.5% hydroxyethyl cellulose 100.000 and various polyvinyl pyrrolidone 17 ( ⁇ ) and polyethylene glycol 2.000 (D) contents.
• Fig. 3-42 Swelling behaviour of xerogels made from hydrogels containing 2.5% hydroxyethyl cellulose 100.000, 10 mM potassium phosphate buffer pH 7.2, 10 mM arginine, 0.1% Tween ® 80, 0.05% polyvinyl pyrrolidone 17; left: dry pad, 2 nd from left: pad is soaking when put in contact with water, 3 rd from left and right: soaked pad stays in shape for 2-3 minutes before flowing starts.
• Fig. 3-45 Relative activity of ACT in 2.5% hydroxypropylmethyl cellulose xerogels with 1OmM potassium phosphate buffer pH 7.2 after reconstitution with 4OmM potassium phosphate buffer pH 7.2 measured by activity assay; control without additional spiking, samples with surfactant and soluble polymer, respectively.
• Fig. 3-46 Relative activity of ACT in xerogels with 1OmM potassium phosphate buffer, 0.1% Tween ® 80, 0.05% PEG 2000 and gelling agent (above) and additional 0.0075% hydroxypropyl- ⁇ -cyclodextrin spike (below) after reconstitution with 4OmM potassium phosphate buffer measured by activity assay; gellants are 2.5% hydroxypropylmethyl-, hydroxypropyl-, hydroxyethyl cellulose 100.000, gellan gum/ hydroxyethyl cellulose 100.000 l%/2% mixture, 3.0% carboxymethyl cellulose sodium 10.000, 5.0% alginate sodium; * sample not measured.
• Fig. 3-47 Recovered relative activities of a xerogel formulation consisting of 60 ⁇ g/ml ACT, 1OmM arginine, 0.1% Poloxamer ® 188, 0.05% PEG 2000, 2.5% hydroxyethyl cellulose 100.000 in a 1OmM potassium phosphate buffer pH 7.2 at 25°C ( ⁇ ) and 40°C (O) in linear scale measured by activity assay.
• Fig. 3-48 Recovered relative activities of a xerogel formulation consisting of 60 ⁇ g/ml ACT, 1OmM arginine, 0.1% Poloxamer ® 188, 0.05% PEG 2000, 2.5% hydroxyethyl cellulose 100.000 in a 1OmM potassium phosphate buffer at 25 0 C ( ⁇ ) and 4O 0 C (G) in logarithmic concentration scale with fitted logarithmic functions resulting in evens in the one-sided logarithmic scale as predicted by first-order time law; measured by activity assay.
• Fig. 3-49 Stability of ACT in formulations after a 3 months storage at 25 °C measured by SDS-PAGE lane 1: formulation (1) with 9.4% 10OkDa aggregate; lane 2: formulation (2) with 4.7% 10OkDa aggregate; lane 3: formulation (3) with 3.5% 10OkDa aggregate; lane 4: formulation (4) with 1.7% 10OkDa aggregate, 7.2% 4OkDa, 10.7% 1OkDa, and other fragments; lane 5: formulation (5) with 3.5% 10OkDa aggregate; lane 6: formulation (6) with 0.7% 10OkDa aggregate; lane 7: marker; control without degradation is visualised in Fig. 3-4.
• Fig. 3-51 Swelling of film matrices made from hydrogels containing 2.5% hydroxyethyl cellulose 100.000, 2.5% hydroxypropyl cellulose 100.000, 2.5% hydroxypropylmethyl cellulose 100.000, 3.5% hydroxypropylmethyl cellulose 15.000, and 5.0% carboxymethyl cellulose sodium 10.000, respectively (left bar); swelling with addition of 0.5% carboxymethyl starch to the basic hydrogels (center bar); swelling with addition of 0.5% crosslinked carboxymethyl cellulose to the basic hydrogels (right bar).
• Fig. 3-52 Measurement of tensile strength with texture analyser (left); force-distance diagram of tension experiments with rupture at 11.8 N (right).
• Fig. 3-54 Response surface calculated from tensile strength values of samples with 2.25% polyethylene glycol 400; hydroxyethyl cellulose 100.000 and polyvinyl pyrrolidone 17 contents vary according to a simplex-lattice design; concentrations refer to the hydrated state before drying.
• Fig. 3-55 Response surface calculated from tensile strength values of samples with 2.25% polyvinyl pyrrolidone 17; polyethylene glycol 400 and hydroxyethyl cellulose 100.000 contents vary according to a simplex-lattice design; concentrations refer to the hydrated state before drying.
• Fig. 3-56 Response surface calculated from tensile strength values of samples with 2.25% hydroxyethyl cellulose 100.000; polyethylene glycol 400 and polyvinyl pyrrolidone 17 contents vary according to a simplex-lattice design; concentrations refer to the hydrated state before drying.
• Fig. 3-57 Response surface calculated from elastic moduli of samples with 2.25% hydroxyethyl cellulose 100.000; polyethylene glycol 400 and polyvinyl pyrrolidone 17 contents vary according to a simplex-lattice design; concentrations refer to the hydrated state before drying.
• Fig. 3-58 Response surface calculated from elastic moduli of samples with 2.25% polyvinyl pyrrolidone 17; polyethylene glycol 400 and hydroxyethyl cellulose 100.000 contents vary according to a simplex-lattice design; concentrations refer to the hydrated state before drying.
• Fig. 3-59 Response surface calculated from tensile strength values of film samples with variable hydroxyethyl cellulose 100.000 and gellan gum F contents according to a simplex- lattice design; films also contain 2.25% polyethylene glycol 400 and 2.25% polyvinyl pyrrolidone 17; concentrations refer to the hydrated state before drying.
• Fig. 3-60 Response surface calculated from elastic moduli of firm samples with variable hydroxyethyl cellulose 100.000 and gellan gum F contents according to a simplex-lattice design; films also contain 2.25% polyethylene glycol (PEG) 400 and 2.25% polyvinyl pyrrolidone (PVP) 17; concentrations refer to the hydrated state before drying.
• PEG polyethylene glycol
• PVP polyvinyl pyrrolidone
• Fig. 3-61 Relative activity of ACT in films with 5mM potassium phosphate buffer, 0.1% Tween ® 80, 1.5% PEG 400 and gelling agent after reconstitution with 45mM potassium phosphate buffer measured by activity assay; gellants are 2.5% hydroxypropylmethyl-, hydroxypropyl-, hydroxyethyl cellulose 100.000, gellan gum/hydroxyethyl cellulose 100.000 l%/2% mixture, 3.0% carboxymethyl cellulose sodium 10.000, 5.0% alginate sodium.
• Fig. 3-63 Stability of ACT in formulations after a 3 months storage at 25 0 C measured by SDS-PAGE: lane 1 formulation (1) with no aggregate detected; lane 2 formulation (2) with 0.7% 5OkDa fragment; lane 3 formulation (3) with no aggregate detected; lane 4 formulation (5) with 0.9% 10OkDa aggregate; lane 5 marker; control without degradation is visualized in Fig. 3-4.
• Fig. 3-64 Modified Loth chamber made of acrylic glass for release studies; the donor chamber (left and above) keeps 1.25ml of donor medium at a layer thickness of 4mm; the acceptor chamber (right and top) provided with a ripple plate supporting the membrane is filled with acceptor medium and is connected to the medium reservoir (not shown); chambers are separated by a cellulose acetate filter membrane with 0.45 ⁇ m pore size; the system is sealed by a polyurethane rubber o-ring.
• Fig. 3-66 Release diagram of Fluorescein Na ( ⁇ ), FITC-Dextran 19kDa (0), FITC- Dextran 7OkDa (D), respectively, from hydrogels containing 2.5% hydroxyethyl cellulose 100.000 in water; release medium is a 1OmM potassium phosphate buffer pH 7.2, 0.1% Poloxamer ® 188.
• FITC- Dextran molecule sizes are stokes diameters - 6.6nm for 19kDa derivative, 12.0nm for 7OkDa derivative200; membrane pore size is given as 450nm, and ACT diameter is estimated as 5.4nm.
• Fig. 3-71 Decrease of relative activity of a 60 ⁇ g/ml ACT solution (1OmM potassium phosphate buffer pH 7.2, 0.1% Poloxamer ® 188) during stirring in the acceptor surplus (D) and during pumping through the tubes and stirring in the surplus ( ⁇ ).
• Fig. 3-72 Residual activity of a 60 ⁇ g/ml ACT solution formulated in a 5OmM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer ® 188 during exposure to tubing material, chamber material, membrane material, and glass, respectively, measured by activity assay; left: control before exposure; centre: exposure for 18 hours, right: exposure for 75 hours.
• Fig. 3-73 Residual total content of a 60 ⁇ g/ml ACT solution formulated in a 5OmM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer ® 188 during exposure to tubing material, chamber material, membrane material, and glass, respectively, measured by ELISA; left: control before exposure; centre: exposure for 18 hours, right: exposure for 75 hours.
• Fig. 3-74 Release diagram of ACT from a xerogel formulation in the static model; release profiles are given as active ACT gained by activity assay (D) and total ACT content measured by ELISA ( ⁇ ); underlying hydrogel contains 60 ⁇ g/ml ACT, 1OmM potassium phosphate buffer pH 7.2, 1OmM arginine, 0.1% Poloxamer ® 188, 0.05% PVP 17, and 2.5% hydroxyethyl cellulose 100.000 in water - xerogel formulation (1) of Tab. 3-18; release medium is a 1OmM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer ® 188.
• Example 1 Preparation of xerogels comprising HEC, gellan gum and ACT in vials
• the preparation is poured at a temperature between 70°C to 9O 0 C into a petri dish to a height of 4 mm. Under these conditions the mass is freely flowing and forms a cylindrical shape with uniform surface and thickness in the dish.
• the film is cut in pieces that exactly cover the bottom of glass vials.
• the film is placed in the vial and is hydrated with the ACT containing solution of step (6) to a filling height of 4 mm In a 2R vial the necessary amount is 0.4ml.
• the mixture is allowed to swell for at least 24 hours to again form a homogeneous hydrogel.
• shear stress which is a critical part in protein handling
• the dry pad has a homogeneous appearance and its mechanical properties are very suitable for proper handling by both patients and personnel. It swells within minutes to form a hydrogel when put in contact with an aqueous solution. Once rehydrated, the gel very soft and feels comfortable when put onto a wound. It also provides intimate contact with the wound ground to ensure the proper release of the active ingredient load.
• Example 2 Preparation of xerogels comprising HEC, gellan gum and ACT in sheets of 2 mm height
• the preparation of the xerogel in sheets follows the same procedure as the preparation of the xerogel in vials from step ACT containing solution of step (6) of example 1 is poured onto the placebo film in the dish to a height of 4 mm and is swollen for at least 24 h.
• the hydrated gel is heated to 40°C to achieve good flowability and is filled into a scraper.
• the gel now ils cast out on a glass base to produce a wet film with a height of 2 mm.
• Step (9) is carried out like step (8) above in example 1.
• Example 3 Preparation of films comprising HEC, gellan gum and ACT in vials
• the preparation of the film follows the same procedure as the preparation of the xerogel from step (1) to (5).
• the film ils cut in pieces that exactly cover the bottom of glass vials.
• the film is placed iln the vial and is hydrated with the ACT containing solution to a filling height of 4 mm.
• the mixture is allowed to swell for at least 24 hours to form a homogeneous hydrogel again.
• the obtained film is very soft and elastic but provides the necessary robustness for good handling qualities. It swells when hydrated to form a clear gel within about 45 minutes. Once rehydrated the gel on the wound is very soft and comfortable. It also provides intimate contact with the wound ground to ensure the proper release of the active ingredient load.
• Example 4 Preparation of films comprising HEC, gellan gum and ACT in sheets
• the preparation of the film follows the same procedure as the preparation of the xerogel from step (1) to (5) in example 1.
• ACT loading simply by hydration a first solution without the protein containing a 5 mM potassium phosphate buffer, 0,1% of Poloxamer 188,0, 1,0% PEG 400, and 1,5% of Kollidon 17PF is prepared and sterilized by autoclavation. Under aseptic conditions the alpha-1-antichymotrypsin is dissolved and the solution is again filtered through a 0.22 ⁇ m unit to provide sterility for the whole solution. (7) The ACT containing solution is poured onto the placebo film in the dish to a height of 4 mm and is swollen for at least 24 h.
• the hydrated gel is heated to 40°C to achieve good flowability and is filled into a scraper.
• the gel now is cast out on a glass base to produce a wet film with a height of 2 mm.
• the matrix is obtained in sheets and can be cut into pieces suitable for further packaging. This simplifies production processes.
• the xerogels are produced like in example 2 but without the steps (4) to (7).
• the product has the same properties than example 2. It can for example be used as a wound dressing.
• the films a produced like in example 4 but without the steps (4) to (7).
• the product has the same properties than example 4. It can for example be used as a wound dressing.
• Example 7 Release of ACT from wound dressing compositions comprising HEC, gellan gum and ACT and determination of stability
• the acceptor consists of 10 niM potassium phosphate buffer pH 7,2.
• the reconstitution of the dried forms is done with phosphate- buffered saline (PBS).
• ACT activity In order to determine the ACT activity, an activity assay based on Cathepsin G binding was performed. 96-well plates were coated with BSA and subsequently with Cathepsin G. After washing, ACT probes were added and incubated for 30 min at 37°C. After washing with PBS-T 3 times, a rabbit anti human ACT antibody was added to the wells and was incubated for 30 min at 37°C. After washing three times with PBS-T buffer, a goat anti rabbit IgG antibody conjugated with horseradish peroxidase was added and was again incubated for 30 min at 37°C. The wells were washed three times with PBS-T buffer.
• OPD (1,2-Diaminobenzene) substrate solution was prepared according to the manufacturer's protocol (Sigma), added to the wells and Incubated at room temperature in the dark. After 10 minutes the reaction was stopped by adding 100 ⁇ m 0,5 M H2SO4 per well. Immediately after stopping the reaction the absorption at 490 nm was determined.
• the percentage of the bioactive fraction of the total ACT amount in each sample was determined and expressed in Figure 1 as percentage in comparison with untreated samples of ACT.
• the release maximum is determined 5 days after start of diffusion when an equilibrium is reached.
• HEC/Gellan gum (Kelcogel) mixtures contain 2% HEC and 1% gellan gum in the wet state.
• HEC gels contain 2,5% of hydroxyethyl cellulose without gellan gum ( Figure 1).
• HEC is hydroxyethyl cellulose (Natrosol® 250 HHXpharm, Dow Chemicals), two different types of Gellan gum were tested: Kelcogel F® (Kelco) as a deacetylated Gellan Gum, and Kelcogel LT 100® (Kelco), as an acetylated Gellan gum.
• compositions of the invention comprising ACT are especially suitable for use in wound healing.
• Example 9 Freeze drying of hydrogels to form xerogels The freeze drying operation is carried out in a Christ® Lab scale freeze dryer. The program is as follows:
• ACT ⁇ i-antichymotrypsin
• ACT has a molecular weight of 68kDa and consists of 423 amino acids (Fig. 2-1). Moreover, it is heavily glycosylated with 40 neutral sugar residues, 35 acetylglucosamine residues, and 14 acetyhieuraminic acid residues per molecule adding up to about 25% sugar content of the total molecular weight.
• AU polymers were purchased as Ph. Eur. 2004 grade where available. Else, pharma grade was ordered.
• Gellan gum is produced by Pseudomonas elodea. It is an anionic polysaccharide consisting of a repeating linear tetrasaccharide unit. The latter is a sugar sequence of ⁇ -D-glucose, ⁇ - D-glucuronic acid, ⁇ -D-glucose, and ⁇ -L-rhamnose.
• Native gellan gum - Kelcogel ® LTlOO - is partly acetylated at the C 6 -atom of the first glucose unit. Moreover, there is a glyceryl at the same glucose unit (Fig. 2-2).
• the acetylated polymers form soft and very elastic gels because the acetyl groups disturb and therefore reduce the intermolecular forces.
• the deacetylated polysaccharides - Kelcogel ® F - form harder but more brittle gels (Fig. 2-3). Both gellan types form thermoreversible gels. Therefore, gel manufacture is described as cooling of a warm gellan solution. Li our case this step is replaced by steam sterilisation.
• Gellan gum molecules are parallel double helices.
• cations like sodium, potassium or calcium are used for their support of interlinking gellan gum polymers.
• the helices are linked via electrostatic interactions between monovalent cations, water molecules and a carboxyl group : double helix - K + - water - K + - double helix
• Bivalent cations form direct complexes with two carboxyl groups.
• an activity assay based on Cathepsin G binding is performed. During the reaction a complex is formed:
• the antibody 2 is linked to horseradish peroxidase catalysing the detectable colour reaction.
• 96-well plates are coated with BSA and subsequently with Cathepsin G (Calbiochem, Darmstadt, Germany). After washing, ACT samples are added and incubated for 30min at 37°C. After three times washing with PBS-T (phosphate buffered saline with 0.05% Tween®) buffer , a rabbit anti human ACT antibody (DAKO, Glostrup, Denmark) is added to the wells and is incubated for 30min at 37°C. After washing three times with PBS-T buffer, a goat anti rabbit IgG antibody conjugated with horseradish peroxidase (DAKO, Glostrup, Denmark) is added and is again incubated for 30 min at 37°C.
• PBS-T phosphate buffered saline with 0.05% Tween®
• OPD 1,2-Diaminobenzene, Sigma, Taufkirchen, Germany
• substrate solution is prepared according to the manufacturer's protocol with hydrogen peroxide (Sigma, Taufkirchen, Germany), added to the wells and incubated at room temperature in the dark. After 10 minutes the reaction is stopped by adding lOO ⁇ l 0.5M sulphuric acid per well. Immediately after stopping the reaction the absorption at 490 run is determined.
• an ELISA is performed. During the reaction a complex is formed: Antibody 1/ACT/Antibody2
• the antibody2 is linked to horseradish peroxidase catalysing the detectable colour reaction.
• 96-well plates are coated with rabbit anti human ACT antibody (DAKO, Glostrup, Denmark) over night at 6 0 C.
• ACT samples are added and incubated for 120min at 37°C.
• a anti human ACT antibody conjugated with horseradish peroxidase Biotrend, Cologne, Germany
• OPD (1,2-Diaminobenzene) substrate solution is prepared according to the manufacturer's protocol (Sigma, Taufkirchen, Germany), added to the wells and incubated at 25°C in the dark. After 15 minutes the reaction is stopped by adding lOO ⁇ l 0,5M BbSO 4 per well. Immediately after stopping the reaction the absorption at 490 nm is determined.
• Protein integrity was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions using an XCeIl II Mini cell system (Novex, San Diego, USA). Samples were diluted in a pH 6.8 Tris-buffer, containing 2% SDS and 2% glycerin for 30 min at 90°C and subsequently loaded into gel wells (NuPAGER Novex 10% Bis-Tris Pre-cast Gel 1.0 mm from Invitrogen, Groningen, Netherlands). Electrophoresis was performed in a constant current mode of 60 mA in a Tris-glycine/SDS running buffer. After staining with coomassie blue staining kit (Novex Colloidal blue stain kit), the gels were dried using a DryEaseR Gel Drying System (Invitrogen).
• SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
• a scraper For the casting of wet films a scraper is used (Erichsen, Hemer, Germany, Model 360, 03216). It is 6cm wide and provided with defined four gaps of 500 ⁇ m, lOOO ⁇ m , 1500 ⁇ m, and 2000 ⁇ m height, respectively. After loading the scraper with approx. 10 ml of gel the apparatus is drawn over a glass surface (retail window glass prepared by float-technique) with a slow constant speed.
• Lyophilisation is operated in a two chamber fireeze-dryer ⁇ l2G (Christ, Osterode, Germany).
• Primary packaging material for production of placebo xerogels are petri dishes, whereas ACT loaded matrices are produced in 2R glass vials, glass type I with Teflon ® coated chlorobutyl rubber stoppers. 2.2.23 Warm air drying
• Drying is performed with dry and tempered nitrogen gas injected through nozzles into heated vials. All temperatures are set to 25°C. Nitrogen flow rate is 1.0 1/min at a pressure of 0.5 bar.
• Viscometric measurements on hydrogels with pseudoplastic behaviour are conducted on a rotating cylinder viscometer DV-II+ (Brookfield, Middleboro, USA). Complex rheology is measured with a MCR 100 cone plate viscometer (PaarPhysika, Ostfildern, Germany). Therefore, a cone (50mm in diameter, 1° angle; CP 50-1) is used at an oscillation frequency of 10 1/s.
• the mechanical properties of the matrices are evaluated using a Texture Analyser model TA.Xtplus (Stable Microsystems, Godalming, UK).
• Compression test method For compression tests on xerogels a cylindrical probe of 0.5" in diameter is used. The compression strain is set to 50% deformation, crosshead speed is lmm/min.
• Tensile test method The test apparatus for measuring the tensile strength of films consists of two mechanic grips and the test procedure is based on the ASTM D822-75 method. The extension speed is lmm/min. Film specimens which break directly at the grips are discarded.
• Puncture test method Films are punctured by a driving ball probe of 0.5" in diameter. Fixation of the specimen is provided by a film holder. The dry rectangular film specimens are positioned between two mounting plates and are fixed with four screws. The plates contain a hole of 22mm in the centre. The ball probe is driven downwards through the mounted film at a crosshead speed of lmm/min. The calculation of the nominal puncture strength and the elongation at rupture is comparable to the tensile test method. However, the elongation is based on the displacement of the radius instead of the length.
• the chamber body consists of Plexiglas ® .
• Associated material is an IPC 12 channel hose pump (Ismatec, Wertheim, Germany), tubing material (Tygon R3603), cellulose acetate membrane filters 0.45 ⁇ m (Sartorius, Gottingen, Germany), and 2ml syringes (HSW, Tuttlingen, Germany).
• Residual moisture is determined via coulorimetric Karl Fischer titration with generating the iodine electrolytically to keep the reaction ongoing. Therefore, a coulorimetric Karl Fischer titrator with a head space oven is used (Analytik Jena AG, Jena, Germany). Sealed samples are heated to 80°C in the oven chamber. For measurement the vaporised water is transported into the coulorimetric cell filled with Hydranal ® Coulomat AG (Riedel-de Haen) via a needle system.
• Measurements (DSC 204, Netzsch, SeIb, Germany) are performed using 5mg to lOmg of sample. Heating and cooling were conducted at a scan rate of 5 K/min from 25°C to 100 0 C.
• ACT Due to its unique mode of action described in chapter 1 and the fact that its native and active state represents only a state with a relative minimum of free energy, ACT is likely to be prone to deactivation by refolding to the non-active energy minimum state and dimerisation or formation of higher order aggregates by entanglement of the loops of two ACT molecules during loop movement.
• Analytical tools therefore have to be chosen to mainly detect inhibitory activity and aggregate formation of ACT. Furthermore, it is desirable to characterise the tertiary structure especially the folding behaviour of ACT during experiments.
• the low concentration of ACT in measurable samples l ⁇ g/ml to 60 ⁇ g/ml
• low sample amounts resulting from high drug prices lead to unsuitability of many standard methods for these tasks due to inappropriate detection limits and minimal sample volumes 135 .
• These methods include separation methods with UV-detection, i.e. reversed phase liquid chromatography, size exclusion chromatography, capillary electrophoresis, and asymmetric flow field-flow fractionation.
• the immunochemical methods outlined in chapter 2 are used for routine detection because of their high specificity and lowest detection limits. However, these methods hold high error and standard deviation compared to e.g. established chromatographic methods.
• the activity assay based on a cathepsin G/ACT/antibody sandwich and a linked colour reaction is used to determine in vitro inhibitory activity of ACT.
• the ELISA is used for determination of total ACT content during release experiments.
• a SDS- PAGE method with coomassie blue detection is used for quantitative detection of aggregation and fragmentation in selected samples.
• SDS-PAGE is suitable for aggregates formed by covalent binding but non-covalently bound aggregates may be undiscovered.
• Samples are prepared in aliquots of 0.1ml with 60 ⁇ g/ml ACT content in eppendorf caps and are stressed either at 40 0 C in an air bath or by freeze/thawing in liquid nitrogen and room temperature, respectively. Readout is done by activity assay and SDS-PAGE for some samples.
• the pH is a very powerful tool to control both the physical stability 136 and the chemical stability of protein drugs by its well known general influence on chemical reactions. Electrolytes including buffer salts can affect a protein solution by indirect mechanisms like changing the solvent properties or a non-specific electrostatic shielding effect. Besides, very specific ion binding phenomena between salts and protein are known . These effects can also stabilise, behave inert and destabilise the protein in solution, respectively, depending on the definite situation in the formulation. Moreover, the content of salts can effect protein stability directly by liydrophilic or hydrophobic interactions depending on their position in the Hoffmeister lyotropic series 138 ' .
• bi- or polyvalent ions can bind to charged positions within the protein molecule, but may also catalyse oxidative reactions or bind catalysers, e.g. by chelation, which can result in both stabilisation and destabilisation, respectively.
• Gel electrophoresis also detects pH dependent degradation. In acidic buffer samples the fragment bands are striking. Lane 2 of Fig. 3-2 show the main fraction of 85% at the monomer band at around 6OkDa, an aggregate band at about 10OkDa to 12OkDa representing 9.7% of total content, and two fragment bands with 4.7% of total content each at approx. 4OkDa and 2OkDa molecular weight. Similar fragmentation is seen in lane 1. In neutral and basic pHs, lane 3 to 6 of Fig. 3-2, solely the aggregate band can be seen.
• TrisHCl and a phosphate/citrate mixture show comparable recovered relative activities and therefore comparable stabilisation characteristics (Tab. 3-2). As a result, these systems act as alternatives to the standard phosphate buffer for future development.
• the phosphate/citrate mixture is further investigated concerning the relation of concentration of the components. Hence, for temperature stress to a 25mM phosphate buffer increasing amounts of citrate resulting in concentrations from 1OmM to 5OmM are added. For F/T treatment the contents are lowered to 5mM phosphate and 2mM to 2OmM citrate.
• phosphate buffer pH 7.2 is chosen as standard buffer, but the TrisHCl system and the equimolar phosphate/citrate mixture can also be considered if advantage or necessity arises during further development. All buffer systems are approved by FDA for injection up to a content of 1% at least which represents solutions of 7OmM to 8OmM.
• osmotic agent for the use as osmotic agent during formulation and for general use, e.g. during drug substance manufacturing, a variety of salts is tested in temperature stress tests. Samples are buffered in 5OmM potassium phosphate buffer pH 7.2 and salts are added resulting in concentrations of 10OmM, except 1OmM for magnesium chloride and EDTA sodium. Other than before, stressing at 40 0 C lasts for 6 days for technical reasons.
• the first relevant factor is the concentration of the potassium phosphate buffer system. It is varied from a minimum of 5mM to a maximum of 10OmM.
• the other factor is the concentration of potassium chloride as osmotic agent, hereby being OmM the low value and 10OmM the high value.
• buffer systems and electrolyte contents of an ACT stabilising solution are investigated.
• optimal pHs, buffer species, buffer contents, compatible salts and suitable buffer/salt combinations are evaluated.
• the basis for the samples are 0.1ml solution of 60 ⁇ g/ml ACT in a 5OmM potassium phosphate buffer pH 7.2 for temperature stressing and a reduced buffer content of 1OmM for the freeze/thaw experiments.
• surfactants are known to bind at hydrophobic surfaces 142 , gas-liquid interfaces 1 and at the protein molecule itself mostly at hydrophobic areas 144 . Therefore, mechanisms of protein protection by surfactants based on a competitive situation for adsorption on denaturing interfaces between protein and surfactant are discussed 145 . Further, a mechanism relating to a direct binding of the surfactant to the protein, marked by a higher necessary concentration of surfactant that depends on the protein content in the solution has been reported 146 . But surfactants are also able to prevent chemical degradation in some cases 147 .
• Ionic surfactants are usually avoided because of their ability to bind to polar as well as to unpolar groups and therefore d deennaattuurree p prrootteins 148 . For that reason only a choice of non-ionic surfactants is investigated in this study.
• Tween ® 80, Poloxamer ® 188, and Solutol ® HS 15 are added to the standard phosphate buffer to a maximum content of 0.2%.
• Solutol ® HS 15 causes a severe decay in ACT activity in all tested concentrations. For that reason, Solutol ® HS 15 appears to be completely incompatible with ACT and the investigation on that surfactant is stopped here. Poloxamer ® 188 and Tween ® 80 are compatible with the protein and moreover can effectively improve its stability particularly when surfactant content exceeds 0.1% (Fig. 3-9).
• Poloxamer ® 188 and Tween ® 80 prove high efficiency in stabilising ACT during temperature and even more in F/T stressing.
• a concentration of 0.1% is recommended representing a compromise between stabilising efficiency and physiological tolerance.
• the regulatory status of these two surfactants is satisfactory.
• Poloxamer ® 188 is approved by FDA to a maximum potency of 0.6% for intravenous injection and 0.3% for subcutaneous injection 149
• Tween ® 80 is approved also by FDA to a maximum potency of 0.2% for intralesional injection and 8% for intravenous injection 149 .
• Solutol ® HS 15 destabilises ACT and is therefore unsuitable for further studies.
• Samples based on 0.1 ml 60 ⁇ g/ml ACT solutions in standard phosphate buffers pH 7.2 are loaded with excipient to a resulting concentration of 5% sugar and polyol, respectively. Stressing is done at 40 0 C for 9 days.
• mannitol and sorbitol though having no stabilising potential can be considered to be added to an ACT formulation for technical reasons. Also, the regulatory status of these substances is satisfactory. Mannitol is approved by FDA to a maximum potency of 13% for intravenous injection and 10.66% for intralesional injection 149 . Sorbitol is approved also by FDA to a maximum potency of 45% for intralesional injection and 30% for intravenous injection 149 .
• Cyclodextrins are cyclic oligosaccharides consisting of six, seven, and eight glucose monomers, respectively. Nomenclature corresponds to the molecule size rising from ⁇ -CD to ⁇ -CD to ⁇ -CD. In the ring the polar hydroxyl groups are located on the outside, whereas the etherlike oxygen atoms in the inside form a nonpolar cavity. The exterior allows cyclodextrins to dissolve in water while the cavity forms inclusion complexes with hydrophobic molecules, e.g. the hydrophobic residues in proteins. Depending on the number of glucose units the cavity grows from ⁇ - to ⁇ - to ⁇ -CD. So, the size relation of cavity and including molecule also influences complex characteristics.
• This way of complexation can affect the protein in two ways. On the one hand, it can stabilise the unfolded state of proteins by intercalating the hydrophobic residues in proteins. On the other hand, by intercalating these hydrophobic residues it can prevent proteins from aggregation 153 ' 154 .
• the non-destabilising cyclodextrins are further tested in F/T studies.
• the testing is conducted in 0.1ml ACT solution 60 ⁇ g/ml in 1OmM phosphate buffer at a stress level of 15 F/T cycles with 2% cyclodextrin.
• ⁇ -cyclodextrin and, most notably, HP- ⁇ - cyclodextrin achieved remarkable stabilising effects.
• ⁇ -CD is not very effective in this stress situation (Fig. 3-13).
• HP- ⁇ -CD can be used as stabiliser mainly for F/T stabilisation. Concentration is tested suitable between 0.015% and 2%. But, HP- ⁇ -CD is approved by FDA for intravenous injection to a maximum potency of 0.4% 149 .
• amino acids are supposed to be able to stabilise proteins by preferential exclusion. Moreover, in special cases some amino acids inhibit chemical degradation, e.g. methionine may work as antioxidant and so reduce oxidative degradation of the protein.
• methionine may work as antioxidant and so reduce oxidative degradation of the protein.
• a selection of amino acids already used in protein stabilisation are investigated 137 .
• the 5OmM phosphate buffers are produced with a 2% addition of the particular amino acid.
• the pH of 7.2 then is adjusted with potassium hydroxide. Stressing is done on 0.1ml 60 ⁇ g/ml ACT solutions at 40°C for 9 days.
• AU tested substances are interoperable with the assay indicated by the untreated samples being in the same range as the buffer control (Fig. 3-16 above).
• the stressed samples glycine, alanine, arginine, and lysine allow hardly any damage to ACT activity during temperature stressing.
• the sulphur containing amino acids methionine and cysteine drop out as well as phenylalanine (Fig. 3-16 below).
• arginine can be used for stabilisation of ACT at an optimal concentration of 1OmM, correspondent to 0.17%. FDA approval is given for up to 88% for intravenous injection .
• a pH of 7.2 turned out to be the best choice for a buffered solution.
• the optimal range of pH values is very narrow.
• low pH levels can harmfully damage, fragment and inactivate ACT.
• a buffer system is recommended in the delivery device to guarantee suitable pH for non-liberated protein. Therefore, phosphate, Tris, and phosphate/citrate buffers can be used.
• phosphate the optimal content and relating osmolarity have been examined.
• surfactants, cyclodextrins, and amino acids have proven stabilising potential on ACT.
• the polymer is already in use in medical products for wound treatment.
• the polymer is readily available to affordable pricings and does not require excipients with toxic or protein destabilising potential for gelling.
• the polymer must not release monomers or oligomers during (bio-) degradation with disturbing activity on protein stability 157 .
• Sterility in general can be produced by several well known techniques, but steam sterilisation is the most effective and safe method. Therefore, gelling agents and the hydrogels made thereof that enable autoclavation without remarkable change in properties are in favour for the choice as formulation ingredient.
• a variety of gels (Tab. 3-5) is evaluated concerning possible sterilisation methods.
• the first to choose technique for sterilisation of the hydrogels is steam sterilisation. Therefore, standard conditions like they are described in the pharmacopoeia are used, i.e. a temperature of 121 0 C at 2 bar steam pressure is applied for 15 min.
• cellulose derivatives From the cellulose derivatives the hydroxyethyl- and the carboxymethyl cellulose sodium species are suitable for autoclavation. Resulting gels are homogeneous, free of air bubbles, and completely swollen. Moreover, changes of viscosities are negligible. Further, xanthan gum and gellan gum can be autoclaved successfully. In addition, autoclavation can replace the heat treatment of these polymers obligatory for gel formation.
• alginate gels are liquefied during autoclavation, while methyl-, hydroxypropyl- and hydroxymethylpropyl cellulose precipitate due to their well known paradox temperature solubility.
• Equ. 3-1 Equ. 3-2 Equ. 3-1&3-2 Power or Ostwald-de Waele law 3-1, in linearised, logarithmic form 3-2 with shear stress ⁇ [dyn/cm 2 lOPa], shear rate ⁇ [1/s], consistency coefficient k [0,1Pa s n ], and flow behavior index or Power-law exponent n [-]; n ⁇ l meaning pseudoplastic and n>l shear thickening behaviour 158 ;
• the viscosity of the hydrogels to be developed are adjusted to the lower In k values of 7.0 to 7.5.
• a set of gels from several gellants is prepared always including a concentration series for every gelling agent.
• steam sterilisation was carried out before measurement.
• the corresponding rheograms for every gel are determined as above. It has been found that In k is directly proportional to the concentration of most tested gelling agents (Fig. 3-21). From that proportion an equation combining In k and the gellant concentration (Equ. 3-3) is gained from linear regression.
• the concentration resulting from the required In k between 7.0 and 7.5 described above is determined for every gelling agent. That is the concentration of the gelling agents to be used in the manufacture of the hydrogel as wound dressing and drug carrier in following studies (Tab. 3-5).
• gels are mobile they offer the advantage of intimate contact with the surface of a wound, but this advantage is, however, tempered by the conflicting needs of making the gel sufficiently mobile for application but not viscous enough to prevent fast flow out of the wound under the influence of gravity.
• the latter disadvantage of free-flowing gels could be overcome by crosslinking of polymers, but this implies major challenges for manufacturing and application.
• gellan gum could be a back door out of this dilemma because it is sensitive to monovalent cations, i.e. gellan gum forms non free-flowing but very brittle hydrogels in the presence of e.g. sodium and potassium salts.
• Equ. 3-4 & Equ. 3-5 & Equ. 3-6 Storage modulus G ⁇ loss modulus G", and loss factor tan ⁇ ; with loss angle ⁇ , amplitude shear stress TA, and shear rate amplitude ⁇ ;
• the storage modulus G' describes the elastic properties of the sample that is responsible for a reversible deformation storing the deformation energy within the system.
• the loss modulus G" is a measure for the plastic or viscous behaviour of the specimen being responsible for irreversible deformation and a loss of deformation energy for the matrix.
• the loss factor tan ⁇ describes the relation of viscous and elastic character in the test sample.
• the gellan moduli curves cross each other after very abrupt change in runs of the curves, describing a break down, of the gel structure at a threshold of shear stress.
• a yield value % ⁇ ge iia n can be calculated.
• hydroxyethyl cellulose is characterised by both very low pronounced elasticity and yield point describing a soft almost tree-flowing gel (Fig. 3-22).
• a higher loss factor expresses a less brittle and softer behaviour of the gel.
• the low loss factor of gellan gum alone can be increased by partly substitution by hydroxyethyl cellulose in isotonic sodium chloride containing gels (Tab. 3-6).
• the 2.0% hydroxyethyl cellulose : 1.0% gellan gum mixture is chosen for further development due to its convenient sensory properties, i.e. how it is felt on skin. Further, it is observed that the gel system containing the two gellants still provides the sensitivity against monovalent cations. Hence, it is castable like a liquid without salt content and forms non-free flowing but soft gels with salt content.
• the desired plastic behaviour of gellan gum can also be activated in the mixture by addition of the usual phosphate buffer.
• the hydroxyethyl cellulose fraction dominates the viscosity of the gel forming a free flowing mass.
• the 2.0% hydroxyethyl cellulose : 1.0% gellan gum mixture conveniently combines the properties of a soft free-flowing gel necessary for manufacture, drug loading, and provision of wound contact with the mechanic advantages of cross-linked matrices.
• this system provides a very attractive alternative to pseudoplastic hydrogels made from a single polymer.
• gellan gum till date is only approved for ophthalmic solutions up to 0.6% by FDA 149 .
• Polymers are a chemically heterogeneous group of substances. Therefore, their effects on proteins can not be outlined in a straight way. Generally, polymer interaction with proteins is of great similarity to other discussed substance classes depending on their underlying chemical structure. So, for example, surface activity, preferential exclusion, steric hindrance, and viscosity limiting structural movement are important stabilising interactions with proteins 137 .
• Polymers are tested not only because of their own protein stabilising potency but also because of their necessity for gel forming. In fact, the polymers are tested for a non- destabilising effect in the formulation instead of an active stabilising effect. That is why the experimental setting is changed concerning sample composition.
• Test samples thus, basically already consist of an improved phosphate buffered (pH 7.2) and surfactant, 0.1% Poloxamer ® 188, containing 60 ⁇ g/ml ACT solution wherein the polymer is added. The polymer content is reduced to 0.5% in these experiments to keep the samples in a liquid state. This is done to provide the possibility of unchanged liquid handling during sample preparation and analytics without further stress factors influencing ACT activity.
• hydroxyethyl cellulose, carboxymethyl cellulose Na, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, alginate Na, gellan gum, gelatine A, polyvinyl pyrrolidone, and polyethylene glycol are compatible with the analytical tool and are not depleting ACT activity in temperature- and F/T stress.
• Gelatine, polyethylene glycol, and carboxymethyl cellulose sodium are approved for intramuscular injection up to a content of 16%, 3 to 65% depending on PEG species, and 0.9% respectively.
• Polyvinyl pyrrolidone and hydroxyethyl cellulose are approved for use in ophthalmic solutions and transdermal delivery systems.
• Hydroxypropyl cellulose is approved for topical, hydroxypropylmethyl cellulose for ophthalmic administration.
• Alginate is solely approved for oral delivery 149 .
• alginate and hydroxyethyl cellulose are used in medical devices for wound healing. So, an approval as drug containing material for parenteral use with blood contact by authorities should be manageable in spite.
• steam sterilisation is conducted on the intermediate drug-free hydrogel products.
• the other polymers are dispersed in 70% ethanol for 15 minutes. After this disinfection time ethanol evaporates and leaves a dry aseptic polymer that gets hydrated with sterile solution to form the hydrogel under aseptic conditions.
• the sterile placebo gel is filled into a first syringe.
• the mixer unit is connected to the syringe and the system is filled with gel to remove air.
• a second syringe containing the concentrated ACT solution is connected without trapping air within the system.
• the mass is pumped back and forth 10 times for complete homogenisation.
• Samples of ACT loaded gels are prepared under aseptic conditions as outlined above.
• Packaging containers are glass vials that are sealed with a Teflon ® -coated rubber stopper.
• the concentration of ACT was 60 ⁇ g/ml, sample volume was 0.4 ml. So prepared samples are stored at three temperature levels, namely 6°C, 25°C, and 40 0 C. After 1, 4, 8, and 12 weeks storage time samples are drawn and stored at -80 0 C until analysis. But, before analysis by activity assay the samples are prediluted 1:8 to form a liquid gel dispensable like a liquid for handling reasons according to the evaluated method.
• results are gained as a set of curves of residual activity over time for every formulation and temperature level.
• An example is shown in Fig. 3-27. From a starting value of about 100% the activity declines over the 3 months of storage time.
• Equ. 3-9 2 »d order ⁇ ) ⁇ ⁇ ) + ⁇ '
• Equ. 3-7 & 3-8 & 3-9 Time laws for reaction kinetics for 1 st , 2 nd , and 3 rd order; c(t) is relative activity at time t, c(0) starting relative activity, ki /2/3 are velocity constants, and t is time in days;
• Fig. 3-28 shows an example for the fitting of the first order time law.
• the datasets are tested on change-over-time, indicated by the p-value of the slope of the fitted line exceeding 0.25 for no change-over-time. And, in the case of no change-over-time variability is tested to confirm statistical conformity of stability data. Further, for an estimation of the shelf life the 95%-confidence intervals are calculated for the curves. The intercept of the lower confidence limit with the lower specification limit - in our case 10 % loss of relative activity - indicates the end of the shelf life. An example is seen in Fig. 3-29.
• Equ. 3-10 Arrhenius equation; Ic 1 is the first order velocity constant, E A is the activation energy [J/mol], R is the gas constant [J/(K*mol)], T is the absolute temperature [K], and A is the collision factor
• Results indicate that reaction kinetics of activity loss of ACT is highly likely to follow a first order equation. Moreover, a commercial formulation of ACT in a wet hydrogel is not possible due to insufficient shelf life. In contrast, all formulations can be applicated into the wound site as far as stability of ACT is concerned. As well, several formulations are applicative as early refrigerated formulation for the purpose of animal experiment or early clinical trial where only limited shelf life is required.
• Samples contain 60 ⁇ g/ml ACT in 2.5% hydroxyethyl cellulose gels. Other ingredients are listed in Tab. 3-8.
• hydrogels are developed for both the concerns of a wound dressing material and the needs of the drug stability of ACT.
• aqueous carriers are suitable for application of ACT on wounds and for early formulations of ACT.
• aqueous carriers are not suitable for long term storage.
• a dry form is to be developed that stabilises ACT during long term storage.
• a hydrogel is to be formed suitable as wound dressing and releasing ACT in suitable period of time. For this task, development starts from the gel bases discussed in the hydrogel section.
• the first technique used for drying of ACT loaded hydrogels during this formulation study is lyophilisation.
• the resulting matrices are xerogels.
• a second technique - the warm air drying - is evaluated. Warm air drying has been successfully used for drying of protein solutions 165 . From this technique swellable, self- supportive polymer films are formed.
• Suprasorb G ® consists of collagen
• Promogran ® contains a mixture of oxidised regenerated cellulose and collagen.
• xerogel as wound dressing and to investigate the compatibility with protein formulation ingredients.
• Xerogels are meant to be reconstituted before or in the moment of application. So, the wound will not be confronted with the dry xerogel pad. It rather will get in contact with the yet hydrated gel state. Therefore, the swelling behaviour is the most important technical feature for xerogels. In fact, a fast and complete swelling is essential for such a product. Moreover, a homogeneous appearance, a convenient and soft consistency is desirable for compliance reasons. Furthermore, a residual moisture of less than 2% is essential for protein drug stability.
• cryostabilising hydrogel formulations described in the prior section are taken as starting points.
• thermograms solely show a freezing and melting peak (Fig. 3-32). Therefore, a standard freeze drying program with a freezing phase leading to temperatures below the crystallisation temperature of around -15°C is adequate.
• the first experiments on lyophilisation of hydrogels were conducted with a standard freeze i drying program according to the preliminary DSC tests. So, the samples are filled in petri dishes to a filling height of 4 mm. These are placed in the drying chamber at room temperature and following freezing is done at very fast rates of 1.1 K/min. After a retaining phase a conservative main drying step is proceeding at — 30°C. The subsequent secondary drying is also conservatively conducted at 20°C (Tab. 3-9).
• This annealing step is carried out as follows: The gels are frozen under the crystallisation point until the described random crystal forming process is completed. After that, the gels are heated up again close below their freezing point (-3°C) to remain there for 90 minutes. Consequently, the gels are cooled again at a very slow rate of 7 K per hour to -45 0 C initialising the primary drying after three hours of balancing time.
• the process is visualised in TbI. 3-10 and Fig. 3-35. In fact, the annealing step at that point is not introduced for the purpose of protein stabilisation but only for matrix considerations.
• lyophilisation and xerogel formation a variety of polymers is screened for eligibility.
• concentration of the gellant in the hydrogel was chosen according to the results of the rheological studies during hydrogel development. After freeze drying with the developed process, the xerogels are examined concerning optical appearance, texture analysis, swelling behaviour, and residual moisture.
• xerogels are prepared from gels with the standard viscosity highlighted in the hydrogel section but with hydroxyethyl cellulose qualities differing in their chain length.
• a series of gels from 2.5% of HEC 100.000 to 8.0% of HEC 300 was manufactured.
• lyophilisation was carried out with the special program with annealing step (see section 3.4.1.1) and analysis was performed by swelling studies, texture analysis and residual moisture detection.
• Equ. 3-11 Swelling value q calculated from dry weight of the pad (m(Xerogel)) and the weight after 3 minutes exposure to water (m(swollen gel);
• the swelling value describes how many times the xerogel can take up its own dry weight of water within three minutes.
• AU samples are acceptable concerning optical appearance measured by sensory valuation.
• the swelling of the tested xerogels instead turned out to be strongly dependent on the chain length of the used polymer. So, xerogels with higher chain length polymers combined with a lower total polymer content swell faster in the given period than xerogels made from polymers with shorter molecules (Fig. 3-37).
• hydroxyethyl cellulose polymers of higher chain length produce xerogels with as well improved hydration properties as mechanical properties.
• the low residual moisture values are constant in all polymer qualities. Therefore, the quality 100.000 appears as best choice for further development. 3.4.1.2.2 Excipients in hydroxyethyl cellulose xerogels
• xerogel matrices After the polymers as main ingredients for xerogels have been characterised the effects of excipients in xerogel matrices are evaluated. Xerogels are prepared as above but the underlying hydrogels contain the additives to be tested.
• the excipients are substances supporting the stability of the protein drug to be loaded on the one hand, i.e. electrolytes including buffer salts and various groups of protein stabilisers.
• substances useful for technical and mechanical reasons, especially improvement of swelling capacities are tested.
• soluble polymers as hydrophilisers and tablet disintegrants for improvement of swelling. So, the first group is tested for compatibility with the matrix and extent of disturbance of the xerogel formation and mechanical features.
• the second group is evaluated for the purpose of improvement of matrix formation and mechanics. Despite the slightly different objective, the influence of the particular excipients on the produced xerogels is evaluated in the same experimental setting, i.e. with regard to compatibility with the matrix, influence on hydration, influence on mechanical properties and residual moisture.
• the test series is conducted with the polymer HEC 100.000.
• buffers, surfactants, and soluble polymers are further tested for their influence on swelling in dependence of their concentration. Also a mixture of components describing a formulation suitable for stabilisation of ACT is tested.
• Buffers Both tested buffer types - potassium phosphate and Tris hydrochloride - decrease swelling in all tested concentrations. Except for very low contents there seems to be only minor dependence on the concentration of the buffer. The disturbance of swelling is far more pronounced with Tris hydrochloride than with the phosphate system (Fig. 3- 39).
• Soluble Polymers Depending on concentration these substances can do both an increase and decrease of xerogel swelling. Very low contents of less than 0.1% effect a high increase in swelling, whereas concentrations exceeding 0.1% lead to a decrease (Fig. 3-41).
• formulations suitable for stabilisation of ACT are evaluated.
• the formulations are made from hydrogels consisting of 1OmM buffer, 1OmM amino acid, 0.1% surfactant, 0.05% soluble polymer, and 2.5% hydroxyethyl cellulose "100.000".
• potassium phosphate, Tris hydrochloride, potassium phosphate/citrate, arginine, Tween ® 80, Poloxamer ® 188, polyvinyl pyrrolidone 17PF, and polyethylene glycol 2000 are combined in all possible variations.
• Fig. 3-42 For visualisation of the reconstitution of placebo xerogel formulations one example is displayed in Fig. 3-42.
• the xerogel pad is soft and has a favourable as well as homogeneous appearance. When put in contact with aqueous solution the pad soaks within seconds until completeness without air bubbles being entrapped. For the next two to three minutes the pad keeps its shape before the gel forming process is finished and the gel starts flowing like it is typical for non-crosslinked gels (Fig. 3-42). So, from a practical point of view this provides enough time after external reconstitution for application into the wound site.
• the mixture of hydroxyethyl cellulose 100.000 with gellan gum F is also investigated for xerogel formation.
• the relation of gellant components in the mixture is tested.
• the concentration range thereby is between 1.5% and 3.0% for hydroxyethyl cellulose and between 0.7% and 1.5% for gellan gum. Due to the sensitivity of the rheology of gellan gum to ionic additives this examination is done both without any further excipients and with the addition of mixtures of formulation excipients, respectively.
• swelling studies are conducted with water and isotonic sodium chloride solution, respectively, as reconstitution media.
• the pads are harder than the pure hydroxyethyl cellulose pads. So, the elastic moduli are measured between 27.5 and 32.1, and restoring energies are found around 45%. Residual moistures are detected around 1%.
• Non-crosslinked carboxymethyl cellulose sodium, alginate sodium, and xanthan gum form harder and more slowly swelling matrices due to the strong ionic binding character of these polymers.
• the crosslinked carboxymethyl cellulose sodium and carboxymethyl starch that are commonly used as tablet disintegrants provide an amazing swelling behaviour. But, the crosslinked carboxymethyl cellulose matrix is not coherent and disintegrates into powdery snatches under slight mechanical stress. Carboxymethyl starch suffers from the hardness of the xerogel structure. A huge swelling capacity is also seen with pure gellan gum, but it as well suffers from hardness of the matrix. Polyacrylate sodium also swells to huge extend but on the contrary forms an only very weak structure due to the low content. Moreover, gelatine forms very hard and hardly swelling matrices.
• Sample gels are prepared in eppendorf caps to 0.1ml volume with 60 ⁇ g/ml ACT and the excipients to be tested. In the further descriptions all concentrations of substances refer to the hydrated state of the particular gel. After swollen homogeneously the gels are freeze- dried using the procedure with annealing step described above whereby the xerogels are formed. These are reconstituted with 4OmM potassium phosphate buffer pH 7.2 immediately after lyophilisation adding up to 5OmM buffer defined as standard in the solution stability testing. Due to the high resulting viscosities samples are diluted for analysis as before with 5OmM potassium phosphate buffer pH 7.2 containing 0.1% Poloxamer ® 188.
• Buffers - the first group of ingredients to be tested are the buffer components.
• 1OmM potassium phosphate, 5/5 mM potassium citrate/phosphate mixture, 1OmM Tris hydrochloride, 1OmM arginine phosphate, and 5/5mM arginine citrate/phosphate mixture are tested in 2.5% hydroxyethyl cellulose matrices (Fig. 3-44).
• Fig. 3-44 hydroxyethyl cellulose matrices
• surfactants and soluble polymers in the next series the influence of surfactants and soluble polymers is studied. Though, generally surfactants are not known to inhibit protein unfolding during dehydration 181 , they are tested for lyoprotection because they have been proven to be beneficial during freezing in the solution stability section.
• the soluble polymers are also known as lyoprotectants of proteins 182 ' 183 .
• Samples with a basic matrix consisting of 2.5% hydroxypropylmethyl cellulose 100.000 and 1OmM potassium phosphate buffer are spiked with 0.1% Poloxamer ® 188, 0.1% Tween ® 80, 0.5% PEG 2000, and 0.5% PVP 17, respectively.
• the control sample next to ACT solely contains gellant and buffer. Freeze drying, dilution and analysis is carried out as above.
• Gelling agents and hydroxypropyl- ⁇ -cyclodextrin - the different gelling agents are tested in already improved formulations. Indeed, they contain 1OmM potassium phosphate buffer pH 7.2, 0.1% Tween ® 80, 0.05% PEG 2000, and variing polymers as gelling agents. These are hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and hydroxyethyl cellulose. Moreover, a gellan gum/hydroxyethyl cellulose mixture, carboxymethyl cellulose sodium, and alginate sodium are tested. A variation with hydroxypropyl- ⁇ -cyclodextrin added to each gellant was conducted in parallel.
• the non-ionic polymers perform better than the ionic and sodium containing gelling agents.
• the gellan gum/HEC mixture surprisingly shows highest recovered ACT activities. It has been reported that this class of substances can provide stabilising capacity on proteins 188 .
• the ionic, sodium containing polymers may interact with the phosphate buffer system and lead to the well known pH shift during freezing. This may lead to the detected loss of activity.
• the series with cyclodextrin addition features generally lower activity values than without cyclodextrin. Most notably, the samples with the more lipophilic agents suffer from the cyclodextrin influence (Fig. 3-46).
• hydroxyethyl cellulose is chosen for its suitability for autoclavation.
• the soluble polymers are varied between polyvinyl pyrrolidone and polyethylene glycol.
• Samples of ACT loaded gels were prepared under aseptic conditions as before.
• Packaging containers are glass vials that are sealed with a Teflon ® -coated lyophilisation rubber stopper.
• the concentration of ACT in hydrated state was 60 ⁇ g/ml, sample volume was 0.4 ml. After swollen homogeneously the gels are freeze-dried using the procedure with annealing step described above whereby the xerogels are formed.
• Xerogels are then stored at two temperature levels, 25°C and 40 0 C. After 4, 8, and 12 weeks storage time samples are drawn and stored at -80 0 C until analysis. Xerogel samples then are reconstituted and prediluted as described above. Readout is again done by activity assay. For the samples stored for three months SDS-PAGE is performed in addition. Calculations described in detail in the hydrogel section are conducted to estimate reaction kinetics and shelf lives under storage conditions. The 25 °C level simulates real conditions for storage at room temperature, 40°C simulates temperature stress conditions. Other than in the hydrogel study the 6°C temperature level was omitted in this setting. This was done because the changes of measured values were estimated to be too small in comparison to the spreading of measured values delivered by the activity assay. In that case reasonable conclusions would be impossible. Moreover, a refrigerated storage of a lyophilised product at this temperature level would be hardly acceptable due to marketing concerns.
• results are gained as a set of activity loss curves over time for every formulation and temperature level.
• An example is shown in Fig. 3-47. From a starting value of about 100% the activity declines over the 3 months of storage time.
• Fig. 3-48 shows an example for the fitting of the first order time law.
• the coefficients for 1 st order are between 0.985 and 0.999, for zero order coefficients are between 0.950 and 0.992, and coefficients for 2 nd order are below 0.96.
• Results indicate that reaction kinetics of activity decay of ACT in xerogels is highly likely to follow a first order equation. Main instability again is the loss of activity and dimer formation. Moreover, a commercial formulation of ACT in a xerogel is possible due to sufficient shelf life. Furthermore, the two suitable formulations after reconstitution can be applicated into the wound site as far as stability of ACT is concerned. As well, these formulations are applicative for the purpose of animal experiment or clinical trial. Results also indicate that a not yet tested excipient combination may be the most effective stabilising formulation. This can be evaluated within later studies, e.g. adjustment of the product for market launch. 3.4.3 Polymer films as drug delivery systems for wound healing
• OpSite ® by Smith&Nephew consists of a polyurethane film with a polyacrylate adhesive.
• polymer films serve as coating material.
• the film is formed by spraying a polymer dispersion onto the tablet core followed by evaporation of the dispersion media.
• the most common method of film manufacture is blown film extrusion. The process involves extrusion of a plastic melt through a circular die to form a thin walled tube, followed by bubble-like expansion by air being introduced via a hole in the centre of the die. The outcome of this process is a film tubing that is cooled and flattened to create a lay-flat tube of film. The regulation of film width and thickness is done by control of the volume of air in the bubble and the output of the extruder.
• Polyethylene is the most common polymer in use for blown film.
• films can be formed by precipitation out of particular baths.
• the method mainly used for water swellable polymers is the dry-cast method. Thereby, a melt or a solution of the polymer is extruded through a die onto a roll. There, the mass cools down to form a film robust enough to be transferred onto a mesh for drying. The drying may be completed in a drying tunnel.
• This procedure is appropriate for many pharmaceutically relevant polymers such as gelatine and the cellulose ethers.
• a special application of this method is the production of transdermal therapeutic systems. Thereby, the polymer matrix is not directly cast onto the roll but onto a backing layer being placed between the roll and the matrix. Maybe, the mass is cast on a web before the backing is added. Further transportation is also supported by the backing layer.
• a method involving a scraper is used. Thereby, the gel is filled in the scraper apparatus which consequently is drawn over a glass plate.
• a wet film of constant thickness is formed (Fig. 3-50).
• This film can be dried under laminar flow at . room temperature or in a cabinet drier at any desired temperature.
• the self supportive film is removed from the plate (Fig. 3-50).
• the appropriate amount of gel is placed on the bottom of an eppendorf cap or a vial. Consequently, the mass is dried under a flow of nitrogen in a special device controlling both gas and product temperature 191 .
• the film forms on the bottom and side walls of the container (Fig. 3-50).
• a variety of polymers is screened for eligibility for warm air drying and film formation.
• concentration of the gellant in the hydrogel is chosen according to the results of the rheological studies during hydrogel development. After drying the films are examined concerning optical appearance, texture analysis, swelling behaviour, and residual moisture. Thereby, as during xerogel development optimised film compositions are to be developed concerning texture properties and embedding of protein stabilisers.
• films consisting of pure gelling agent are screened.
• texture properties of film compositions containing hydroxyethyl cellulose as gelling agent are optimised.
• formation of a film matrix made from two gellants — hydroxyethyl cellulose and gellan gum - is described. The compatibility of excipients with the particular film matrices is investigated subsequently.
• the screening of gelling agents is conducted in similar manner as during xerogel development described above.
• plasticiser In the film structure solely consisting of gelling agent, the individual molecules of the polymer lack mobility because of their mutual interference which is much higher when ionic binding is involved.
• the use of a plasticiser implicates the introduction of a lower molecular weight substance into the structure that acts as a molecular lubricant, physically separating the chains and allowing them some mobility, thus giving flexibility. Obviously, the larger the volume of plasticiser is, the greater is the flexibility and softness.
• Common plasticisers in pharmaceutical products are esters of organic acids, e.g. citric acid and phthalic acid, polyalcohols and esters thereof, as well as polyethylene glycol derivatives.
• the application of films is supposed to be different from the application of xerogels.
• the films can not swell in that short period of time.
• the film is to be wetted before placing onto the wound.
• reconstitution is started and will proceed in the wound with wound fluid or solution provided externally beneath and on top of the film.
• the wound will be confronted with a semi-reconstituted film not yet completely transformed into a hydrogel.
• the texture properties of films play a very important role for the applicability of these matrices. Consequently, these are optimised by texture analysis outlined below.
• Characterisation is done by texture analysis. Thereby, the tensile strength at rupture is gained from a tensile test method where the film is placed between two grips and is extended until rupture (Fig. 3-52).
• the tensile strength value is calculated from applied force at rupture and the cross section area of the film specimen 192 (Equ. 3-12).
• the elastic modulus is tested in a puncture test where the sample is expanded and penetrated by a ball probe 192 (Fig. 3-53). From the resulting load-displacement diagrams and the physical dimensions of the construction elastic moduli are calculated (Equ. 3-13).
• Equ. 3-13 Young's modulus of elasticity E [N/mm 2 ]; F is applied force [N], A is cross- sectional area [mm ], dL is change in length [mm], L is unstressed length [mm];
• the tensile strength of the system depends on all components of the ternary mixture.
• a set of gels is examined with a constant PEG content and varying HEC and PVP contents.
• the tensile strength of the film increases with rising HEC contents.
• tensile strength values of about 4.5 N/mm 2 gained for the practical concentrations of HEC between 2% to 3% are not satisfactory.
• PVP an increase in tensile strength can be detected.
• values exceeding 10 N/mm 2 can be achieved a reasonable content should be between 2.0% to 3.5% leading to strength values of 8.0 to 9.0 N/mm 2 (Fig. 3-54).
• Fig. 3-55 The content of PVP in this setting is kept at constant level - 2.25% in the hydrogel state.
• Variables are the HEC and PEG content.
• a clear optimum for the PEG content is detected, i. e. from 0% until 2% PEG content an increase of strength values can be seen.
• 2% to higher concentrations of PEG a decrease of tensile strength is following.
• the described maximum of tensile strength at an optimal PEG concentration is also visible in the third group of interacting partners. Because, for varying PVP contents and a constant HEC concentration the same maximum around 2% PEG content is obtained (Fig. 3-56).
• the elastic modulus E which is a measure for stiffness and a reciprocal measure for flexibility is calculated from texture analysis as given in Equ. 3-13.
• the elastic modulus is mainly dependent on the PEG content. Only at very low concentrations of PEG influences by the other components are noticeable in the tested concentration range. Indeed, at the PEG level detected as optimal for tensile strength - around 2% concentration in hydrogel state - the elastic moduli of the systems are at a constant low level nearly independent of the gellant (Fig. 3-57) and strengthener content (Fig. 3-58).
• the mixture of hydroxyethyl cellulose 100.000 with gellan gum is also investigated for film formation.
• the relation of gellant components in the mixture is tested with the concentration range being between 1.5% and 5.0% for hydroxyethyl cellulose and between 0% and 1% for gellan gum.
• Each film additionally contains 2.25% PEG 400 and 2.25% PVP 17 according to the results with pure hydroxyethyl cellulose films outlined above. These films are tested by texture analysis and swelling studies that are conducted with water and isotonic sodium chloride solution, respectively, as reconstitution media.
• the elastic modulus of the system is also slightly increased with higher gellan gum contents. Despite, all measured values are in acceptable low range (Fig. 3-60). Besides, as found in the section above the main dependence should be on the plasticiser contents rather than on gellants which was not further tested. Therefore, as far as flexibility is concerned all tested formulations can be used.
• the mixture of hydroxyethyl cellulose and gellan gum is suitable for film formation.
• the relation of gellants found during the hydrogel studies can be taken as film composition as well. But, for a higher robustness a bisection or even further reduction of the gellan gum content maybe favourable. Although, further studies are conducted with a mixture of 2.0% hydroxyethyl cellulose 100.000 / 1.0% gellan gum mixture.
• Phosphate buffer is suitable up to a concentration of 5mM.
• higher contents lead to crystallisation on the film surface. Crystals on the film surface are also seen with the following substances given with their tested concentration: sodium chloride (1%), potassium chloride (1%), sodium sulphate (1%), mannitol QPA), and glycine (2%).
• surfactants just combined with gellant provoke shrinking of the film during drying at room temperature in every tested concentration up to 0.2%. Therefore this class can be used only in combination with other excipients, e.g. the shrinking can be partly impeded by the use of Tris as buffer component.
• sucrose 2%
• high molecular weight polyethylene glycol 2%
• crosslinked carboxymethyl cellulose 5%
• Tris hydrochloride 5OmM
• arginine 2%
• calcium and magnesium chloride 0.5%)
• EDTA sodium 0.5%)
• ⁇ -cyclodextrin 1%)
• the warm air drying apparatus 165 is used for the drying of the prepared hydrogel samples. Thereby, drying is carried out with flowing nitrogen for 12 hours, the gas stream and the gel containing eppendorf caps being tempered to 25°C. Reconstitution and dilution for analysis are conducted as described in the xerogel section.
• the formulations contain the substances according to Table. 3-16 with ACT in a hydroxyethyl cellulose matrix.
• the preferable excipients are detected by comparison of the recovered ACT activity in the different formulations.
• Arginine - results indicate that arginine has a stabilising effect on ACT during film manufacture. This is gained by comparison of formulations (l)-(3) against (4)-(6) and (7)- (9) against (1O)-(12), respectively. The stabilising potential of arginine on proteins during vacuum drying has been reported previously 155 (Tab. 3-16).
• Polyethylene glycol is used as plasticiser in the film.
• Poloxamer ® 188 containing samples an additional stabilisation, on ACT is shown. This results from the comparison of formulation (1) with (3) and (4) with (8). On the contrary, comparing formulation (7) with (9) and (10) with (12) indicates that in the Tween ® containing samples there is no additional stabilisation proven by polyethylene glycol (Tab. 3-16).
• Gelling agent - for evaluation of the gelling agents a standard formulation was chosen - 60 ⁇ g/ml ACT, 5mM potassium phosphate pH 7.2, 0.1% Tween ® 80, 1,5% PEG 400 - with variable polymer types as gellants. These are hydroxypropyl cellulose, hydroxypropyl- methyl cellulose, and hydroxyethyl cellulose. Moreover, a gellan gum/hydroxyethyl cellulose mixture, carboxymethyl cellulose sodium, and alginate sodium are tested. Unlike with xerogels, the more hydrophilic and ionic polymers perform better than the more lipophilic gelling agents - HPC and HPMC The gellan gum/HEC mixture again shows highest recovered ACT activities (Fig. 3-61).
• the surfactant Tween ® 80 performs better than the Poloxamer ® 188 alternative. See comparison of formulation (1) with (3). Moreover, the HEC/gellan gum F gellant system is favourable over pure hydroxyethyl cellulose and the HEC/gellan gum LTlOO alternatives. This results from comparison of formulation (1) with (4) and (5) (Tab. 3-17).
• the formulations (3) and (5) can be recommended for further development.
• the greatest stabilising potential of a film formulation is to be estimated for a combination of Tween ® 80 and HEC/gellan gum F.
• this combination is as well not yet tested for the polymer films and therefore cannot be directly recommended supported by data for immediate further development. But, in future studies, e.g. adjustment of the formulation for market launch, this can be taken into account.
• Results indicate that reaction kinetics of activity decay of ACT in films is highly likely to follow a first order equation. Main instability again is the loss of activity. In contrast to xerogel studies dimer formation is not found during film studies. Moreover, a commercial formulation of ACT in a swellable polymer film is possible due to sufficient shelf life. Furthermore, the two suitable formulations after reconstitution can be applicated into the wound site as far as stability of ACT is concerned. As well, these formulations are applicative for the purpose of animal experiment or clinical trial. Results also indicate that a not yet tested excipient combination may be the most effective stabilising formulation. This can be evaluated within later studies, e.g. adjustment of the product for market launch.
• Two xerogel formulations are identified that meet all the requirements. They have a homogeneous and soft appearance, they swell spontaneously when hydrated to a hydrogel, and they deliver shelf lives for ACT exceeding 18 months.
• the release period a time frame of one to five days is desirable from the medical point of view because a fast release and absorption of the drug is favourable.
• the common change of dressing intervals in clinical practice of wound care are between three to five days. Therefore, for highest effectiveness, the maximum release period preferably should not exceed five days.
• the in vivo release site to be simulated is an open wound. Although a topical delivery is described, wounds are not supposed to provide a major diffusion barrier for a drug substance. Thus, there is no physiological diffusion barrier like skin to be mimicked in vitro. Rather, a system providing a very low resistance to diffusion of the drug out of the matrix is preferable.
• the release temperature is set to 32°C representing the USP specification for dermal release 195 .
• a separation of donor and acceptor chamber is obligatory. Therefore, a membrane is necessary that is not used for simulation of a physiological barrier, e.g. skin, rather it should mechanically separate the media.
• a membrane is evaluated that allows an unimpeded diffusion of model substances but keeps the major part of the gelling agent on the donor side.
• a cellulose acetate filter membrane of 0.45 ⁇ m pore size meets the named requirements at best 196 (data not shown).
• Cellulose acetate is non-lipophilic and water permeable. And, according to manufacturers' instructions this material has also a low protein adsorption tendency.
• an ACT stabilising solution consisting of 1OmM potassium phosphate buffer pH 7.2 and 0.1% Poloxamer ® 188 is chosen as acceptor. This solution is proven to be suitable for stabilisation in solution state during the release experiment as well as for stabilisation during storage of samples as frozen solution (section 3.2.2).
• the release medium has to provide sink conditions for the diffusion of the drug substance. That means a sufficient amount of acceptor medium has to be offered in relation to the amounts of drug substance and donor medium.
• sink conditions for a substance are provided if its concentration at the end of the experiment does not exceed 10% of its saturation solubility in the medium.
• sink is provided even with rather low acceptor volumes. Therefore, the minimum of acceptor medium is determined by the sample volume to be taken out of the reservoir until the experiment is finished.
• the release tests are performed in a modified Loth model 134 .
• the gel containing chamber is mechanically fixed. Due to the higher osmolarity of the donor medium a slight permanent pressure results in the donor chamber that is absorbed mechanically by the bending membrane and the chamber body. Moreover, the model is modified in size that the donor sample cavity is circular in shape with 2.0cm in diameter and has a height of 0.4cm, resulting donor volume is only 1.25ml.
• the acceptor moreover, provides a ripple plate supporting the membrane and providing contact between acceptor solution and the membrane. Particular drill holes in the body enables the acceptor medium to be pumped through the chamber (Fig. 3-64). The whole apparatus is tempered to 32°C for measurement in a cabinet heater.
• the dynamic model the acceptor medium is pumped in closed circuit by a hose pump with a flow rate of lOml/min. The total volume is 20ml. Samples are taken from the reservoir without replacement according to a sample plan.
• the static model two syringes are connected to the acceptor chamber (Fig. 3-65). A 2ml portion of acceptor medium is filled in the syringes and provided to the diffusion site. Following a sample plan, sample collection is done by complete exchange of the portion against fresh medium. 3.5.4 Theoretical background and data interpretation
• Equation 3-14 describes the whole process. But, due to its complexity a simplified form (Equ. 3-15) of this relation is used for calculations 199 .
• Equ. 3-14&3-15 Model functions after Higuchi describing the diffusion of a substance totally in solution out of a semi-solid matrix into a sink; equ. 3-14 describes the correlation more exactly; equ. 3-15 is a simplification strictly valid in the first third of the process; hereby, is
• the diffusion coefficient of a substance is also related to its molecular weight, the relation of the release rate to the diffusion coefficient becomes apparent. This also is predicted by theory (Equ. 3-15).
• the model can be used for release studies of molecules of the size of proteins, especially ACT with 68kDa molecular weight.
• Fig. 3-67 The molecule diameters of FITC-Dextran derivatives are given as Stokes diameters 200 .
• the diameter of ACT is calculated by an approximation according to Equ. 3-16 201 .
• Equ. 3-16 Approximation of the volume of a protein molecule V pro tein molecule [10 "3 nm 3 ] by its molecular weight M prote i n mole cule [Da]; the correction factor is related to the average partial specific volume of proteins being 0.73 cm 3 /g 201 ;
• the membrane because the pore structure effects a reduction of the diffusional area in the system compared to e.g. an in vivo situation where the gel has direct and intimate contact with the wound ground. Therefore, the release in vivo is supposed to be faster to some degree compared to in vitro studies due to the lack of the membrane.
• the different application forms under development during this work are tested with a standard load of FITC-Dextran 7OkDa.
• Xerogel and film samples are prepared as described for the ACT loaded specimen (section 3.3.2.2.). Samples are reconstituted with water for one minute before the release test in the dynamic model is started. The release curves are again linearised and displayed in Fig. 3-69.
• the release rate slightly exceeds the rate from the corresponding wet hydrogel. This should be rooted in a higher concentration gradient of the drug in the film experiment. Unlike gels, films start the diffusion with a very low volume. Though it of course increases during the hydration period, at least in the first part the concentration in the film is higher than in the gel. That results in a higher driving force for the diffusion and with that in a higher release rate from films.
• model studies indicate that the present system is suitable for in vitro release studies of drugs with the required molecular weight from wet or reconstituted gel preparations.
• the reason for the decline of activity in the acceptor medium is found in the system of the dynamic release model.
• the medium is pumped through plastic tubing during the entire testing time by a hose pump.
• the medium in the supply is homogenised by a magnetic stirrer.
• a further improvement of the release system e.g. by using glass instead of plastic materials for the chamber or pre-treatment of the membrane, would have been a promising possibility to improve ACT recovery results but was not possible for technical reasons.
• a simple mathematical adjustment with a correction factor is problematic because of the measurement uncertainty of the analytical methods being amplified in such an operation.
• a result gained after correction would imply an unsatisfactory inaccuracy.
• the total released amount of ACT values of all tested formulations vary in the range of 70% to 96%. Thereby, the lower values, also correlated with lower release rates, are detected in Tween ® 80 - formulation (2), Fig. 3-76 - and gellan gum containing samples - formulations (3), Fig. 3-77 and formulation (5), Fig. 3-79.
• formulation (2) and formulation (5) are to be assessed as best candidates because they offer the highest bioactive fraction released with acceptable release rates and sufficient total amounts liberated.

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