Patent Application
20240197954
This is a U.S. non provisional patent application claiming priority of European patent application EP22202774.0 filed on Oct. 20, 2022.
The sequence listing titled “S300-3_corrected.XML”, created on Mar. 3 2024, and having a file size of 2019 bytes is hereby incorporated by reference pursuant to 37 C.F.R. section 1.52 (e) (5).
The present disclosure relates to stabilized pharmaceutical wound dressing compositions comprising calreticulin protein for use in medicine, in particular for topical treatment of acute and chronic wounds. More specifically, the present invention is directed to hydrogel wound dressings that can be sprayed onto a wound as a liquid or applied topically in the form of gel.
Wound may be described as disruption of anatomical structure and function of an organ, starting from the skin and depending on the injury depth and area might include soft tissues, subcutaneous tissue, muscles, tendons, nerves, vessels, even to the bones. The major mechanisms of injuries are related to mechanical (trauma or surgical interference), chemical, thermal and radiation factors. Based on healing dynamics wounds are sorted in to the two types—acute and chronic.
Normally wound healing or tissue regeneration process is short and dynamic process that goes through several stages: coagulation and hemostasis phase (immediately after injury), inflammation, cell tissue and vascular proliferation, tissue granulation and maturation. Chronic wounds or ulcers have tendency to heal significantly longer compared to acute wounds.
Wound healing is facilitated using wound dressings that perform one or more of the following functions: a) maintain moist environment, b) enhance epidermal migration, c) promote angiogenesis and connective tissue synthesis, d) allow gas exchange between wounded tissue and environment, e) maintain appropriate tissue temperature to improve the blood flow to the wound bed and enhance epidermal migration, f) provide protection against bacterial infection, h) provide debridement action to enhance leucocytes migration and support accumulation of enzymes. Furthermore, it is desirable that wound dressings are non-adherent to the wound and are easily removed after healing. They must be sterile, non-toxic and non-allergenic.
Several wound-dressing technologies are known in the art for use in wound-healing such as traditional dressings, advanced wound dressings for delivery of active substances, antimicrobial dressings, and anti-inflammatory and analgesic dressings.
In traditional dressings, cotton, wool, bandages, and gauzes are mostly used to cover the wound to restore its function underneath. This is still a common approach because of their ease of use, accessibility to most surgical hospitals, and low cost. Such dressings are classified as passive, as they are non-occlusive.
Wound dressings for delivery of active substances are used to transport substances such as antimicrobials, anti-inflammatory agents, and analgesics for wound-healing and are classified as interactive dressings that are semi-occlusive or occlusive. These dressings act as a barrier against penetration of bacteria to the wound environment. In the case of antimicrobial dressings, they are used to kill bacteria or fungi that are present in infected wounds and can reduce the risk of reinfection during wound-healing, surgical procedures, or when changing the dressing. Finally, anti-inflammatory and analgesic dressings can help to reduce the pain and inflammation in wounded areas.
Bioactive wound dressings containing biological agents are also known in the art and may include proteins, such as growth factors, nucleic acids and stem cells, all of which accelerate the regeneration of cells at the site of a wound or adjust the level of substances to be produced when natural wound-healing occurs.
In case of wound dressings for delivery of active substances and bioactive wound dressings, polymeric materials such as hydrogels, hydrocolloids, foams, films, and wafers can be used to transport the substances.
Hydrogels are cross-linked networks of macromolecular compounds characterized by high water absorption capacity. Hydrogels are three-dimensional polymeric networks held together by cross-linked covalent bonds and weak cohesive forces in the form of either hydrogen bonds or ionic bonds. This class of hydrophilic polymeric materials shows an inherent ability to swell in water and other suitable solvents and is capable of imbibing and retaining more than 10% of their weight in water within the gel structure. Hydrogels, together with other chemical compounds, can constitute cosmetic or pharmaceutical formulations, which can find several topical applications on the body and hair surface.
Hydrogels are prepared using a variety of polymeric materials, which can be divided broadly into two categories according to their origin: natural or synthetic polymers. Natural polymers for hydrogel preparation include hyaluronic acid, collagen, chitosan, heparin, alginate, dextran, fibrin.
Synthetic polymers such as poly (vinyl alcohol), polyacrylamide, poly (ethylene oxide), poly (ethylene glycol), sodium polyacrylate, acrylate polymers and copolymers thereof have been used for hydrogel formation. These hydrogels are mainly used in biomedical applications. Natural polymers usually present higher biocompatibility when compared to synthetic polymers. But, on the other hand, synthetic ones are chemically stronger than natural ones. Hydrogels may also be in the form of liquid spray compositions comprising monomers, macromers, and/or polymers that polymerize or otherwise thicken upon or shortly after delivery, to form a hydrogel dressing on a wound as disclosed in WO2003063923A1, U.S. Ser. No. 10/449,272B2.
Examples of synthetic polymers known to be of particular relevance are carbomer, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and xanthan gum, all of which are commercially available from numerous vendors. Carbomer is a term used for a series of polymers primarily made from acrylic acid. Carbomers are white, fluffy powders that are frequently used as gels in cosmetics and personal care products. Hydroxyethyl cellulose and hydroxypropyl methyl cellulose are gelling and thickening agents made chemically through the modification of cellulose. Xanthan gum is an extracellular polymer produced mainly by the bacterium Xanthomonas campestris, a polysaccharide with many industrial uses, which can be produced industrially from simple sugars using fermentation process. Traditionally it plays an important role in industrial applications as thickener, emulsion stabilizer and it has been added to water-based drilling fluids due to its pseudoplastic behavior and thermal stability. The structural properties of xanthan in solution can be tuned by the temperature and ionic strength; under high ionic strength or low temperature, xanthan chains are arranged in helical conformation, whereas under low ionic strength or high temperature, xanthan chains are coiled. Xanthan high molecular weight favors the building up of physical and chemical networks, which have been used as carriers for drugs and proteins and as scaffolds for cells. In combination with other polymers xanthan has been applied as excipient in tablets or as supporting hydrogels for drug release applications, particularly due to its acid resistance.
Many biological agents have been tested as bioactive additives to hydrogels for pharmaceutical applications. It is known that exogenous application of growth factors benefits the wound healing process, and this was proven by numerous studies. Among different growth factors, platelet derived growth factor (PDGF) is the most commonly used growth factor which promotes chemotactic recruitment and proliferation of cells and increases angiogenesis. Besides, PDGF, fibroblast growth factor (FGF), epidermal growth factor (EGF), and autologous platelet thrombin are also studied extensively for their application in wound healing process. PDGF and EGF are already approved by FDA for medical use in human applications (as described in Cosmetic Science and Technology, Elsevier, 2017, p. 767-780). However, accumulation of EGF and PDGF is associated with proliferation of certain cancer cells, therefore, it is desirable to provide biologically active proteins that facilitate tissue regeneration.
Enzymatic debridement of necrotic tissues without harming healthy tissue is also a crucial part to promote normal healing process. Ointments comprising papain and collagenase are currently used to digest necrotic tissue. Collagenase acts on the collagen by attacking native collagen and gentle on viable collagen by gradual breakdown of tissue whereas papain attacks cystein residue and associated with inflammatory response. Debridace™ is a commercially available wound dressing which increases proteolytic action.
Unexpected effects on the wound healing were observed when testing recombinant human calreticulin protein (CLT) on the chronic diabetic and acute wound healing models. Calreticulin is a highly conserved chaperone protein which has no relationship with the growth factors protein family. In humans the calreticulin protein is a 400 amino acid protein having a molecular weight of 46 kDa. It resides primarily in the endoplasmic reticulum, and is involved in a variety of cellular processes, among them, cell adhesion. Additionally, it functions in protein folding quality control and calcium homeostasis.
In extracellular space calreticulin protein activities are associated with autoimmune regulation, cancer cell inactivation, blood clothing/thrombosis inhibition and facilitation of tissue regeneration.
Calreticulin proteins may be produced as recombinant using bacterial (E. coli) or yeast (Pichia sp.) production systems. Stability and biological activity are the most important quality attributes for recombinant calreticulin protein which depend on the expression host dependent post-translational modifications. It was shown that calreticulin protein produced in Pichia production system using its native signal sequence exhibits biological activity similar to that of native protein most likely because of similar profile of post-translational modifications (Čiplys E, Slibinskas R, Sasnauskas K, Michalak M, Gold L I: Generation of native recombinant secreted human endoplasmic reticulum chaperones by using their native signal sequences in yeast expression systems. PCT patent application WO 2014/011723 A1. Pub. Date: 2014 Jan. 16. Čiplys E, Žitkus E, Gold L I, Daubriac J, Pavlides S C, Højrup P, Houen G, Wang W A, Michalak M, Slibinskas R: High-level secretion of native recombinant human calreticulin in yeast. Microb Cell Fact. 2015 Oct. 15; 14: 165). Furthermore, it was demonstrated that calreticulin protein exhibits high biological activity in wound healing in various models (Siebert J W, Garg H G, Gold L I. Beneficial wound healing applications of calreticulin and other hyaluronan-associated proteins, U.S. Pat. No. 5,591,716. Pub. date Jan. 7, 1997; Gold L I: Therapeutic and cosmetic uses and applications of calreticulin, WO 2011/160082A2. Pub. Date: 2011 Dec. 22).
However, all above described publications in their experiments use aqueous calreticulin protein solutions containing 3.0 mM calcium buffered with either Tris, or PBS that need to be stored either at minus 80° C., or at plus 4° C. to maintain proper conformation of this calcium-binding molecule. Such compositions are inconvenient for routine medical treatment of patients both in hospital environments and as over-the-counter preparations.
Therefore, it is desirable to provide stabilized compositions comprising calreticulin protein which are convenient for therapeutical use in hospital environments and as over-the-counter preparations.
In a first aspect the present disclosure provides stabilized pharmaceutical compositions in the form of hydrogel comprising recombinant human calreticulin protein or a functionally active fragment or derivative thereof for use in topical treatment of chronic and acute wounds. Stabilized pharmaceutical compositions of the present invention may comprise calreticulin or a functionally active fragment or derivative thereof in a concentration ranging from about 1 μg/mL to 10 mg/mL, more preferably in the range from 5 μg/mL to 1 mg/mL, most preferably in the range between about from 5 μg/mL to 500 μg/mL. Stabilized pharmaceutical compositions of the present invention may be formulated as liquid hydrogel spray, or a gel for administration to body surfaces such as the skin or mucous membranes.
In a second aspect the stabilized pharmaceutical composition of present invention comprises recombinant human calreticulin protein or a functionally active fragment or derivative thereof and one or more polymeric materials that form hydrogel. Stabilized pharmaceutical compositions of the present invention may comprise polymeric materials in a concentration ranging between about from 0.5% to 10% w/v of the composition. In further aspects the stabilized pharmaceutical composition of present invention may comprise polymeric materials such as carbomer, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and xanthan gum. In further aspects the stabilized pharmaceutical composition of present invention may be in the form of liquid spray, or a gel.
In some aspects the stabilized pharmaceutical composition of present invention may further comprise serum albumin proteins, such as bovine serum albumin or human serum albumin. In further aspects the stabilized pharmaceutical composition of present invention may comprise serum albumin proteins in a concentration ranging from about 1 mg/mL to 10 mg/mL.
In some aspects the stabilized pharmaceutical composition of present invention may further comprise preservatives, such as potassium sorbate or sodium benzoate.
In some aspects the stabilized pharmaceutical composition of present invention may further comprise a cytokine, a growth factor, any agonist of wound healing (or effective wound healing agent), including but not limited to small molecule agonists, peptide agonists, chemical agonists, or mixtures thereof. A growth factor according to the present invention can be, for example, platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, TGF-ß, and mixtures thereof.
Stabilized pharmaceutical compositions of the present invention may be formulated for topical treatment of chronic and acute wounds by administering such composition topically to a wound of a patient suffering from an acute wound, or from delayed wound healing. Wounds treatable by the pharmaceutical compositions of the present invention include acute wounds, such as originating from thermal and chemical burns, trauma, or surgical interference, and chronic wounds, such as ulcers and diabetic wounds.
The present disclosure relates to the medical use of stabilized pharmaceutical compositions comprising calreticulin in treating acute and chronical wounds of a subject. Similarly, the present disclosure provides a method of treating chronic and acute wounds of a subject, comprising administering to the wound the composition of the present invention. The subject can be any mammal, including a human, non-human primate, or a domesticated mammal such as a cat or a dog. Preferably the subject is a human.
The present invention teaches that calreticulin protein, when formulated into formulations with pharmaceutically acceptable polymeric materials that are used to form hydrogels, exhibit increased stability and functional activity at room temperature down to plus 4° C.. Pharmaceutically acceptable means compounds that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and neither biologically nor otherwise undesirable and that are acceptable for human pharmaceutical use as well as for veterinary use. Addition of serum albumins to the formulations also contributed to the formulation stability. Serum albumins suitable for use in the compositions of the present invention may be selected from human serum albumin (HSA), or beef serum albumin (BSA). BSA and HSA are available commercially from several vendors (e.g. Sigma Aldrich) both in native and recombinant forms. Preferably serum albumins for the present inventive compositions are recombinant to conform with pharmacopeia regulations.
Calreticulin is an endoplasmic reticulum protein that is found in a wide range of species and has a highly conserved sequence. In particular, in humans the calreticulin protein is a 400 amino acid protein having a molecular weight of 46 kDa.
The calreticulin may be described as a mature protein (and not the protein precursor), i.e. one which has undergone post-translational modification and in particular has had the translocation signal removed. In humans the translocation signal is a 17 amino acid hydrophobic N-terminal signal sequence which is cleaved off the 417 amino acid protein precursor. The sequence of human calreticulin precursor (i.e. which includes the 17 amino acid translocation signal) can be found in the UniProt Database at UniProtKB-P27797. The mature human calreticulin protein has SEQ ID NO: 1, which is the sequence of UniProtKB-P27797 minus the 17 amino acid translocation sequence.
In some examples of the present invention the calreticulin may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85% identical or at least 90% to SEQ ID NO: 1. Preferably the calreticulin comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. More preferably the calreticulin comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 98% or at least 99% identical to SEQ ID NO: 1. In particular, variants of calreticulin are known from homologous sequences from different animals and from non-disease causing polymorphisms already known in the art. Suitable variants can also be made based on single amino acid substitutions, particularly conservative substitutions, that retain the function described herein (for example as determined by the in vitro assay(s) indicated above).
The calreticulin may be obtained from eukaryotic cells, and in particular may be obtained by recombinant expression in either E. coli or eukaryotic cells. For example, recombinant calreticulin expressed in E. coli, may be obtained from Sigma Aldrich (#SRP8001, His tagged, >90% (SDS-PAGE)
Preferably the calreticulin is prepared by recombinant expression in yeast cells, such as Saccharomyces cerevisiae or Pichia pastoris.
In particular, the calreticulin may be prepared using recombinant protein production technology based on the secretion of native recombinant protein to the culture medium after expression of human calreticulin precursor including its native signal sequence as described in Čiplys et al, 2014 and 2015, both of which are incorporated herein by reference in their entirety.
In preferred embodiments of the invention, one or more pharmaceutically acceptable polymers are used in the pharmaceutical composition and form hydrogels which provide stabilization functionality. The stabilized pharmaceutical compositions of the present invention may comprise polymeric materials in a concentration ranging from about 0.5% to 10% w/v of the composition, preferably 0.5% to 5% of the composition. In the preferred embodiments, the stabilized pharmaceutical composition may comprise one or more of the polymeric materials of the group: carbomer, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and xanthan gum.
In preferred embodiments, the stabilized pharmaceutical composition may be in the form of liquid spray or a gel and may be stored at temperatures up to 37° C., more preferably stored at room temperature, 22-25° C.
In another embodiment of the invention, the stabilized pharmaceutical composition may further comprise serum albumin proteins, such as bovine serum albumin or human serum albumin. The stabilized pharmaceutical composition of present invention may comprise serum albumin proteins in a concentration ranging from about 1 mg/mL to 10 mg/mL.
In some embodiments, the stabilized pharmaceutical composition may further comprise preservatives, such as potassium sorbate or sodium benzoate.
In yet other embodiments, the stabilized pharmaceutical composition may further comprise a cytokine, a growth factor, any agonist of wound healing (or effective wound healing agent), including but not limited to small molecule agonists, peptide agonists, chemical agonists, or mixtures thereof. A growth factor according to the present invention can be, for example, platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, TGF-β, and mixtures thereof.
Stabilized pharmaceutical compositions of the present invention may be formulated for topical treatment of chronic and acute wounds by administering such composition topically to a wound of a patient suffering from an acute wound, or from delayed wound healing. Wounds treatable by the pharmaceutical compositions of the present invention include acute wounds, such as originating from thermal and chemical burns, trauma, or surgical interference, and chronic wounds, such as ulcers and diabetic wounds.
PBS (phosphate buffered saline) was added to all preparations to a 0.1× working concentration to maintain pH of the preparations at about 6.5.
Wound healing (or scratch) assay was carried out to determine the wound healing abilities of human skin epithelium after the exposure to calreticulin protein alone or in the presence of hydrogen peroxide (H2O2), which renders cells unable to cover the scratch. All experiments were carried out at 37° C. Human skin epithelium cells were seeded into 6-well plates and grown until 80-90% confluence. The cell monolayer was scratched with a sterile 1 mL pipette tip and then the preparation containing calreticulin was applied to the scratch surface. Subsequent cell migration and wound area coverage was monitored daily by means of microscopy imaging. The cell-free area was measured by ImageJ software.
Data presented in
Wound healing (or scratch) assay was carried out in the same way as in Example 1 to determine the wound healing abilities of human skin epithelium after the exposure to hydrogels comprising calreticulin, except that no evaluation of hydrogels effect on H2O2-induced damage was performed. Obtained data show that all hydrogel preparations comprising calreticulin, or calreticulin in combination with serum albumin have positive effect on wound healing at 72 hours' time point and were able to restore cell ability to cover the scratch area.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22202774 | Oct 2022 | EP | regional |
