Peripheral blood mononuclear cells (PBMCS) supernatant for use in the treatment of wounds

Abstract

The present invention relates to a pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell (PBMC) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure, wherein the composition applied topically per cm2 wound area comprises the components present in 1 ml of the supernatant, which is obtainable by cultivating PBMCs in a culture medium at a concentration of 20x106 to 50x106 PBMCs / ml culture medium for at least 4 h, wherein the PBMCs are subjected to ionizing radiation at a dose of at least 10 Gy before or during cultivation.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR TREATING WOUNDS

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of wound treatment .

[0004] BACKGROUND ART

[0005] Wound healing is a complex physiological process that involves the coordinated interplay of various biological mechanisms, including hemostasis, inflammation, proliferation, and remodeling. Significant advances in wound care management have been made over the past few decades, particularly due to the development of advanced dressings, biologies, and other therapeutic interventions. However, despite these improvements, there remains a substantial unmet need for effective methods and therapeutic approaches in wound healing, especially in the context of chronic wounds such as diabetic ulcers and pressure sores. Chronic wounds affect millions of individuals worldwide and lead to significant healthcare costs and diminished quality of life.

[0006] Transplantation of adult stem cells has been proposed and tested to repair different kinds of tissue damage, since stem cells are known to transdifferentiate into various cells types, including skin cells. Despite very promising pre- clinical results, meticulously performed first clinical trials did not fully meet the expectations and the use of adult stem cell therapy for organ regeneration has not entered the routine clinical practice.

[0007] In a more recent approach, Timmers et al. (Stem Cell Res 1 (2007) : 129-137) demonstrated that fractionated conditioned medium products of mesenchymal stem cells and containing only soluble factors smaller that 100-200 nm in size provide cardio protection in an ischemia reperfusion (I / R) injury model. Thus, it was thought that such preparations may contribute to wound healing processes.

[0008] Hacker S et al. (Sci Rep 6(2016) : 25168) found that paracrine factors present in the supernatant of irradiated peripheral blood mononuclear cells (PBMCs) improve skin regeneration and angiogenesis in a porcine burn model demonstrating that such supernatants may be used in the treatment of wounds.

[0009] Clinical studies have shown that no wound closure progression in wounds treated with supernatants of irradiated PBMCs compared to wounds treated with placebo could be achieved under the conditions chosen (Simader E et al. Sci Rep. (2017) 7 : 6216) .

[0010] Wagner T et al. (Sci Reports 8 (2018) : 18016) show that the secretome derived from y-irradiated peripheral blood mononuclear cells (PBMCs) exhibits enhanced pro-angiogenic and regenerative properties compared to its isolated subfractions, such as extracellular vesicles alone. It further shows that using the complete PBMC secretome leads to superior wound healing in a diabetic mouse model.

[0011] Beer L et al. (Apoptosis 21 (2016) : 1336-1353) show the regenerative potential of an apoptotic PBMC secretome, comprising proteins, lipids, microRNAs and extracellular vesicles, and presents preclinical evidence of its efficacy in models of myocardial infarction, chronic heart failure, stroke, spinal cord injury, and wound healing.

[0012] Beer L et al. (Sci Reports 5 (2015) : 16662) characterize the secretome of apoptotic PBMCs, identifying proteins and exosomal components that significantly enhance tissue regeneration, including angiogenesis and cytoprotection .

[0013] WO 2022 / 219073 discloses a composition comprising a supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment of skin scars.

[0014] US 2022 / 118020 relates to a topical pharmaceutical preparation for treating an inflammatory skin condition, preferably a condition associated with ischemia, comprising a supernatant of a PBMC cell culture.

[0015] Hence, it is an object of the present invention to provide conditions under which supernatants of irradiated PBMCs result in a wound closure and, thus, in an effective treatment of wounds .

[0016] SUMMARY OF THE INVENTION Thus , the present invention relates to a pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure , wherein the composition applied topically per cm2wound area comprises the components present in 1 ml of the supernatant , which is obtainable by cultivating PBMCs in a culture medium at a concentration of 20xl 06to 50xl 06PBMCs / ml culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation .

[0017] It turned surprisingly out that the application of a certain dose of a supernatant of a PBMC culture on a wound promotes wound healing and wound closure and, thus , results in the treatment of wounds . The application of a composition comprising the supernatant of the present invention leads to a wound closure which cannot be achieved using standard of care products or other compositions known to promote the healing process of wounds . It turned out that the composition of the present invention enhances signi ficantly the healing process i f applied on a wound at a certain dose .

[0018] The present invention relates also to a pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure , wherein the supernatant is obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, wherein the composition is applied topically on the wound at a dose equivalent to a supernatant of 20xl 06to 50xl 06PBMCs / ml culture medium per cm2wound area .

[0019] A further aspect of the present invention relates to a pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure , wherein the supernatant is obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and wherein the composition is applied topically on the wound in combination with a wound dressing comprising or consisting of alginate fibers .

[0020] It turned surprisingly out that the supernatant obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, shows enhanced ef fects in regard to the treatment of wounds and in promoting wound healing and wound closure compared to the use of amorphous hydrogels comprising, e . g . , alginate . The use of alginate fibers for the application of the pharmaceutical composition and the supernatant of the present invention, respectively, turned out to be advantageous since it leads to a faster healing and a faster closure of wounds compared to the use of amorphous hydrogels , in particular amorphous alginate . This is an unexpected and surprising finding .

[0021] Alginate is a well-known naturally derived polysaccharide with excellent biocompatibility, hemostatic properties and high fluid absorption capacity . Alginate fibers processed into a fleece , mat or nonwoven structure provide a mechanically stable yet conformable dressing that absorbs wound fluid, swells into a soft gel , and ensures close wound contact while preventing lateral leakage and maceration .

[0022] A particular advantage of the alginate fiber dressing is its suitability as a carrier for biologically active substances present in the supernatant of the present invention . The dressing serves as a delivery matrix for the PBMC cell culture supernatant of the invention . The fibrous structure enables uni form loading and controlled release of the supernatant , thereby delivering cytokines , growth factors , and other active mediators directly to the wound bed . This combination promotes moist wound healing, reduces inflammation, and accelerates tissue regeneration much more ef ficient compared to amorphous hydrogel .

[0023] Another aspect of the present invention relates to a method for treating a wound and / or for promoting wound healing or wound closure comprising the step of applying topically per cm2wound area a composition comprising the components present in 1 ml of a supernatant , which is obtainable by cultivating PBMCs in a culture medium at a concentration of 20xl 06to 50xl 06PBMCs / ml culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation .

[0024] An aspect of the present invention relates to a pharmaceutical combination comprising a ) a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and b ) a wound dressing comprising or consisting of alginate fibers .

[0025] The present invention relates also to a pharmaceutical combination comprising the supernatant of the present invention and a wound dressing comprising or consisting essentially of alginate fibers . Thus , the invention provides a wound dressing with superior handling and clinical performance compared to amorphous hydrogels , while of fering an advanced therapeutic platform for topical delivery of the PBMC derived secretome of the invention . A further aspect of the present invention relates to a kit comprising a ) a container comprising a composition, preferably a lyophili zed composition, comprising or consisting of a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture , wherein the supernatant is obtainable by cultivating a PBMC cell culture for at least 4 h, wherein said PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and b ) a container comprising an aqueous solution comprising 1 to 3 wt% of an alginate or a package comprising a wound dressing comprising or consisting of alginate fibers . It is advantageous to store the composition, preferably the lyophilized composition, comprising or consisting of the supernatant of the PBMC culture separate from the aqueous solution comprising the alginate or the wound dressing in order to increase the shelf life of the pharmaceutically active supernatant. Furthermore, the separate storage allows to adjust the concentration of the supernatant present in the pharmaceutical composition to be applied to a wound.

[0026] Another aspect of the present invention relates to a method for producing a pharmaceutical composition to treat a skin lesion by combining a) a composition comprising or consisting of a supernatant of a peripheral blood mononuclear cell (PBMC) culture, wherein the supernatant is obtainable by cultivating a PBMC cell culture for at least 4 h, wherein said PBMC cell culture is subjected to ionizing radiation at a dose of at least 10 Gy before or during cultivation, with b) an aqueous solution comprising 1 to 3 wt% of an alginate in a ratio from 1:1 to 1:5, preferably from 1:2 to 1:4, more preferably about 1:3 or a package comprising a wound dressing comprising or consisting of alginate fibers.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] Fig. 1 shows patients with >80% reduction in wound area from the start of the treatment.

[0029] Fig. 2 shows the percentage of complete wound closure of wounds of patients treated with various concentrations of APO- 2 and placebo.

[0030] Fig. 3 shows the experimental workflow of Example 2. Female diabetic db / db mice received a standardized 1 x 1 cm2fullthickness wound and were allocated to different treatment groups. Two healing conditions were investigated: open wound healing (amorphous hydrogel and control groups) and healing under occlusion (Kaltostat™ (alginate) dressing groups) . Fig. 4 shows the percentage wound area over time for each treatment group of example 2, presented as the mean value within each group.

[0031] Fig. 5 shows the mean epidermal thickness in wound and skin regions across treatment groups (see example 2) .

[0032] DESCRIPTION OF EMBODIMENTS

[0033] The term "treat" or "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition, disorder or injury, substantially ameliorating clinical or esthetical symptoms of a disease, condition, disorder or injury, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, disorder or injury, and protecting from harmful or annoying symptoms. The term "treat" or "treating", as used herein, further refers to accomplishing one or more of the following: (a) reducing the severity of the disease, condition, disorder or injury; (b) limiting development of symptoms characteristic of the disease, condition, disorder or injury being treated; (c) limiting worsening of symptoms characteristic of the disease, condition, disorder or injury being treated; (d) limiting recurrence of the disease, condition, disorder or injury in patients that have previously had the disease, condition, disorder or injury; and (e) limiting recurrence of symptoms in patients that were previously symptomatic for the disease, condition, disorder or inj ury .

[0034] The term "wound", as used herein, refers to a disruption of the normal continuity of structures caused by a physical (e.g. mechanical) force, a biological or a chemical means. The term "wound" includes, but is not limited to, incisional wounds, excisional wounds, traumatic wounds, lacerations, punctures, cuts and the like.

[0035] The term "wound healing", as used herein, refers to a regenerative process with the induction of a temporal and spatial healing program, including, but not limited to, the processes of inflammation, granulation, neovascularization, migration of fibroblast, endothelial and epithelial cells, extracellular matrix deposition, reepithelialization, and remodeling. "Promoting wound healing" means that these processes are started or enhanced by applying the composition of the present invention on the wound.

[0036] The term "wound closure", as used herein, refers to the healing of a wound such that the edges of the wound are rejoined to form a continuous barrier.

[0037] "A dose per cm2wound area equivalent to 1 ml of a supernatant obtainable by cultivating 20xl06to 50xl06PBMCs / ml culture medium", "a dose equivalent to a supernatant of 20xl06to 50xl06PBMCs / ml culture medium per cm2wound area" or , as used herein, means that the pharmaceutical composition of the present invention, that is applied per cm2of a wound, comprises the components of a supernatant or of 1 ml of a supernatant which is obtainable by cultivating 20xl06to 50xl06PBMCs per ml culture medium under the conditions set forth herein. Thus, the pharmaceutical composition applied to one cm2of a wound comprises the components present in 1 ml of a supernatant obtainable by cultivating 20xl06to 50xl06PBMCs per ml culture medium under the conditions set forth herein. If the supernatant is obtained by cultivating, for instance, more than 50xl06PBMCs per ml culture medium, the supernatant will be diluted accordingly or, in case the supernatant has been lyophilized, dissolved in an appropriate amount of an aqueous pharmaceutically acceptable composition.

[0038] The term "wound size" or "wound area", as used herein, refers to a physical measure of disruption of the normal continuity of structures caused by a physical (e.g., mechanical) force, a biological or a chemical means. The wound size and the wound area can be determined by methods known in the art. Particularly preferred to determine the wound size and the wound area is the use of a camera system, preferably a 3-dimensional camera system. Exemplary methods are described in Jorgensen LB et al. (Int Wound J. 2016 Aug; 13 (4) : 540- 553) . A particularly preferred method for determining the wound size / wound area is described in Gugerell A et al. (Trials . 2021; 22 : 10) . PBMCs used to produce the supernatant of the present invention can be obtained from whole blood using methods known in the art, such as ficoll gradient, hypotonic lysis, etc. These methods are well known in the art (see e.g. WO 2010 / 079086) .

[0039] The PBMCs present in the culture medium are subjected to ionizing radiation during or before cultivation or even before being added to the culture medium. The ionizing radiation can be gamma radiation (e.g. Caesium 137 gamma radiation) or photon radiation (e.g. from a light source, from a laser etc.) , at a dose of at least 10 Gy.

[0040] The pharmaceutical composition of the present invention is preferably applied topically on the wound. However, the pharmaceutical composition can also be applied by injection whereby the injection occurs preferably into the tissue / skin surrounding the wound.

[0041] The wound to be treated can be a mammalian or human wound (e.g. a wound affecting the skin of a mammal or a human individual) , preferably a human wound. It turned out that the pharmaceutical composition of the present invention can not only be used to treat a human wound or promote the healing of a human wound, but also wounds of mammals.

[0042] According to a preferred embodiment of the present invention the composition of the present invention is applied on the wound in combination with a wound dressing comprising or consisting of alginate fibers.

[0043] The term "wound dressing comprising or consisting of alginate fibers", as used herein, refers to a solid wound covering material that is manufactured from fibers prepared from alginic acid or salts thereof, preferably sodium alginate, calcium alginate or mixtures thereof. Alginate fibers are elongated, flexible structures produced by extrusion or wet-spinning of alginate solutions into a coagulation bath containing calcium or other divalent cations, which induce ionic cross-linking of the alginate chains and formation of insoluble calcium alginate filaments (Thomas S., J. Wound Care 9(2000) : 56-60) . The fibers are subsequently collected, washed, and dried, and may be cut, carded, and processed into nonwoven fabrics , fleeces , or mats by conventional textile methods such as needling or hydroentangling .

[0044] A wound dressing according to this definition consists essentially of such alginate fibers in a coherent fibrous layer . The dressing may be sterili zed and cut to si ze , and optionally combined with backing layers , adhesive borders , or secondary support materials . Upon contact with wound exudate , the calcium ions in the alginate fibers exchange with sodium ions in the wound fluid, resulting in partial solubili zation and conversion of the fibers into a soft , cohesive hydrogel that conforms intimately to the wound bed, absorbs exudate , and maintains a moist healing environment .

[0045] The phrase "comprising alginate fibers" encompasses dressings in which alginate fibers form the principal absorbent layer, optionally blended with other biocompatible fibers ( e . g . , carboxymethylcellulose , viscose , or chitosan) . The phrase "consisting of alginate fibers" is intended to cover dressings in which the fibrous material is exclusively alginate , without addition of other fiber types .

[0046] A typical method of manufacture includes the steps of dissolving sodium alginate in puri fied water to obtain a viscous spinning solution ( typically 1-10% w / v) , extruding the solution through a spinneret into a coagulation bath containing calcium chloride ( 0 . 1-1 M) to precipitate calcium alginate filaments , collecting, stretching and washing the fibers to remove excess salts , Drying the fibers under controlled conditions , and cutting the fibers to desired staple lengths and processing them into nonwoven fleeces or mats by carding, cross-laying, and needle-punching or hydroentangling .

[0047] A well-known commercial example is Kaltostat® ( ConvaTec Ltd . , UK) , which comprises calcium-sodium alginate fibers processed into a nonwoven dressing . Kaltostat® is manufactured by wet-spinning alginate into calcium chloride , producing calcium alginate fibers subsequently processed into a dressing . The product demonstrates strong absorbency, gel formation, hemostatic activity, and atraumatic removal , properties that exempli fy the advantages of fiber-based alginate dressings compared to amorphous hydrogels .

[0048] Thus , a wound dressing comprising or consisting of alginate fibers is a fibrous alginate structure produced preferably by wet-spinning and textile processing, which upon contact with wound exudate forms a gel while retaining structural integrity, thereby providing an ef fective , biocompatible , and clinically established platform for advanced wound care .

[0049] The supernatant of the present invention is applied preferably directly or after reconstitution with an aqueous solution from a lyophili zed supernatant on such a wound dressing before being contacted with the wound to be treated . In order to protect the wound dressing comprising the supernatant of the present invention in the desired concentration from external mechanical influences , from germs or from dehydration, for instance, the wound dressing itsel f can be covered on the side opposite to the wound with a film layer .

[0050] As used herein, the term " film layer" refers to a thin, continuous sheet of a polymeric material , preferably polyurethane , which provides a barrier function . The film layer may be transparent or translucent , semipermeable to gases and water vapor, and substantially impermeable to liquids and microorganisms . The film layer may be used as an outer cover, barrier, or fixation component in the wound dressing of the present invention .

[0051] The film layer functions as a cover or fixation component for the wound dressing of the present invention comprising or consisting of alginate . In such a configuration, the hydrogel provides moisture donation, exudate absorption and therapeutic activity, while the transparent film layer prevents desiccation, stabili zes the hydrogel in intimate wound contact , and protects the wound from external contamination . The combination of a hydrogel with a semipermeable film layer thus results in a composite wound dressing that ensures optimal moisture balance , mechanical stability, and barrier protection, while allowing atraumatic removal and improved patient comfort . According to a preferred embodiment of the present invention the wound is a chronic wound or an acute wound .

[0052] The composition of the present invention can be used for treating chronic or acute wounds or for promoting their healing process and wound closure . In particular, chronic wounds need special attention since they are known to be treated usually over a long period of time .

[0053] According to a further preferred embodiment of the present invention the wound is a skin wound, preferably an ulcer, a burn, an abrasion, a laceration, a puncture wound, an incision or an avulsion .

[0054] The composition of the present invention can be used for treating any kind of wound irrespective of their cause . This is a surprising finding since it is known that wounds being the result of di f ferent causes may involve di f ferent biological processes . It is , however, most preferred to use the composition of the present invention in the treatment of ulcers .

[0055] "Ulcers" , as used herein, refer to a lesion of the skin that is characteri zed by the formation of pus and necrosis ( death of surrounding tissue ) usually resulting from inflammation or ischemia .

[0056] Hence , according to a particularly preferred embodiment of the present invention the chronic wound is an ulcer, preferably a diabetic ulcer, more preferably a diabetic foot ulcer, or a pressure ulcer ( decubitus ) .

[0057] Diabetic wounds and impaired wound healing represent clinical challenges with an unmet need . The process of wound healing involves well-orchestrated biological events , including macrophage invasion, reepitheli zation, fibroblast migration and proli feration, extracellular matrix deposition, and neo-angiogenesis . However, in diabetic wounds / ulcers , the healing process is impaired, which can lead to chronic nonhealing wounds , such as diabetic foot ulcers , and, in the worst case , requires limb amputation . It was surprisingly found that the composition of the present invention applied on such wounds / ulcers at the defined dose leads to a complete wound closure . It turned out that the composition can be used for the treatment of wounds of any size. However, the healing process is particularly fast, if the wound covers, according to a preferred embodiment of the present invention, an area of less than 10 cm2, preferably of less than 8 cm2, more preferably of less than 5 cm2, more preferably of less than 4 cm2.

[0058] According to another preferred embodiment of the present invention the wound covers an area of more than 0.1 cm2, preferably of more than 0.2 cm2, more preferably of more than 0.5 cm2, more preferably of more than 0.8 cm2, more preferably of more than 1 cm2.

[0059] According to a further preferred embodiment of the present invention the wound covers an area from from 0.1 cm2to 10 cm2, preferably from 0.1 cm2to 8 cm2, preferably from 0.1 cm2to 5 cm2, preferably from 0.1 cm2to 4 cm2, preferably from 0.2 cm2to 4 cm2, preferably from 0.2 cm2to 5 cm2, preferably from 0.2 cm2to 8 cm2, preferably from 0.2 cm2to 10 cm2, preferably from 0.5 cm2to 4 cm2, preferably from 0.5 cm2to 5 cm2, 0.5 cm2to 8 cm2, preferably from 0.5 cm2to 10 cm2, preferably from 0.8 cm2to 4 cm2, 0.8 cm2to 5 cm2, preferably from 0.8 cm2to 8 cm2, preferably from 0.8 cm2to 10 cm2, preferably from 1 cm2to 4 cm2, preferably from 1 cm2to 5 cm2, preferably from 1 cm2to 8 cm2, preferably from 1 cm2to 10 cm2. According to a particularly preferred embodiment of the present invention the wound covers an area from 0.2 cm2to 5 cm2, preferably from 0.2 cm2to 4 cm2, preferably from 0.8 cm2to 4 cm2, 0.8 cm2to 5 cm2.

[0060] According to a preferred embodiment of the present invention the composition is applied to a wound for at least 8 days, preferably for at least 10 days, more preferably for at least 14 days, more preferably for at least 21 days, more preferably for at least 30 days.

[0061] The application of the composition of the present invention on wounds over several days is advantageous and results in a complete closure of the treated wound. Hence, it is preferred to apply the composition on a wound for at least 8 days.

[0062] According to a further preferred embodiment of the present invention the composition is applied to the wound twice a day, preferably once a day, more preferably every second day, more preferably every third day, more preferably every fourth day, more preferably every fifth day, more preferably every sixth day, more preferably every seventh day.

[0063] According to another preferred embodiment of the present invention the composition is applied to the wound for at least 8 days, twice a day; preferably for at least 8 days, once a day; preferably for at least 8 days, every second day; preferably for at least 8 days, every third day; preferably for at least 8 days, every fourth day; preferably for at least 8 days, every fifth day; preferably for at least 8 days, every sixth day; preferably for at least 8 days, every seventh day; preferably for at least 10 days, twice a day; preferably for at least 10 days, once a day; preferably for at least 10 days, every second day; preferably for at least 10 days, every third day; preferably for at least 10 days, every fourth day; preferably for at least 10 days, every fifth day; preferably for at least 10 days, every sixth day; preferably for at least 10 days, every seventh day; preferably for at least 14 days, twice a day; preferably for at least 14 days, once a day; preferably for at least 14 days, every second day; preferably for at least 14 days, every third day; preferably for at least 14 days, every fourth day; preferably for at least 14 days, every fifth day; preferably for at least 14 days, every sixth day; preferably for at least 14 days, every seventh day; preferably for at least 21 days, twice a day; preferably for at least 21 days, once a day; preferably for at least 21 days, every second day; preferably for at least 21 days, every third day; preferably for at least 21 days, every fourth day; preferably for at least 21 days, every fifth day; preferably for at least 21 days, every sixth day; preferably for at least 21 days, every seventh day; preferably for at least 30 days, twice a day; preferably for at least 30 days, once a day; preferably for at least 30 days, every second day; preferably for at least 30 days, every third day; preferably for at least 30 days, every fourth day; preferably for at least 30 days, every fifth day; preferably for at least 30 days, every sixth day; and preferably for at least 30 days, every seventh day. According to a preferred embodiment of the present invention the composition is administered to the wound once a week, preferably twice a week, more preferably three times a week, more preferably four times a week, more preferably five times a week, more preferably six times a week, more preferably daily .

[0064] According to a particularly preferred embodiment of the present invention the composition applied topically to one cm2wound area comprises the components present in 1 ml of a supernatant obtainable by cultivating 20xl 06to 40xl 06PBMCs / ml , preferably 20xl 06to 30xl 06PBMCs / ml , more preferably 22xl 06to 28xl 06PBMCs / ml , more preferably 24xl 06to 26xl 06PBMCs / ml , more preferably about 25xl 06PBMCs / ml , culture medium .

[0065] According to a preferred embodiment of the present invention the composition comprises 0 . 5 to 5 wt% , preferably 1 to 4 wt% , more preferably 2 to 3 wt% , more preferably about 2 . 25 wt% , of an alginate , preferably an amorphous alginate .

[0066] The addition of alginate to the composition of the present invention is advantageous since the presence of alginate in the composition enhances the ef fect on the treatment of the wound to be treated and may additionally promote wound healing and, thus , wound closure . Furthermore , the presence of alginate in the composition of the present invention increases its viscosity which is advantageous when applying the composition on the wound to be treated .

[0067] As used herein, the term "amorphous alginate" or "amorphous hydrogel" refers to a semi-solid, non- fibrous hydrogel composition comprising alginate as a gelling component . The amorphous form denotes that the alginate is not present as organi zed fibers or filaments , but rather as a non-structured, paste-like or gel-like mass . Such hydrogels are generally prepared by hydrating alginic acid salts , such as sodium alginate and / or calcium alginate , in aqueous solution under conditions that promote partial cross-linking, thereby yielding a viscous , conformable material without defined fiber morphology . The amorphous alginate hydrogel typically contains a high proportion of water ( greater than 80% by weight ) , with alginate polymers dispersed therein to form a three- dimensional network . The gel may additionally comprise other hydrophilic polymers ( e . g . , carboxymethylcellulose , polyvinyl alcohol , polyethylene glycol ) , humectants , stabili zers , or pharmaceutically active agents . The material is conformable , fills irregular wound cavities , donates or maintains moisture , and is capable of absorbing limited amounts of exudate .

[0068] In contrast thereto , alginate fibers refer to fibers of alginate , typically prepared by wet-spinning alginate solutions into a calcium ion bath to yield calcium alginate filaments . These fibers are processed into nonwoven fleeces , mats , or textiles , which form a coherent , solid dressing . Upon contact with wound exudate , ion exchange between calcium and sodium ions leads to controlled gelling of the fibers , forming a cohesive gel layer that conforms to the wound bed while retaining mechanical strength .

[0069] While both amorphous alginate and alginate fibers share the property of maintaining a moist wound environment through alginate-based gelling, amorphous alginate / hydrogels are defined by their non- fibrous , paste-like character, whereas alginate fiber dressings are defined by their fibrous structure and solid form .

[0070] According to a preferred embodiment of the present invention the alginate fibers comprise or consist of calcium alginate and / or sodium alginate , preferably calcium alginate and sodium alginate .

[0071] Calcium alginate fibers are known to exhibit superior gelforming capacity upon contact with wound exudate due to ionic exchange of calcium and sodium ions , resulting in the formation of a soft , cohesive hydrogel layer at the wound interface . Sodium alginate , by contrast , provides enhanced flexibility, swelling capacity, and contributes to improved conformability of the dressing . By combining calcium alginate and sodium alginate in defined proportions , an optimi zed balance between gel strength, absorption, and mechanical integrity is achieved . According to another prferred embodiment of the present invention the wound dressing comprises or consists of 70 to 90 wt% , preferably 75 to 85 wt% , more preferably around 80 wt% , calcium alginate and 10 to 30 wt% , preferably 15 to 25 wt% , more preferably around 20 wt% , sodium alginate .

[0072] This speci fic ratio has been found to provide advantageous ef fects in terms of wound exudate absorption, gel cohesiveness , atraumatic removability of the dressing and release of components of the supernatant of the present invention absorbed thereto . The predominance of calcium alginate ensures ef fective hemostasis and strong gel formation, while the defined proportion of sodium alginate contributes to increased hydration, softness , and patient comfort .

[0073] Such fiber compositions may be produced by preparing alginate spinning solutions of calcium and sodium alginate salts , extruding these solutions through a spinneret into a coagulation bath containing divalent cations , and subsequently processing the resulting fibers into nonwoven mats , fleeces , or textile structures . The dressing thus obtained may be sterili zed and cut to appropriate si ze for clinical use .

[0074] The defined mixture of calcium alginate and sodium alginate fibers provides a pharmaceutical composition that is particularly suitable as a wound dressing for exudative wounds , burns , ulcers , or surgical sites , combining the desirable properties of both alginate species while overcoming the limitations of dressings containing only one alginate form .

[0075] According to another preferred embodiment of the present invention the amorphous alginate is a sodium alginate and / or a potassium alginate .

[0076] According to a further preferred embodiment of the present invention the alginate can be obtained from various sources . Alginate is a natural polysaccharide that is primarily obtained from the cell walls of brown seaweeds . Some of the main sources of alginate include species from the genus Laminaria, Ascophyll um, Macrocysti s and Sargassum . Other sources of alginate can be other marine algae. Also certain microorganisms can be a source of alginate. Certain bacteria, for instance, can produce alginate as an exopolymer, whereby bacteria from the genus Pseudomonas and Azotobacter are known producers of alginate. However, the preferred alginate is of a marine source.

[0077] According to a preferred embodiment of the present invention the pharmaceutical composition comprises further 0.1 to 3 wt%, preferably 0.5 to 2 wt%, more preferably 0.5 to 1.5 wt%, more preferably 0.5 to 1 wt%, more preferably about 0.825 wt%, cellulose or at least one cellulose derivative.

[0078] Cellulose and cellulose derivatives in the composition of the present invention are helpful to adjust viscosity of the composition. A concentration of 0.1 to 3 wt% cellulose or at least one cellulose derivative in the composition of the present invention renders the composition sufficiently viscose to be applied topically on the wound.

[0079] According to another preferred embodiment of the present invention the at least on cellulose derivative is carboxymethyl cellulose (CMC) and / or hydroxyethyl cellulose.

[0080] According to a preferred embodiment of the present invention the pharmaceutical composition comprises further 10 to 25 wt%, preferably 15 to 20 wt%, more preferably about 18.25 wt%, of at least one diol, preferably propane-1 , 2-diol .

[0081] Diols and in particular propylene glycol serve in the composition of the present invention as moistening agent, as solvent for active components present in the supernatant and as a penetration enhancer for facilitating the delivery of active ingredients into the wound.

[0082] According to a further preferred embodiment of the present invention the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells.

[0083] According to a preferred embodiment of the present invention the PBMCs are cultivated in a culture mediuem, preferably in a cell culture medium selected from the group consisting of a cell growth medium, preferably CellGro medium, more preferably Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture . The PBMCs may be cultivated in one of these culture media. It is also possible to use an alternative culture medium to obtain the supernatant of the present invention. Using alternative methods and culture media shall result in the production of a supernatant showing similar, identical or even better properties as supernatants obtained by cultivating PBMCs with cell culture media typically used for mammalian cell cultures (e.g. CellGro medium, Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture as used to obtain PBMC supernatants) . The properties are measured using a potency assay as described in EP 3 729 079 Bl, preferably potency assays involving the measurement of AP-1 promoter activities and / or the amount of phosphorylated HSP-27 as described therein .

[0084] A preferred method for cultivating PBMCs may comprise the step of incubating the PBMCs in a culture medium comprising at least one sodium salt, at least one potassium salt and at least calcium salt under an atmosphere comprising less than 1% carbon dioxide.

[0085] It turned out that the PBMCs can be cultivated under an atmosphere comprising less than 1% CO2 using a cell culture medium comprising at least one sodium salt, at least one potassium salt and at least calcium salt. The use of such a culture medium allows to keep the pH in a range suitable for mammalian cells although the CO2 concentration in the atmosphere, where the cells are cultivated, is low compared to typically used atmospheres / gases comprising 4 to 7% or approx. 5%, CO2 • This is particularly advantageous since a CO2 incubator, which is typically used to cultivate mammalian cells, is not needed and even air (i.e. atmospheric air) can be used to cultivate mammalian cells without adding additional CO2.

[0086] The atmosphere under which the PBMCs are cultivated my comprise less than 0.5%, preferably less than 0.2%, more preferably less than 0.1%, more preferably less than 0.05%, carbon dioxide. The atmosphere may also comprise oxygen and / or nitrogen, wherein said atmosphere comprises preferably 0.5 to 21%, more preferably 1 to 21%, even more preferably 5 to 21%, even more preferably 10 to 21%, oxygen and / or preferably 60 to 78%, more preferably 65 zo 78%, more preferably 70 to 78%, nitrogen. The atmosphere is particularly preferred air.

[0087] The culture medium preferably used to obtain the supernatant of the present invention may comprise 50 to 200 mmol / L, preferably 100 to 150 mmol / L, more preferably 125 to 135 mmol / L, more preferably 130 to 132 mmol / L, more preferably around 131 mmol / L, sodium ions.

[0088] "Around", as used herein, means that the concentration to which the term is associated to can be 10% higher or lower, preferably 5 % higher or lower, more preferably 4 % higher or lower, more preferably 3 % higher or lower, more preferably 2 % higher or lower, more preferably 1 % higher or lower.

[0089] The at least one sodium salt is preferably sodium chloride.

[0090] The culture medium may comprise 2 to 8 mmol / L, preferably 3 to 6 mmol / L, more preferably 4 to 6 mmol / L, more preferably 5 to 6 mmol / L, more preferably around 5.36 mmol / L, potassium ions, which are added to the culture medium as potassium chloride, for instance.

[0091] The culture medium may comprise 0.5 to 3 mmol / L, preferably 1 to 2 mmol / L, more preferably 1.5 to 2 mmol / L, more preferably around 1.84 mmol / L, calcium ions, which can be added as calcium chloride.

[0092] The culture medium to cultivate the PBMCs is preferably Ringer's solution (USP43-NF38 (2020) , Monograph "Ringer's Injection", S. 3887) .Ringer's solution may comprise further at least one salt of a C2 to C5 carbonic acid, wherein the C2 to C5 carbonic acid is preferably lactate, more preferably L- lactate, and may be added to the culture medium in a concentration of 10 to 50 mmol / L, preferably 15 to 40 mmol / L, more preferably 20 to 30 mmol / L, more preferably around 28 mmol / L. Hence, the culture medium is particularly preferred lactated Ringer's solution (USP43-NF38 (2020) , Monograph "Lactated Ringer's Injection", S. 3891) .

[0093] The Ringer' s solution used as culture medium comprises preferably at least one polypeptide, preferably at least one blood polypeptide, most preferably albumin (e.g. serum albumin) or insulin, at a concentration of 1 mg / L to 50 g / L, preferably 2 mg / L to 40 g / L, more preferably 4 mg / L to 30 g / L, more preferably 1 g / L to 20 g / L, more preferably 2 g / L to 10 g / L .

[0094] According to a preferred embodiment of the present invention the PBMCs are incubated / cultivated in the culture medium at a temperature of 30 ° C to 38 ° C, preferably 35 ° C to 38 ° C, in order to obtain the supernatant of the present invention .

[0095] According to a further preferred embodiment of the present invention the PBMCs are subj ected to an ioni zing radiation at a dose of at least 20 Gy, preferably at least 30 Gy, more preferably at least 40 Gy, more preferably at least 50 Gy .

[0096] According to another preferred embodiment of the present invention the PBMCs are cultivated for at least 6 h, preferably for at least 12 h, more preferably for at least 24 h, most preferably for around 24 h, before isolating its supernatant . The maximum cultivation time does preferably not exceed 72 h, preferably 60 h, more preferably 48 h, more preferably 36 h .

[0097] After the incubation of the stressed PBMCs under the conditions set forth herein the cells and cellular debris can be removed by centri fugation to obtain the supernatant and / or by filtrating the cultured cell culture through a filter ( e . g . 0 . 2 pm filter ) .

[0098] Another aspect of the present invention relates tp a pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure , wherein the supernatant is obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and wherein the composition is applied topically on the wound in combination with a wound dressing comprising or consisting of alginate fibers as defined above .

[0099] Another aspect of the present invention relates to a method for treating a wound and / or for promoting wound healing or wound closure comprising the step of applying topically per cm2wound area a composition comprising the components present in 1 ml of a supernatant , which is obtainable by cultivating PBMCs in a culture medium at a concentration of 20xl 06to 50xl 06PBMCs / ml culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation .

[0100] Another aspect of the present invention relates to a kit comprising a ) container comprising a composition, preferably a lyophili zed composition, comprising or consisting of a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture , wherein the supernatant is obtainable by cultivating a PBMC cell culture for at least 4 h, wherein said PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and b ) a container comprising an aqueous solution comprising 1 to 3 wt% of an alginate and optionally further components as described above or a package comprising a wound dressing comprising or consisting of alginate fibers as defined herein .

[0101] A further aspect of the present invention relates to a method for producing a pharmaceutical composition to treat a wound and / or for promoting wound healing or wound closure by mixing a ) a composition comprising or consisting of a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture , wherein the supernatant is obtainable by cultivating a PBMC cell culture for at least 4 h, wherein said PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, with b ) an aqueous solution comprising 1 to 3 wt% of an alginate and optionally further components as described above in a ratio from 1 : 1 to 1 : 5 , preferably from 1 : 2 to 1 : 4 , more preferably about 1 : 3 or a package comprising a wound dressing comprising or consisting of alginate fibers . EXAMPLES

[0102] Example 1

[0103] Materials & Methods

[0104] Production of APO-2 (the pharmaceutical composition of the present invention) and placebo

[0105] Blood obtained from the Austrian Red Cross Blood Transfusion Service of Upper Austria was used to produce APO- 2. PBMCs were separated from whole blood samples by density centrifugation using LSM 1077 (Lymphocyte Separation Medium, Lonza, Switzerland) . Removal of LSM was achieved by two washing steps using Dulbecco's phosphate-buff ered saline (Lonza, Switzerland) . PBMCs were resuspended in phenol red- free CellGro GMP DC medium (CellGenix, Freiburg, Germany) containing no xenogeneic proteins. A sample was drawn for complete blood count to adjust white blood cells to a concentration of 25x l06cells / ml. Irradiation with 60 Gy (gamma radiation) induced apoptosis of PBMCs. By cultivation of apoptotic PBMCs in CellGro GMP DC medium, release of the secretome was achieved. After incubation for 24h±2h, cells were removed by centrifugation. The supernatant containing the secretome was sterile filtered at a pore size of 0.22pm. The adequate production of APO-2 was defined by appropriate secretion of the following important cytokines: interleukin (IL) -8 ( 0-5214 pg / ml ) , epidermal growth factor (EGF; 25- 226pg / ml) , and transforming growth factor-p (TGF-p; 2575- 21732pg / ml) . The supernatant was finally lyophilised.

[0106] Lyophilized culture medium not containing any cells (CellGro, CellGenix, Freiburg, Germany) served as placebo.

[0107] Induction of apoptosis was determined before irradiation and after cultivation of the cells by fluorescence-activated cell sorting analysis using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, US) . Concentrations of IL-8 / CxCL8 (C x C-motive-chemokine 8) , EGF, and TGF-p were determined with enzyme-linked immunosorbent assay (ELISA) to verify successful production of APO-2 according to GMP definitions. Finally, the product underwent endotoxin, mycoplasma and sterility testing. Cell culture supernatant samples were additionally screened for herpes virus contamination via polymerase chain reaction.

[0108] Production of the preparation to be applied on wounds

[0109] The preparation to be applied on the wound of the patients comprised 50 U supernatant / ml, 100 U supernatant / ml and 200 U supernatant / ml, respectively. 1 U corresponds to a supernatant obtained by cultivating 106PBMCs . The respective lyophilized product obtained as described above was dissolved in a 0.9% NaCl solution to the required concentration and combined in a ratio of 1:3 with a solution comprising 70 wt% water, 25 wt% propylene glycol, 3 wt% sodium alginate, 1 wt% carboxymethyl cellulose, 0.1 wt% hydroxyethyl cellulose and 0.1 wt% NaCl .

[0110] Clinical Assessment

[0111] To assess the effectiveness of the APO-2 preparation on wound healing four groups of patients with diabetic foot ulcer present for >4 weeks, a foot ulcer Wagner Grade I-II or ARMSTRONG Grade I-A or II-A, and an estimated foot ulcer surface area >0.8 cm2and <8 cm2, received three different doses of supernatant in a hydrogel matrix (12.5 U / ml / cm2wound area, 25 U / ml / cm2wound area and 50 U / ml / cm2wound area APO-2) and placebo (hydrogel matrix and fresh cell culture used to culture the PMBCs) . At least 20% of the patients of each patient group had wounds with a wound area >4 cm2.

[0112] In total 122 patients (27 [12.5 U / mL] , 38 [25 U / mL] , and 26 [50 U / mL] patients in the APO 2 groups; 31 patients in the placebo group) were treated with the respective preparation for 4 weeks.

[0113] Results

[0114] The wound area in patients treated with 25 U / ml per cm2wound area could be decreased within 8 weeks of treatment by over 60% in average compared to the lower and even higher doses and the placebo group (see Table 1) . Interestingly, in patients having wound areas >5 cm2no significant difference in the decrease of the wound area could be observed in the 12.5 / 25 / 50 U / ml groups compared to the placebo group. A minor effect in wound decrease could be observed in patients with wounds having an area of >4 cm2and <5 cm2. Table 1: Percentage reduction in wound area from baseline (at the beginning of the treatment) to week 4 (patients with adjudicated wound areas >0.8 cm2to <4 cm2at baseline, n = 69)

[0115] APO-2 APO-2 APO-2 Placebo

[0116] 12.5 U / mL / cm225.0 U / mL / cm250.0 U / mL / cm2

[0117] Mean 53.85 60.19 46.24 18.65

[0118] As shown in Fig. 1 85.7 % of the patients treated with 25 U / ml per cm2wound area showed after 4 weeks treatment an at least 80% reduction in wound area.

[0119] In Fig. 2 the observed complete wound closure in 90 patients who agreed to a follow-up visit after 12 weeks after the last treatment is shown. It turned out that the application of 25 U / ml per cm2wound area showed the best results .

[0120] Example 2: Comparative evaluation of hydrogel- and alginate- mediated Apo-2 application in diabetic wound healing

[0121] Delayed wound healing is a major complication of diabetes mellitus and represents a significant therapeutic challenge. Apo-2 (see Example 1) , the secretome derived from stressed peripheral blood mononuclear cells has shown regenerative and immunomodulatory effects in preclinical models and early translational studies. In this example a GMP-grade Apo-2 formulation was investigated in female diabetic BKS db / db mice. Two application strategies were compared: delivery in a hydrogel matrix and administration via an alginate dressing. The aim was to determine whether the mode of delivery influences wound closure and histological parameters of tissue regeneration .

[0122] Materials & Methods

[0123] Animal model

[0124] Female diabetic BKS db / db mice were acclimatized for two weeks prior to surgery. Two days before wounding the dorsal area was shaved and depilated with Veet® cream to improve Tegaderm (3M, Austria) dressing adhesion. On the day of surgery mice were anesthetized with an MMF / K regimen and received perioperative analgesia with buprenorphine as well as a subcutaneous glucose depot for fluid support. A standardized 1 x 1 cm2full-thickness excision (epidermis and dermis) wound was then created in the interscapular region and anesthesia was subsequently antagonized to ensure rapid recovery. Postoperative pain management was provided by adding piritramid to the drinking water for the following days.

[0125] Treatment groups (each group consisted of five female diabetic BKS db / db mice)

[0126] Healing under occlusion (Kaltostat™, Convatec, UK; fibrous fleece comprising 80% calcium alginate and 20% sodium alginate) :

[0127] Wounds received either Apo-2 and Kaltostat™ or NaCl and Kaltostat™ (control) . For Apo-2 and Kaltostat™, lyophilizate (100 U) was dissolved in 1.2 ml 0.9% NaCl, 0.3 ml of this solution (corresponding to 25 U per animal as the final wound dose) was applied to a 1x1 cm Kaltostat™-dressing and covered with Tegaderm. The NaCl and Kaltostat™ control used a Kaltostat™ piece soaked with 0.3 ml 0.9% NaCl, likewise covered with Tegaderm.

[0128] Open wound healing (amorphous hydrogel) :

[0129] Mice received either Apogel (Apo-2 and hydrogel, i.e. an amorphous alginate comprising hydrogel) or a hydrogel control (NaCl and hydrogel, i.e. an amorphous alginate comprising hydrogel) . For Apogel, Apo-2 lyophilizate (100 U) was dissolved in 1.2 ml 0.9% NaCl; 0.5 ml of this solution (corresponding to 25 U per animal as the final wound dose) was mixed with 1.5 ml hydrogel immediately before application. From the final preparation, 0.5 ml of gel mixture was applied per wound. The hydrogel control was prepared in the same way, using 1.5 ml Hydrogel mixed with 0.5 ml 0.9% NaCl, of which 0.5 ml was applied per wound.

[0130] In all intervention groups treatments were renewed every third day (days 0, 3, 6, 9, and 12) . Wounds in the Apogel and Hydrogel groups were photographed daily, whereas wounds in the two Kalotostat™-dressing groups (Apo-2 and Kaltostat™; NaCl and Kaltostat™) were photographed at each renewal (every third day) . Because complete wound closure was achieved across groups by day 14, the study was terminated at this time point earlier than the originally planned day 25.

[0131] The experimental workflow is summarized in Fig. 3, illustrating wound induction, treatment allocation, and follow-up until the endpoint at day 14. Two distinct healing settings were applied: (A) open wound healing without occlusion as in the hydrogel and control groups, and (B) healing under occlusion achieved by combining Kaltostat™ dressings with Tegaderm film.

[0132] Wound area and epidermal thickness were measured using ImageJ / Fiji (Version 2.16.0 / 1.54p) . Quantification of CD34+vessel area was performed in QuPath (Version 0.6.0) . Statistical analyses were carried out with SPSS Statistics, Version 30.0 (IBM, Armonk, NY, USA) . Group comparisons were performed separately within the two healing models (open wound healing (with amorphous hydrogel) and healing under occlusion (with Kaltostat™) ) . For each model, the treatment group was compared with the respective control group using the Mann- Whitney U test. A p-value < 0.05 was considered statistically significant .

[0133] Results

[0134] Healing under occlusion (alginate-based treatment)

[0135] Wounds treated under occlusion with Kaltostat™ dressings covered by Tegaderm showed progressive reduction in wound area over time. By day 6 wound sizes were reduced below baseline, and from day 9 onward the Apo-2 and Kaltostat™ group displayed smaller wound areas compared to NaCl and Kaltostat™ (control) . At day 12 Apo-2 and Kaltostat™ wounds measured 1.7% of baseline, while NaCl and Kaltostat™ wounds measured 12.6%. At day 14, wound areas were 0.3% in the Apo-2 and Kaltostat™ group and 4.7% in the NaCl and Kaltostat™ group (Fig. 4) .

[0136] Open wound healing (hydrogel-based treatment)

[0137] In the open setting with amorphous hydrogel, wounds were treated either with Apogel (Apo-2 and hydrogel) or with hydrogel control (NaCl and hydrogel) . Both groups exhibited an initial increase in wound size during the first days after surgery followed by a continuous reduction. By day 14 residual wound areas measured 10.6% of baseline in the Apogel group and 6.6% in the NaCl Hydrogel group (Fig. 4) .

[0138] In the open wound setting (amorphous hydrogel groups) a significant difference was observed at day 1 (p = 0.008) , with larger wound areas in the Apogel group compared with NaCl hydrogel (control) . At all subsequent time points wound closure progressed along a similar course and no further statistically significant differences were detected (p > 0.151) . In contrast, under occlusion (Kaltostat™ groups) wound reduction advanced steadily, and from day 12 onwards Apo-2 + Kaltostat™ wounds were significantly smaller than NaCl + Kaltostat™ controls (day 12: p = 0.032; day 14: p = 0.016) .

[0139] In addition to macroscopic wound closure histological analyses were performed. H&E staining was used to assess tissue morphology and epidermal thickness was measured both within the wound area and in adjacent skin. Furthermore, CD34 staining was carried out as a marker for vascular structures in the wound region.

[0140] H&E staining was performed and epidermal thickness was quantified in the wound region using ImageJ and compared with adjacent skin to further characterize tissue regeneration. In the Apo-2 and Kaltostat™ group wound epidermal thickness (70.9 pm) was comparable to the surrounding skin (75.3 pm) suggesting a near-complete restoration of epidermal structure. In contrast, in the NaCl and Kaltostat™ group the wound epidermis remained very thin (15.7 pm) compared with the adjacent skin (49.4 pm) . The comparison of wound / skin ratios between both alginate groups showed no statistically significant difference (p = 0.117) , although a trend toward higher values was observed in the AposecNew+alginate group (see Fig . 5 ) .

[0141] In the open healing model, both amorphous hydrogel-based groups displayed markedly thicker epidermis in the wound compared with the corresponding skin with values of 65.5 pm versus 26.9 pm in the Apogel group and 85.5 pm versus 25.4 pm in the hydrogel control group. The comparison between Apogel and NaCl plus hydrogel showed no significant difference (p = 0.754) (see Fig. 5) . These observations show different patterns between open and occlusive healing conditions. In both models, no statistically significant differences were found between treatment and control group.

Claims

CLAIMS :

1. Pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell (PBMC) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure, wherein the composition applied topically per cm2wound area comprises the components present in 1 ml of the supernatant, which is obtainable by cultivating PBMCs in a culture medium at a concentration of 20xl06to 50xl06PBMCs / ml culture medium for at least 4 h, wherein the PBMCs are subjected to ionizing radiation at a dose of at least 10 Gy before or during cultivation.

2. Pharmaceutical composition for the use according to claim 1, wherein the composition is applied on the wound in combination with a wound dressing comprising or consisting of alginate fibers.

3. Pharmaceutical composition for the use according to claim 1, wherein the composition comprises 0.5 to 5 wt%, preferably 1 to 4 wt%, more preferably 2 to 3 wt%, more preferably about 2.25 wt%, of an alginate.

4. Pharmaceutical composition for the use according to claim 3, wherein the alginate is a sodium alginate and / or a potassium alginate.

5. Pharmaceutical composition for the use according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises further 0.1 to 3 wt%, preferably 0.5 to 2 wt%, more preferably about 0.825 wt%, cellulose or at least one cellulose derivative.

6. Pharmaceutical composition for the use according to claim 5, wherein the at least on cellulose derivative is carboxymethyl cellulose (CMC) and / or hydroxyethyl cellulose.7 . Pharmaceutical composition for the use according to any one of claims 1 to 6 , wherein the pharmaceutical composition comprises further 10 to 25 wt% , preferably 15 to 20 wt% , more preferably about 18 . 25 wt% , of at least one diol , preferably propane- 1 , 2 -diol .8 . Pharmaceutical composition comprising a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture for use in the treatment of a wound and / or for promoting wound healing or wound closure , wherein the supernatant is obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMCs are subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and wherein the composition is applied topically on the wound in combination with a wound dressing comprising or consisting of alginate fibers .9 . Pharmaceutical composition for the use according to any one of claims 1 to 8 , wherein the wound is a chronic wound or an acute wound .10 . Pharmaceutical composition for the use according to any one of claims 1 to 9 , wherein the wound is a skin wound, preferably an ulcer, a burn, an abrasion, a laceration, a puncture wound, an incision or an avulsion .11 . Pharmaceutical composition for the use according to claim 9 or 10 , wherein the chronic wound is an ulcer, preferably a diabetic ulcer, more preferably a diabetic foot ulcer .12 . Pharmaceutical composition for the use according to any one of claims 1 to 11 , wherein the wound covers an area of less than 10 cm2, preferably of less than 8 cm2, more preferably of less than 5 cm2, more preferably of less than 4 cm2.13 . Pharmaceutical composition for the use according to any one of claims 1 to 12 , wherein the wound covers an area ofmore than 0.1 cm2, preferably of more than 0.2 cm2, more preferably of more than 0.5 cm2, more preferably of more than 0.8 cm2, more preferably of more than 1 cm2.

14. Pharmaceutical composition for the use according to any one of claims 1 to 13, wherein the composition is administered to the wound for at least 8 days, preferably for at least 10 days, more preferably for at least 14 days, more preferably for at least 21 days, more preferably for at least 30 days.

15. Pharmaceutical composition for the use according to any one of claims 1 to 14, wherein the composition is applied to the wound twice a day, preferably once a day, more preferably every second day, more preferably every third day, more preferably every fourth day, more preferably every fifth day, more preferably every sixth day, more preferably every seventh day .

16. Pharmaceutical composition for the use according to any one of claims 1 to 15, wherein the composition is applied to the wound once a week, preferably twice a week, more preferably three times a week, more preferably four times a week, more preferably five times a week, more preferably six times a week, more preferably daily.

17. Pharmaceutical composition for the use according to any one of claims 1 to 16, wherein the composition applied topically per cm2wound area comprises the components present in 1 ml of a supernatant obtainable by cultivating20x106to 40xl06PBMCs / ml, preferably 20xl06to 30xl06PBMCs / ml, more preferably 22xl06to 28xl06PBMCs / ml, more preferably 24xl06to 26xl06PBMCs / ml, more preferably about 25xl06PBMCs / ml, culture medium.

18. Pharmaceutical composition for the use according to any one of claims 2 to 17, wherein the alginate fibers comprise or consist of calcium alginate and / or sodium alginate, preferably calcium alginate and sodium alginate.19 . Pharmaceutical composition for the use according to any one of claims 2 or 18 , wherein the wound dressing comprises or consists of 70 to 90 wt% , preferably 75 to 85 wt% , more preferably around 80 wt% , calcium alginate and 10 to 30 wt% , preferably 15 to 25 wt% , more preferably around 20 wt% , sodium alginate .20 . Pharmaceutical combination comprising a ) a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture obtainable by cultivating PBMCs in a culture medium for at least 4 h, wherein the PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and b ) a wound dressing comprising or consisting of alginate fibers .21 . Pharmaceutical composition according to claim 16 , wherein the composition has a lyophilised form .22 . Kit comprising a ) a container comprising a composition, preferably a lyophili zed composition, comprising or consisting of a supernatant of a peripheral blood mononuclear cell ( PBMC ) culture , wherein the supernatant is obtainable by cultivating a PBMC cell culture for at least 4 h, wherein said PBMC cell culture is subj ected to ioni zing radiation at a dose of at least 10 Gy before or during cultivation, and b ) a container comprising an aqueous solution comprising 1 to 3 wt% of an alginate or a package comprising a wound dressing comprising or consisting of alginate fibers .

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