Activated leukocyte composition

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns a method for making an activated leukocyte composition involving human leukocyte incubation, exposure to hypotonic shock and addition of physiologically acceptable saline in an amount sufficient for the reconstitution of isotonicity, to the leukocytes. The group of inventions also concerns using: the activated leukocyte composition in preparing a drug for wound healing; a wound healing dressing containing the above composition.

EFFECT: group of inventions provides preparing the composition containing a 90 times increase of the leukocyte count as compared to the known state-of-the-art methods.

16 cl, 3 dwg, 6 tbl, 3 ex



[1] This application declares priority of the application under serial number 61/209298 filed March 5, 2009 and entitled "Activated leukocyte composition", and the application under serial number 61/211587 filed April 1, 2009 and entitled "Activated leukocyte composition for vascular disease", the disclosure of which is given in the present text by reference.


[2] the Process of wound healing involves the participation of white blood cells, also known as leukocytes. Leukocytes include lymphocytes, granulocytes and monocytes. There are three main types of lymphocytes: T-cells, B-cells and natural killer cells. T-cells and B-cells play an important role in the recognition of antigens in the body (Parkin, 2001). Natural killer cells (NK) identify infected cells by changes in the levels of major histocompatibility complex (GCG) and destroy the infected cells (Moretta, 2008). The involvement of lymphocytes in the healing process is largely due to their production of cytokines and growth factors (Keen, 2008). In the skin has been described a new class of gamma-Delta T-cells (Jameson, 2002. Havran, 2005). Among the various types of granulocytes significant are the neutrophils, basophils and eosinophils. Monocytes differentiate into macrophages, which are responsible for the destruction of�other defects of the organic substances of the tissues or penetration of foreign substances. Macrophages also secrete molecules that control inflammation and healing (Riches, 1996).

[3] the Process of wound healing occurs in three overlapping phases (Li, 2007; Broughton, 2006; Tsirogianni, 2006; Singer, 1999; Martin, 1997). The first phase is a stage of inflammation. It is characterized by the involvement of neutrophils and then monocytes in the wound area, where they kill and phagocytose bacteria (Agaiby, 1999).

[4] the Second phase of wound healing, known as the proliferative phase, includes the formation of new granulation tissue. Fibroblasts proliferate and migrate into the wound space and synthesize collagen and other components of the extracellular matrix (Greiling, 1997). At the same time there is angiogenesis, providing metabolically active new granulation tissue with nutrients and oxygen (Tonnesen, 2000). Keratinocytes from intact epidermis begin to migrate through a temporary matrix, and to multiply, which leads to the formation of new epithelial tissue (Kim, 1992).

[5] Remodeling is the third and final phase in wound healing. This phase is characterized by the differentiation of fibroblasts into myofibroblasts that contract and bring the wound edges closer to each other (Tomasek, 2002). Remodeling of the collagen fibers through the degradation and re-synthesis allows the wound to gain momentum La�due to the reorientation of collagen fibers (securely process is controlled by growth factors (Werner, 2003).

[6] the problem of the treatment of wounds is often exacerbated in patients with multiple pathologies, such as diabetes, coronary heart disease and hypertension. These diseases have a common effect of acute vascular complications due to different physiological States. Complications of wounds can lead to increased morbidity and mortality (Doshi, 2008).

[7] Conventional methods of treatment of wounds include debridement, antibiotic therapy and the imposition of various dressings (Magap, 2008; Fonder, 2008). Wounds that are resistant to traditional therapy, also called refractory wounds. These wounds lead to reduced quality of life and can lead to increased morbidity and mortality. Thus continues to exist a need in the compositions and methods effective in the healing of wounds.

Summary of the INVENTION

[8] One aspect of the present invention relates to a method of manufacturing an activated leukocyte composition (ALA) derived from blood (e.g., derived or obtained from a sample of whole blood). The method includes stages, at which the white blood cells, which can be obtained from samples of whole human blood, are first incubation during the period of time and at a temperature that allows the leukocytes become activated, which in preferred embodiments, sostavljae� from about 8 hours to about 20 hours, at room temperature. After incubation of the leukocytes are contacted with a physiologically acceptable aqueous solution such as, for example, sterile distilled water to initiate hypoosmotic shock, with subsequent contact of leukocytes in a state of shock with a physiologically acceptable saline solution to restore isotonicity. This activated leukocyte composition (ALA) can be used therapeutically. However, in some embodiments, separately, and essentially in parallel with the first incubation of leukocytes, plasma specimen, which can be obtained from the same or from a different sample of whole blood (e.g., from the same or from another person), in contact with the coagulant at a temperature approximately equal to 37°C simultaneously with the leukocyte incubation, which, in preferred embodiments, is from about 8 hours to 20 hours, followed by separation of the serum from the coagulated plasma specimen. Leukocytes was resuspended in serum samples collected from coagulated plasma, thus forming ALA. After the first incubation of the leukocytes can be further subjected to a second incubation for about 60 to 120 minutes at a temperature approximately equal to 37°C.

[9] Another aspect of the present invention relates to ALK, received�by the auditors from the blood. Activated leukocyte composition of the present invention includes, from the point of view of the leukocyte populations present in it, from about 40% to 90% granulocytes, from about 5% to 20% of monocytes and from about 5% to 30% of lymphocytes from the total number of leukocytes in ALA. As shown in the working examples, invent ALA can also be characterized and different from the known compositions from the standpoint of minimum yield of cells (relative to the sample of whole blood), the viability of cells, as well as minimum levels of activation of granulocytes, for example, as indicated CD lib. ALA may further contain residual levels of platelets (in an amount approximately equal to 46.8+/-39,2 (103/µl)), and erythrocytes (approximately equal to 0.1+/-0,06 (106/ál)) ALA. The population of lymphocytes may include from about 7% to 25% of b-cells (CD 19+), from about 20% to 30% of natural killer cells (CD3-/CD56+), from about 40% to 60% of T-cells (CD3+), from about 0.1% to about 30% of NKT cells CD3+/CD56+, from about 8% to about 20% of T-helper cells (CD4+/CD3+), and from about 20% to about 30% cells CD8+/CD3+. Cells can be suspended in a medium such as serum (which may be autologous or allogeneic to the recipient), or in other physiologist�Cesky acceptable isnormally liquids, suitable for storage and injection of the cells, as, for example, the solution used to restore isotonicity.

[10] Another aspect of the present invention relates to a method of stimulating healing of wounds which comprises administering or applying ALA to the wound in a different way.

[11] the Disclosed invention achieves several unexpected results against at least one known composition for wound healing containing the leukocytes. As demonstrated in the working examples of the present invention, the results include an increase in the yield and viability of cells and a higher percentage of activated granulocytes. Also believe that the described invention includes an unexpectedly higher percentage of activated monocytes and a relatively higher percentage of T CD4 cells compared with T cells CD8.

BRIEF description of the DRAWINGS

[12] Fig. 1 schematically depicts a first part of a representative system for the production of ALA compositions of the present invention, including bags A-C (Set-1) representing the bags or containers for storage of blood, where a bag contains red cell mass, collected from a donor; bag b contains the plasma, and the bag contains leukocytes (which, after initial separation from the whole blood to form a layer, which is usually called�tsya leukocyte film).

[13] Fig. 2 schematically depicts the second part of a representative system for the production of ALA compositions of the present invention, which includes a set of 7 bags, once deleted from the system bag And with erythrocytes, and the bags b and C of Fig. 1 is connected with bags 1-5 (Set of 2).

[14] Fig. 3A and 3B are graphs illustrating General trends in the expression of both CD62L and CD42b, which are indicators of cell activation.

DETAILED description of the INVENTION

[15] the Blood is defined here as whole blood or any of its components (e.g., plasma, leukocytes, platelets or red blood cells). The number of platelets and red blood cells that may be present in the ALA of the present invention, may be lower than the number that is present in whole blood.

[16] the Term "approximately" as used here in connection with any values (including the lower and upper limits of numerical ranges), as having any value acceptable range of deviation of +/-0.5% to +/-20% (and all values in between, e.g.,±1%, ±1,5%, ±2%, ±2,5%, ±3%, ±3,5%, ±4%, ±4,5%, ±5%, ±5,5%, ±6%, ±6,5%, ±7%, ±7,5%, ±8%, ±8,5%, ±9%, ±9,5%, ±10%, ±10,5%, ±11%, ±11,5%, ±12%, ±12,5%, ±13, ±13,5%, ±14%, ±14,5%, ±15%, ±15,5%, ±16%, ±16,5%, ±17%, ±17,5%, ±18%, ±18,5%, ±19%, ±19,5% and ±20%).

[17] the starting materials for the production of innovating ALA can be obtained from several sources. Whole blood and�and one or more of its components (for example, leukocytes and plasma) can be obtained from autologous or allogeneic sources. In one embodiment of the present invention, the blood sample taken from the patient, which ultimately will be treated with ALA, called in this document the autologous sample or source of the blood. In embodiments in which the source(s), i.e. blood or its components, obtained from individuals that are not planned by the recipients of ALA, which(e) is(are) allogeneic sample or a blood source, these starting materials can be obtained from the blood Bank. Samples can be investigated by blood Bank for blood group and rhesus factor (ABO, Rh), irregular antibodies to antigens of red blood cells and diseases transmitted through blood transfusion. In particular, the study can be conducted with use of the blood analyzer Abbott PRISM with antibodies against: hepatitis B and C, HIV 1/2, CTL and syphilis (HCV, HbsAg, anti-HIV ½ 0+; and anti-HTLV I/II). Samples can also be tested for HIV, HCV and HBV molecular methods (nucleic acid testing - TNC). Molecular screening can be performed using commercially available instruments, for example, TIGRIS system (Chiron).

[18] In these embodiments involving allogeneic sources samples can be obtained from donors with the same blood type as prepolar�ed recipient of ALA. Alternatively, as further set forth herein, the plasma samples may be obtained from blood donors AB+, leukocytes can be obtained from patients with blood group 0-. Patients with blood type AB+ are universal plasma donors, and patients with blood group 0 are universal donors of cells. Regardless of the source, all necessary processing of specimen(s) can be carried out without the need to use highly specialized equipment.

[19] the Preferred method of producing ALA compositions of the present invention is now described with reference to Fig. 1 and 2, which show a system that contains two sets of interrelated sterile infusion bags. The system is sealed so that the external environment are absent. In particular, the tube connecting the two sets will be connected into one system, using a sterile connecting device (for example, TSCD®-II, catalog number ME-AN, manufacturer Agito). In particular, to ensure compliance with standards of sterility connection(welding) and cutting of the tubes are made using special plates, preheated to approximately 300°C. Such a high temperature enhances the sterility of the weld procedure. To further ensure the sterility of St�RCA can be performed in a biological safety Cabinet class 100 inside the region of localization of class 100000.

[20] As shown in Fig. 1 and 2, the system contains two sterile kit bags. Kit 1 includes bags A, B and C, is a standard, commercially available set of three bags, typically used for blood transfusion. A sample of human blood, usually in the amount of approximately from 400 ml to about 550 ml, is collected in the blood Bank via venipuncture into vacutainers and placed in a bag, and then fractionary into its component parts using standard methods in bags A, B and C. for Example, a bag containing the blood sample was centrifuged. After centrifugation, the blood components are separated, for example, using the extractor of blood components production Baxter. Leukocyte film, which contains white blood cells, placed in a bag C, the plasma is placed in bag B, and the red blood cells remain in the bag A. Thus, as a result of this process the bag contains A red cell mass; bag b contains the plasma; and bag C contains leukocyte tape containing the leukocytes (and possibly residual plasma and red blood cells). In addition, blood components can be separated from whole blood using apheresis method known in the art.

[21] the Bag then A pulling away from the set consisting of three bags. As shown in Fig.2, the bags B and C are then attached to a custom-made infusion bags 1-5 Set-2) for the formation of the system, used for the manufacturing of activated leukocyte composition. As described above, welding is performed with a sterile connection device. The bag 1 comprises a first aqueous solution (for example, 200 ml of sterile distilled water) and used to expose cells leukocyte film hypoosmotic shock. This serves to lyse residual red blood cells that may be present. Bag 2 contains the second solution (e.g., 20 ml of buffered sodium chloride solution (8,91% NaCl, USP, or any other physiologically acceptable solution containing inorganic ions, organic osmolyte such as sucrose, or any combination thereof, for example, a solution of ringer's lactate (Hartmann), which serves to restore leukocytes isotonicity after hypoosmotic shock. When the sodium chloride solution was added to 200 ml of distilled water, it becomes a 0.9% solution of sodium chloride. Bag 3 contains the third solution (for example, approximately 60 ml of buffered solution of calcium chloride (1,17% CaCl dehydrate, USP), which affects the coagulation of plasma in the bag and to facilitate the separation of platelets and serum. Bag 4 and bag 5, each contains sterile filtered air, approximately 60 ml and 500 ml, respectively. Set UPA�byvaut as a single unit and sterilized with the use of high pressure steam, which greatly reduces the risk of developing a secondary infection for the patient.

[22] Leukocytes are then transferred from bag to bag 4 (or bag 5) and incubated, preferably in a vertical position to enable them to be activated. For the purposes of the present invention, the activation of the cells is defined as the process by which cells (leukocytes) provide the opportunity to become activated, but rather as a transition from a quiescent to a functionally active state, which is accompanied by the synthesis of biologically active substances or relocation of pre-synthesized substances from the cytoplasm to the cell membrane, or their release from cells. These substances may include proteins or polypeptides, lipids, sugars, oxygen radicals and other biochemical fragments that function as adhesion molecules, cytokines, growth factors, enzymes, transcription factors and receptors cell signaling and mediators. Upon detection and identification inside the cells or on the cell surface these molecules are called markers of activation.

[23] In some embodiments, the leukocytes were incubated, simply leaving them to stand at room temperature. For the purposes of the present invention, the room temperature refers to a temperature in the range from about 12°C to approximately 28°C, and in some vari�ntah from about 16°C to about 25°C. The period of incubation may vary depending on the temperature. The incubation time will be lower at elevated temperatures. The incubation time required for the activation of leukocytes, will be approximately inversely proportional to the temperature at which incubation is carried out. For example, in embodiments where the leukocytes leave to stand at room temperature, the incubation time is usually from about 90 minutes, and more from 2, 3, 4, 5, 6, 7 or 8 hours to about 20 hours. In preferred embodiments, the incubation time ranges from about 8 to about 20 hours. In a more preferred embodiment, incubation of cells occurs at a temperature from about 18°C to 24°C for from about 8 hours to 12 hours. In other embodiments, the incubation of cells includes exposure to heat, e.g. at temperatures above room temperature to about 37°C. the Period of incubation at elevated temperature is generally from 5 hours to about 24 hours.

[24] Referring again to Fig.2, leukocytes out of the bag C is moved to the bag 4, which is usually less than a bag of C (e.g., about 250 ml, about 500 ml). Also, the cells can be moved into another bag 500 ml (bag 5). To grant the ability to activate leukocytes, bag of 4 (or bag 5) �amemait in a vertical position and incubated for about 8 hours to about 20 hours at room temperature.

[25] After incubation of the leukocytes are subjected hypoosmotic shock that lyses the red blood cells. In preferred embodiments, hypoosmotic shock is performed immediately (i.e., after the completion of the previous stage without any intermediate stage or unnecessary delay, usually less than 2 minutes). Hypoosmotic shock may be initiated by moving the distilled water from the bag 1 in the bag 4 (or bag 5) containing leukocytes. Processing using hypoosmotic shock, as a rule, is carried out for approximately 45 seconds. Following this stage and, preferably, immediately thereafter, restore izotonichnost leukocytes by moving the sodium chloride solution from bag 2 to bag 4 (or bag 5). The ratio of the volume of sodium chloride solution to the cell suspension in water is typically about 1:10. The contents of the bag 4 is then transferred into the bag 1 (or leave in the bag 5) that is larger in volume than the bag 4. As described above, this composition is patentable in the methods can be used therapeutically.

[26] a Preferred embodiment includes at least one additional stage. Thus, in a separate step, which can be done simultaneously with the leukocyte incubation, the plasma is separated into platelets and serum using a coagulant, such l 2with subsequent centrifugation. Thus, in this typical embodiment, l2out of the bag 3 is transferred to bag B. the Bag B, which now contains the composition of plasma and l2as a rule , provide an opportunity to coagulate at a temperature of approximately 37°C. the Plasma remains in contact with the coagulant substantially the same period of time, during which leukocytes were incubated.

[27] After hypoosmotic shock and restore isotonicity of leukocytes and subsequent transfer of the contents of the bag 4 bag in 1 (or leave in the bag 5), and after coagulation of plasma in the bag B, the entire system consisting of 7 bags, centrifuge. In preferred embodiments, the centrifugation is carried out immediately after the leukocytes move in bag 1 (or leave in the bag 5) so as not to expose the cells to the effects of gemolizata. After centrifugation the supernatant from the bag 1 or bag 5) move in bag C, and leukocyte precipitate formed in the bag 1 or bag 5) as a result of centrifugation, was resuspended in approximately 20 ml to about 50 ml of serum from the bag B.

[28] In another step of a preferred embodiment of the present invention, the activated leukocytes can be incubated in the above-mentioned coagulated plasma before ETS�Ana final composition. Typically, this second period of incubation is carried out for 1-2 hours at 37°C.

[29] In other embodiments, the composition of ALA can be obtained from smaller volumes of blood samples, with a proportionate reduction in the volumes of all solutions and use smaller bags. Furthermore, the use of bags of different sizes produce different compositions of ALA. Even in these embodiments, the starting materials can be used for allogenic or autologous blood samples. The use of smaller volumes provides the Clinician with the ability to perform the blood collection offline, without using an external blood Bank, for example, in emergency situations, when treating patients with a healthy throughout the rest of the immune system, but suffering from some types of traumatic wounds (e.g., on the battlefield and in combat). In these embodiments, testing for infectious diseases and antigens may be omitted. However, in such cases patients with refractory wounds are not clinically acceptable blood donors for efficient production of ALA. If this situation occurs, alcohol will be produced from allogeneic donors with the tools described here.

[30] ALK of the present invention include leukocytes, such as granulocytes, monocytes and lymphocytes. Granulocytes include not�trofile, eosinophils and basophils. In the broadest sense, the population of leukocytes alcohol generally contains from about 40% to about 90% granulocytes, from about 5% to about 20% monocytes and from about 5% to about 30% lymphocytes. The specific number of cells may differ depending on the applied analysis techniques. If the analysis is carried out using a FAX (for example, using the analysis of scatter plot of light scattering at an angle in comparison with the forward scattering), the composition of leukocytes typically contains from about 55% to about 80% granulocytes, from about 5% to about 15% monocytes and from about 5% to about 30% lymphocytes, and in some embodiments is approximately 58-76% granulocytes; approximately 5-11% of monocytes and approximately 9-23% of lymphocytes. If the analysis is carried out using the analyzer Cell Dyn, composition of leukocytes typically contains from about 50% to about 90% granulocytes; from about 5% to about 15% monocytes; and from about 10% to about 25% lymphocytes. The lymphocyte subpopulation in ALA may support the following cells in the total limits as follows: from about 7% to about 25% B-cells (CD19+); from about 20% to about 30% NK cells (CD3-/CD5+), approximately 40% to approximately 60% of T-cells (CD3+), from about 0.1% to about 30% of NKT cells CD3+/CD56+, from about 8% to about 20% of the cells T-helper cells (CD4+/CD3+), and approximately 20% to approximately 30% of the cells CD8+/CD3+. In preferred embodiments, the lymphocyte subpopulation is enriched by at least 9% of CD56+ cells (CD3-/CD56+; CD3+/CD56+; CD3+/CD56+/CD8+), the number of T-lymphocyte helper cells (CD4+/CD3+) is reduced to less than 20% and/or proportion of cells of T-helper to T-suppressor (CD4+/CD3+: CD8+/CD3+) is less than 0.8.

[31] Patients suffering from wounds can be with physiological complications or healthy in all other respects. For example, in connection with an already weakened metabolic systems of patients with diabetes and other medical complications are candidates for ALA produced from heterologous genes, as their own leukocytes may not be optimal for this procedure. Nevertheless, a healthy throughout the rest of the patients, as in patients with injuries, are also good candidates for ALA compositions of the present invention.

[32] the Present invention is suitable for stimulating the healing of many types of wounds. Although in practice the invention may be used in combination with other treatment modalities, it does not require the use of additional� ways to achieve effective healing. The inventors propose the use of ALA for any type of wound and don't foresee any constraints on the types of injuries that can be treated. Ease of application, for example, using a standard syringe or similar device for use makes inventing songs ALA safe and easy to use.

[33] Injuries that are treatable by the invention as a rule are the punctures, cuts or ruptures of living tissues. Wounds of the skin can penetrate into the epidermis, dermis or in the event of an injury to full-thickness skin, it can penetrate into subcutaneous tissue. Thus, typical examples of injuries that are treatable by the compositions and methods of the present invention include decubital ulcers or bedsores, diabetic ulcers, deep wounds of the sternum, for example, after the operation open heart (to the great saphenous vein after coronary revascularization and selection of the great saphenous vein), and postoperative wound after abdominal surgery and any other type of surgery. Other wounds resulting from trauma, such as from firearms, stab wounds, or wounds from the effects of any other object capable of causing a cut or tear of the skin. The wounds of the oral cavity (e.g., teeth), as well as injuries that occur as a side� the effect of treatment or as a symptom of various pathologies (e.g., sores associated with Kaposi's sarcoma), as well as internal wounds (e.g., anal fissures and wounds or injuries of the gastrointestinal tract, such as ulcers in the stomach or intestines) may also be treatable using the present invention.

[34] ALA may also be used to treat any wounds complicated by vascular insufficiency. Vascular insufficiency, for the purposes of the present invention, refers to the insufficient circulation of blood as a result of inadequate perfusion in the affected areas. This failure may be caused by trauma (for example, damage to the vasculature adjacent to the bone fracture), or various pathologies (e.g., diabetes mellitus, and atherosclerosis). In any case, vascular insufficiency induced by injury or disease, reduces the likelihood of effective wound healing. To improve wound healing in these patients may be suitable alcohol, and it should be administered in accordance with the methods described herein. In addition, the algorithms of treatment should not be limited to the severity and type of wound or the extent of vascular insufficiency. ALA may be more effective in patients with the most severe wounds and vascular insufficiency.

[35] In General, application of the activated leukocyte composition OS�carried out using one or more injections of ALA directly into the wound or tissue, surrounding the wound. ALA can be directly applied to an open wound.

[36] For injection into a wound it is preferable to use a syringe Luer-Lok or any other commercially available type of syringe, which has the locking mechanism between the syringe and the needle. The biological space of the wound, in particular bedsores, are often limited. When injected into the wound there is a risk of pressure, which causes the disengagement of the syringe from the needle. The use of the syringe fixing mechanism eliminates this risk.

[37] If an injection into the tissue of the wound is not possible, alcohol can be applied directly to the wound. Applique can be performed using direct application with a syringe or pipe.

[38] ALA may be deposited on the wound or around the wound with bandages. Dry dressings include gauze and bandages, non-adhesive mesh, membranes and films, foams, and tissue adhesives. Water-retaining barrier dressings include pastes, creams and ointments, impermeable or semi-permeable membrane or film, hydrocolloids, hydrogels and combinations of these products. Biologically active dressings include antimicrobial dressings, interactive dressings, one-component biological dressings and combined products. In some variations�tah wound tamponiruut sterile gauze, soaked in alcohol. Dressings, for example, such as sterile gauze pads may be impregnated with such a composition as a solution of ringer's lactate (Hartmann), originaldatabase bandage, polyurethane dressings or bandage of carboxymethylcellulose are used to cover wounds with subsequent application of a dry dressing. If the wound of the subject is extremely infected, it can be applied dressings with silver, such as Silverlon. The choice postinjection dressings based on the doctor's decision. Commercial availability, previous history of clinical success and patient tolerance are all factors to consider when choosing a bandage on the wound. Dressing can be periodically removed, for example, usually, after about 24 hours, in order to irrigate the wound, for example, sterile water and soap.

[39] In another embodiment, the composition may be placed on physiologically inert and/or resorbable matrix or frame (e.g., collagen) and inserted into the wound by pressing (tamponade). This ensures continuous delivery of ALA to the place that provides the benefits to the patient so that the cells have a longer period on the site.

[40] ALA compositions can be introduced into the wound once or several times, for example, after 4 weeks, as�Ko doctor determines will there be a re-application. The factors that could be taken into account include the increased size of the wound (width, length and depth), presence of sepsis, hyperthermia, or any other sign or symptom indicating untreatable infection, using which justified re-treatment. In addition to repeated therapy at any time that the physician considers appropriate may be appointed in the direction of surgical treatment.

[41] ALA may be used in conjunction with any other traditional treatment of wounds, such as warming (therapeutic heat), electrical stimulation, magnetism, laser phototherapy, cycloidal vibration therapy and ultrasound. It can also be used with biological therapy, such as licencetype, skin substitutes, culture keratinocytes (Epicel, Genzyme biosurgery), the replacement of human skin (Dermagraft, Smith and Nephew Inc.), the treated dermis obtained from a cadaver (Alloderm, Life Cell Corporation), double-layered skin equivalent (Apligraf, Organogenesis Inc.), TransCyte (Smith and Nephew Inc.), growth factors (PDGF (platelet growth factor) is currently the sole growth factor, a licensed for external use), and fibrin sealant (glue). In some embodiments, ALK is used in conjunction with vacuum therapy (VAC), which is a commercial�ski available therapy RAS production KCI. VAC promotes wound healing by activating the negative pressure wound. In these embodiments, ALK is preferably applied to the wound before VAC therapy. In other embodiments, ALK is used in conjunction with hyperbaric therapy (Thackham, 2008). For example, ALA may be applied to the wound before the patient will receive hyperbaric therapy. ALA may also be used in combination with low-energy shock wave therapy (e.g., pulses approximately equal to 0.1 MJ/mm2; 5 Hz) per cm of length of the wound). See, for example, Dumfarth, et al., Ann. Thorac. Surg. 86:1909-13 (2008).

[42] After treatment of the wound can be assessed by measuring the length, width and height. Typically, the wound is considered healed when all measurements of these parameters are insignificant. ALA can also provide an analgesic effect.

[43] Activated leukocyte composition is particularly suitable for the treatment of wounds, including diabetic foot ulcers and decubital ulcers. Decubital ulcers are pressure ulcers, caused by obstruction of blood flow, usually due to prolonged pressure on a single area (Berlowitz, 2007). Decubital ulcers cause morbidity and mortality in the elderly. At least 48% pressure ulcers grade IV remain unhealed after one year of treatment (Girouard, 2008). Patients suffering from d�cubital ulcers, also often have comorbidities, such as diabetes and hypertension. These pathology further complicate successful treatment decubital ulcers.

[44] In one embodiment for the treatment decubital ulcers composition aspirated into a sterile syringe of any size, using needles 18-gauge (18G). Aspiration is carried out slowly in order to minimize cell damage. Although the size of the syringe and needle are not a restriction, a large-gauge needle is preferred for aspiration. This facilitates movement and reduces cell damage.

[45] Application of the activated leukocyte composition on the ulcer comprises injecting the composition into the wound. Can be used the entire sample from the syringe and, if on the basis of clinical parameters need to be installed, the physician can select the introduction of additional ALC.

[46] 18G Needle used for aspiration, exchanged for the needle size in the range of 22-35G. ALA may be introduced by injection into the wound in different locations. In one embodiment, the injection is administered at a distance of approximately one to approximately three inches apart throughout the length of the wound. At each injection site are 0.1 to 0.3 ml ALC.

[47] In another embodiment, the entire contents of the syringe can be entered at one time in the only place within the wound.

[48] the Aspect(s) of the present invention will be described in accordance with the following non-limiting examples.

[49] Example 1 - Analysis of cell activation

[50] Activated leukocyte composition made in accordance with a preferred variant implementation of the present invention was quantified by analysis of different markers on the cell surface. The increase in aggregation of platelets or monocytes, or granulocytes is a sign of activation of monocytes and granulocytes through the expression of selectin P. CD62L is a plasma membrane protein that is lost during the activation and, consequently, decreases during cellular activation. CD42b is a marker of platelet activation, is involved in the coagulation process as a factor of aggregation. It interacts with extracellular matrix and adhesion molecules, and is also used in the present invention as an indicator of activation of monocytes and granulocytes.

[51] For samples at three time points selected cells: fresh leukocyte film (Swlp); incubated leukocyte film (Lai); and a final activated leukocyte composition (OP) (table 1).

Table 1
The cell surface markers indicating activation of leukocytes
% CD62L positive cells
% CD42b positive cells

[52] Svlp taken immediately after preparation of leukocyte film. Lai was taken after the first incubation period and the OP are taken from the final product. At each time point, cells mark specific monoclonal antibodies (allophycocyanin (APC), conjugated anti-CDH, phycoerythrine (PE), conjugated anti-042b and fluorescein-isothiocyanate (FITZ), conjugated anti-62b antibodies, and then analyzed using a FAX.

[53] To�ETCI from each time point were washed FAX with staining solution (PBS, 2% normal mouse serum; 0.02% of sodium azide), selected the sample and stained according to the staining Protocol. Briefly, a 0.5× (106/ál) of cells were incubated with the appropriate monoclonal antibodies for 30 min at room temperature (CT) or at +4°C in the dark. After incubation, the cell composition treated with buffer for lysis of red blood cells, washed, and then, finally, was resuspended in FSB for susceptibility to FAX. For CD62L positive (+) staining, cell composition were incubated with anti-SA (AFC) and anti-human CD62L (FITZ) antibodies at +4°C. For staining of CD42b+ cellular composition were incubated with anti-human ER (AFC) and anti-human CD42b (PE) at room temperature (CT). A negative control of cell composition were incubated with anti-human CD 14 and control the corresponding isotype in appropriate conditions. Associated with cell scattering and fluorescence analyzed using the analyzer "FAX Calibur" (Becton Dickinson). Monocytes defined as CD14-positive cells, and granulocytes identified by their characteristic properties of light scattering. CD62L or CD42b positive cells is defined as the percentage of monocytes and granulocytes with fluorescence greater than the threshold set by monocytes and �ranolazine, incubated with the appropriate antibody isotype control.

[54] As shown in Fig. 3A and 3B and Table 1, the results are consistent with the expectation that the expression of CD62L will decrease with the progression of activation and the level of CD42b will increase with the progression of activation. These data demonstrate that the leukocytes become activated.


[55] Tables 2 and 3 show the cellular structure of the final ALA, as determined by analysis by the analyzer Cell Dyn. Cells analyzed after resuspension of activated leukocytes in serum. Viable cells were stained using the exclusion trypan blue and observed under the microscope.

Table 2
The composition of the activated leukocyte composition
Platelets (103/ál)Erythrocytes (106/ál)Leukocytes (103/ál)
The final concentration in ALA46,8od
Standard deviation39,20,063,8
Table 3
The composition of leukocytes in ALA
Neutrophils,%Basophils, %Eosinophils, %Monocytes, %Lymphocytes, %
% end of ALA65,51,64,69,118,5
Standard deviation8,20,332,14,1
Ranges 52-781-21-96-1213-24

[0056] the results of the study by the analyzer Cell Dyn, as shown in Table 2, show that alcohol contains platelets in the amount to 46.8+/-39,2 (103/μl), red blood cells in an amount of 0.1+/-0,03 (106/ál) and leukocytes in the amount of 6.8+/-3,8(103/µl). Based on the analysis of Cell Dyn, it was also found that the composition of leukocyte ALK (as shown in Table 3), contains 52%-78% neutrophils, 1-2% basophils, 1-9% eosinophils, 6%-12% of monocytes, and 13% -24% of lymphocytes, including the standard deviation. The ranges shown in these tables represent the high and low results after you run 8 separate analyses using the analyzer Cell Dyn.

[57] Example 3

[58] the Comparison of the proposed method and the existing prior art process.

[59] to highlight various unexpected advantages associated with the present invention, its variants is compared with the process described in U.S. patent 6146890, with Danone ("Danone").

[60] the Variants of embodiment of the invention

[61] Referring to Fig. 1 and 2, immediately after the separation of whole blood on its main components, i.e. red cells, plasma and leukocyte film, leukocyte film is moved from bag to bag 4, and incubated for 12 cha�s ±2 hours at room temperature. This phase is followed by the displacement of distilled water from the bag 1 in the bag 4 (or bag 5) for the purpose of the procedure hypoosmotic shock (resulting in hemolysate). This procedure is carried out for approximately 45 seconds. Immediately after a buffered sodium chloride solution contained in the bag 2, is moved to the bag 4 (or bag 5) to restore isotonicity to the leukocyte membrane (leukocytes).

[62] Together with the leukocyte incubation of the film described above, a buffered solution of calcium chloride out of the bag 3 is moved to the bag containing the portion of the plasma to ensure the coagulation of plasma. This process provides the opportunity to take 12 hours ±2 hours, plus an additional short period of time, during which the leukocytes are subjected hypoosmotic shock, and then to restore isotonicity.

[63] Immediately after the restoration of isotonicity, the whole set of bags are centrifuged (typically, from about 8 to 10 minutes), followed by the separation of cells from gemolizata. The cells exposed to gemolizata for a minimal period of time, i.e. for about 10 minutes. After centrifugation the supernatant gemolizata from the bag 1 or bag 5) throw away and add a fresh environment to bag 1 or bag 5), followed by incubation for at�to be about 1-2 hours at 37°C. After incubation, the cells are subjected to a stage of the washing.

[64] Comparative (current level of technology) procedure.

[65] In accordance with the teachings of Danone, leukocyte film is subjected hypoosmotic shock immediately after processing whole blood and split it into 3 main components, i.e., red cells, plasma and leukocyte film. Thus, in sharp contrast to the present invention, the procedure Danone does not entail leukocyte incubation of the film after the separation of the main components of blood and before leukocyte film is subjected hypoosmotic shock. Hypoosmotic shock is carried out while leukocyte film contained in bag PB3.

[66] In a separate step, and after hypoosmotic shock buffered solution of calcium chloride moves out of the bag PB6in bag PB2that contains the plasma, with subsequent deep freezing of plasma in the bag PB2within 10 minutes, and then put it in a water bath at 37°C for an additional 30-minute incubation period. During this time the faction of leukocyte film, which was subjected hypoosmotic shock, provide an opportunity to settle, whereas the leukocytes leave under the influence gemolizata.

[67] After the completion of casanovapockets coagulation, which in total lasts approximately 40 minutes, the whole system bags centrifuged, and the hemolysate is discarded. Thus, cells exposed to gemolizata for at least approximately 55 minutes (i.e. time period, which includes a 40-minute settling period, which coincides with the coagulation of plasma, and an additional 15 minutes centrifugation). In contrast, in the method of the invention, leukocytes was centrifuged immediately for about 5 minutes and, thus, the hemolysate was subjected to a much shorter time, i.e., less than 10 minutes.

[68] After the supernatant from the bag PB3throw, it adds a fresh environment, and then move all the suspension back in RV7which is then incubated for approximately 17 hours at 37°C. After incubation cells were washed 3 times. In contrast, in embodiments of the present invention, the incubation period is carried out in coagulated plasma within 1-2 hours, and washed only once.

[69] Before conducting the above-mentioned incubation, all of the previous steps is carried out in bags for transfusion, which are bags that were collected whole blood. In contrast, embodiments of the present invention is fixed and�the use of infusion bags, generally used for the administration of intravenous solutions such as saline.

[70] Procedure Danone was performed 4 times and the obtained results were compared with the results obtained in the variants of implementation of the present invention. The results presented below are averaged.


[72] the Total number of white blood cells (leukocytes) obtained from standard units of blood, calculated as the final concentration multiplied by the final volume of the cell suspension, is determined for each batch of whole blood. Average results for 111 batches of whole blood, processed in accordance with the method of the present invention, and four parties that have been processed in accordance with the procedure disclosed in Danone, are shown in Table 4.


The present invention (n=111)
Table 4
The output of the leukocytes per unit of blood (the present invention gave the number of cells ~ 90 times higher)
The number of batchesLeukocytes (×106)
AverageStandard deviation (SO)
Danone (n=4)54

The results show that in the result of the method of the present invention from a standard unit of blood (about 450 ml), get the total number of leukocytes in approximately 90-fold increase compared with the procedure Danone. In General, as a result of the present method is used for the output of white blood cells, at least about 100×106, 125×106, 150×106, 175×106, 200×106, 225×106, 250×106, 275×106, 300×106, 325×106, 350×106, 375×106, 400×106, 425×106, 450×106, 475×106, 500×106or higher on a standard unit of blood (including all sub-bands).

[74] the Number of viable cells contained in the total cell population, expressed in percentages, measured by the method of exclusion trypan blue. Cells are counted in hemocytometer Newbauer after weighing in trypan blue (in the ratio 1:1) to estimate the number of cells and percent viability. The results are presented in Table 5.


Table 5
Procedure Viability (% live cells)
AverageStandard deviation (SO)
The present invention (n=111)98%0.02%
Danone (n=4)77%6%

The data of Table 5 demonstrate that, in addition to lower output (as shown in Table 4), the procedure Danone resulted in a cell suspension with a significantly lower viability. That is, almost a quarter (23%) product consisted of dead leukocytes (more than 10-fold excess of the percentage of dead cells). In contrast, almost all leukocytes treated in accordance with the method of the invention, were deemed viable. In General, patentable ALA may contain at least, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more viable leukocytes on the basis of the total number of these cells leukocytes in ALA (including all sub-bands).

[76] the Expression of CD11b marker of activated granulocytes measured by flow cytometry and presented as percentage of CD11b positive cells in the population of granulocytes (CD15-positive cells). Cells selected from to�final product together stained with anti-CD11b, conjugated with fluorescein isothiocyanate (FITZ), and anti-CD15, conjugated with phycoerythrin (PE), and analyzed by flow cytometer "FAX Calibur" (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Analyze CD11b expression on granulocytes in the cells of the 81 party of the final product produced in accordance with the proposed method, and 3 batches of the final product produced in accordance with the procedure Danone.


Table 6
The expression of CD11b on granulocytes

Procedure% granulocytes expressing the activation marker CD lib
AverageWITH (Standard deviation)
The present invention (n=81)84,66,2
Danone (n=3)46,99,2

According to the data contained in Table 6, the proposed method of the invention provides for�year-twice the yield of activated granulocytes in percentage compared with the procedure Danone. Overall, alcohol of the present invention can contain at least, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% or higher, CD11b(+) granulocytes, relative to the overall population of granulocytes in ALA (including all sub-bands).

[78] the Results of comparative experiments show that the present disclosure of the invention achieves at least 3 unexpected results in comparison with the process disclosed in the work of Danone, namely greater yield of viable cells, a higher percentage of viable cells, as well as a higher level of activation of granulocytes, as demonstrated by higher expression of the activation marker CD11b. These increases are substantial and unexpected.

[79] List of publications cited in the application

[80] J. Li, et al., Pathophysiology of Acute Wound Healing. Clinical Dermatol. 2007 Jan-Feb; 25(1):9-18.

[81] G. Broughton 2nd, et al., Wound Healing: An Overview. Plast Reconstr Surg. 2006 Jun; 117(7 Suppl):le-S-32e-S.

[82] AK Tsirogianni et al., Wound Healing: Immunological Aspects. Injury. 2006 Apr; 37 Suppl 1:S5-12. Epub 2006

[83] Singer AJ, et al., Cutaneous Wound Healing. New Eng. J. Med. 1999; 341(10):738-46.

[84] P. Martin, Wound Healing - Aiming For Perfect Skin Regeneration. Science 1997; 276(5309):75-81.

[85] Agaiby AD, et al., Immuno-Inflammatory Cell Dynamics During Cutaneous Wound Healing. J. Anat. 1999 Nov; 195 (Pt 4): 531-42.

[86] Riches DWH, Macrophage Involvement In Wound Repair, Remodeling and ®brosis. In The Molecular and Cellular Biology of Wound Repair, (1996) lnd edn (ed. Clark RAF), pp.95±141. New York, London: Plenum Press.

[87] D. Greilin, et al., Fibronectin Provides a Conduit for Fibroblast Transmigration from Collagenous Stroma into Fibrin Clot Provisional Matrix. J. Cell. Sci. 1997;110:861-70

[88] Tonnesen MG, et al., Angiogenesis In Wound Healing. J. Investig. Dermatol. Symp. Proc. 2000; 5(1):40-6.

[89] Kim JP, et al., Mechanisms of Human Keratinocyte Migration on Fibronectin: Unique Role of RGD Site and Integrins. J. Cell. Physiol. 1992; 151:443-50.

[90] Tomasek JJ, et al., Myofibroblasts and Mechano-Regulation of Connective Tissue Remodelling. Nat. Rev. Mol. Cell. Biol. (2002) 3:349-363

[91] Werner S, et al., Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev. 2003 Jul; 83(3):835-70.

[92] D. A. Keen, Review of Research Examining the Regulatory Role of Lymphocytes in Normal Wound Healing. J. Wound Care. 2008

[93] J Jameson, et al., A Role for Skin - T Cells in Wound Repair. Science 296: 747-749, 1992.

[94] Havran WL, et al., Epithelial Cells and Their Neighbors. III. Interactions Between Intraepithelial Lymphocytes and Neighboring Epithelial Cells. Am. J. Physiol. Gastrointest Liver Physiol. 2005 Oct; 289 (4):G627-30.

[95] J Parkin, et al., An Overview of the Immune System. Lancet. 2001; 357:1777-1789.

[96] Moretta A, et al., Human NK Cells: From HLA Class I - Specific Killer Ig-like Receptors to therapy of Acute Leukemias. Immunol. Rev. 2008 Aug; 224:58-69.

[97] BM Doshi, et al., Wound Healing From a Cellular Stress Response Perspective. Cell Stress Chaperones. 2008 Dec; 13 (4):393-9.

[98] Moran GJ, et al., Antimicrobial Prophylaxis for Wounds and Procedures in the Emergency Department. Infect. Dis. Clin. North Am. 2008.

[99] Fonder MA, et al., Treating the Chronic Wound: A Practical Approach to the Care of Nonhealing Wounds and Wound Care Dressings. J. Am. Acad. Dermatol. 2008 Feb; 58 (2): 185-206.

[100] Thackham JA, et al., The Use of Hyperbaric Oxygen Therapy to Treat Chronic Wounds: A Review. Wound Repair Regen. 2008, 1998 May-Jun;16(3):321-30.

[101] DR Berlowitz, et al., Are all pressure ulcers the result of deep tissue injury? A Review of the Literature. Ostomy Wound Manage. 2007 Oct; 53 (10):34-8.

[102] K. Girouard, et al., The symptom of pain with pressure ulcers: a review of the literature. Ostomy Wound Manage. May 2008; 54(5):30-40, 32.

[103] All publications cited in the specification, both patent publications and non-patent publications, indicate the level of qualification of specialists in the field of technology to which this invention belongs. All of these publications are included in this document as a reference to the same extent as if each individual publication was specifically and individually indicated included as a reference.

[104] Although the present invention has been described with reference to specific options, you should understand that these options are only illustrative of the principles and applications of the present invention. Therefore, it should be understood that can be made numerous modifications to the illustrative embodiments and that can be developed other schemes without deviation from the essence and scope of the present invention, as defined by the appended claims of the invention.

1. A method of manufacturing an activated leukocyte composition, including:
(a) incubation of human leukocytes with:
(i) a temperature from approximately 12°C to 28°C for 90 minutes 2, 3, 4, 5, 6, 7, 8 or 12-20 hours, or
(ii) the temperature to approximately 37°C for about 5-24 hours;
(b) exposure of cells from step a) hypoosmotic shock; and
c) adding to the leukocytes of step b) physiologically acceptable salt Sol�RA, containing inorganic ions, organic osmolyte or combinations thereof, in an amount sufficient to restore isotonicity.

2. A method according to claim 1, further comprising contacting a separate sample of human plasma with a coagulant to obtain serum, where the white blood cells and plasma can be obtained from a single donor or from multiple donors, and mixing of cells of step (C) with serum.

3. A method according to claim 1, characterized in that the leukocytes are getting from a donor with blood group 0, RH negative, get a plasma from a donor with blood group AB, RH positive.

4. A method according to any one of claims. 1-3, characterized in that hypoosmotic shock comprises contacting leukocytes with water, where physiologically acceptable saline solution is a solution of sodium chloride.

5. A method according to claims. 1-3, characterized in that it further comprises the incubation of cells in serum at a temperature of from 29.6°C to 44.4°C.

6. Activated leukocyte composition for the treatment of wounds, obtained by the method according to claim 1, containing:
a) from 40% to 90% granulocytes;
b) from 5% to 20% monocytes; and
c) from 5% to 30% lymphocytes.

7. Activated leukocyte composition for the treatment of wounds, obtained by the method according to claim 1, containing:
a) at least 95% of viable cells at the basis of the total number of these� cells leukocytes to activated leukocyte composition; and/or
(b) at least 75% of CD11b(+) granulocytes relative to the overall population of granulocytes in an activated leukocyte composition.

8. A composition according to any one of claims. 6 or 7, where the method of obtaining the composition additionally comprises contacting a separate sample of human plasma with a coagulant to obtain serum, where the white blood cells and plasma can be obtained from a single donor or from multiple donors, and mixing the leukocytes of step c) with the serum.

9. A composition according to any one of claims. 6-7, where granulocytes contain:
(i) from 52% to 78% neutrophils;
ii) from 1% to 9% eosinophils; and
iii) from 1% to 2% basophils.

10. A composition according to claim 6, where the lymphocytes contain:
(i) from 7% to 25% of b-cells (CD 19+);
ii) from 20% to 30% NK cells (CD3-/CD56+);
(iii) 40% to 60% of T cells (CD3+);
iv) from 0% to 30% of NKT cells (CD3+/CD56+);
(v) from 8% to 20% of the cells T-helper cells, (CD4+/CD3+); and
(vi) from 20% to 30% of the cells CD8+/CD3+.

11. A composition according to any one of claims. 6-7 for use as a medicament.

12. A composition according to any one of claims. 6-7 for use as an agent for wound healing.

13. The use of an activated leukocyte composition according to any one of claims. 6-12 in the manufacture of drugs for the treatment of wounds.

14. The use according to claim 13, wherein the wound is decubital ulcer, pressure ulcer, ulcer of lower limb diabetic patient, deep sternum wound, postoperative�innovative wound, refractory postoperative wound area of the body, the wound of the great saphenous vein after selection of the great saphenous vein, the wound resulting from trauma, anal fissure or a venous ulcer.

15. Bandage for treatment of wounds containing a composition according to any one of claims. 6-10.

16. Physiologically inert and/or resorbable matrix or scaffold for the treatment of wounds containing a composition according to any one of claims. 6-10.


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2 tbl, 2 ex

FIELD: medicine.

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2 ex

FIELD: medicine, pharmaceutics.

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2 cl, 2 tbl, 1 ex

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EFFECT: method makes it possible to increase the output of fibrin-monomer.

2 ex

FIELD: medicine.

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1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: with underlying conventional treatment, a leukocyte serum preparation is used that is produced by incubation of 20-30 ml of a patient's whole blood bottled in three sealed flasks in a thermostat at a temperature of 37-38°C for 24, 48 and 66-68 hours respectively; then the flasks are removed from the thermostat; the leukocyte serum is aspirated by a sterile syringe; the leukocyte serum preparation prepared at different times is introduced into the patient three times subcutaneously in a dose of 2-5 ml daily or triduan.

EFFECT: method provides higher clinical effectiveness and reduced length of treatment.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: mixture of 0.5% Novocaine or 0.25% Lidocaine 19.0ml and venous autoblood 1.0 ml is administered into the acupuncture points fu-tu and zu-san-li once every day or every second day in total number of blocks 8-16, in an amount of 5 ml into each point at a penetration depth of 1.5-2.0 cm.

EFFECT: reducing pain syndrome and oedema of paraarticular tissues, improving support ability of the lower extremities and knee joint functions by improving microcirculation in the lower extremities.

4 ex