Method for therapy of hair loss by intradermal administration of homogenized fibrin matrix

Abstract

FIELD: dermatology.SUBSTANCE: used to treat hair loss. The patient's blood is collected into a sterile test tube, the blood is centrifuged, and a homogenized fibrin matrix is introduced. In this case, venous blood is taken from the patient into a vacuum cylindrical test tube made of borosilicate glass. A single centrifugation of the obtained venous blood is performed at a relative centrifugal force of 900–1200 for 5–10 minutes. The fibrin-platelet mass is separated from erythrocytes under aseptic conditions. The fibrin clot is homogenized. The finished homogenized fibrin matrix is collected and subsequently injected intradermally or subcutaneously into the hair loss area 1–2 times a month, for a course of 8–12 procedures.EFFECT: method ensures the induction of neoangiogenesis, activation of proliferative processes in the cells of the dermal papilla and stimulation of the transition of hair follicles from the telogen phase to anagen due to the controlled release of growth factors from a finely dispersed fibrin matrix obtained by mechanical homogenization and centrifugation of autologous venous blood with the most optimal parameters.3 cl, 14 dwg, 3 ex

Description

[0001] The invention relates to the field of medicine, specifically to dermatovenereology, cosmetology, trichology, restorative medicine, and plastic surgery, specifically to a method for producing a cellular fibrin substance and a method for administering it. The proposed method relates to cellular biotechnology and can be used for injection therapy of non-scarring (androgenetic alopecia, seborrheic alopecia, diffuse alopecia, and focal alopecia) and scarring hair loss, as well as for improving the survival rate of hair grafts after hair transplant procedures.

[0002] Alopecia (hair loss) is a group of diseases characterized by:

[0003] 1) visible excess (more than usual) amount of hair loss per day (lost hair is more often visualized by patients in the bathtub drain, on a comb when combing, when drying hair with a hair dryer, on clothes during the day);

[0004] 2) uniform thinning of hair, its thinning and translucence of the scalp;

[0005] 3) areas of hair loss.

[0006] Alopecia can be accompanied by hair loss, hair diameter, and changes in hair structure. Currently, the generally accepted classification of alopecia is cicatricial and non-cicatricial. Among the non-cicatricial alopecias, the most common are androgenetic alopecia, diffuse alopecia, seborrheic alopecia, and alopecia areata. Specialists in this field know that alopecias lead to a decrease in the thickness of the subcutaneous fat in the scalp, decreased vascularity, thinning of the dermis, thickening, replacement by fibrous tissue, and decreased skin mobility and movement.

[0007] Currently, oral and oral therapies, topical therapy, and laser physiotherapy are widely used for alopecia treatment. However, treatment results vary, and the need for daily medications, decreased quality of life, and the risk of developing side effects are contraindications for many patients. Injectable therapy methods have now been standardized, and cell-based technologies—injections of the patient's autologous plasma, including platelet-poor or platelet-rich plasma, with a high or low leukocyte count, and calcium-activated or non-activated—are widely used in both monotherapy and combination therapy.

[0008] The physiological effect is due to the liquid portion of autologous plasma, which contains proteins, carbohydrates, vitamins, macro- and microelements, and platelet secretory products, namely platelet growth factors. Under the influence of growth factors, active cell division of fibroblasts, endothelial cells, and epithelial cells occurs in the skin. This results in the formation of an extracellular matrix, the growth of new vessels, and the maturation and remodeling of connective tissue. The severity of the effect varies depending on the platelet count in the prepared autologous plasma preparation. It has been proven that the best stimulating effect of autologous plasma is achieved with autologous platelet-rich plasma therapy, when the platelet concentration in the plasma is greater than 1,000,000 per µl. However, the fibrin matrix remains the connecting link between the cells.Platelet-rich plasma is injected intradermally via papular, superficial, median or deep nappage, ensuring short-term presence of the obtained material in the skin.

[0009] State of the art. Several basic methods for obtaining autologous plasma from a patient's blood are currently known in global medical clinical practice:

[0010] 1) using a single centrifugation at high speed to obtain blood plasma without cellular elements. This method;

[0011] 2) using a single centrifugation at low centrifuge speeds to obtain platelet-rich plasma without their further activation (the concentration of platelet cells is 1.5-2 times higher than their concentration in the patient’s blood);

[0012] 3) using a single centrifugation at low speeds to obtain plasma enriched with platelets with their subsequent activation with calcium ions;

[0013] 4) using double centrifugation to obtain platelet-rich plasma without their further activation (the concentration of platelet cells is 3-5 times higher than their concentration in the patient’s blood);

[0014] 5) using double centrifugation to obtain plasma rich in platelets with their subsequent activation with calcium ions (the concentration of platelet cells is 3-5 times higher than their concentration in the patient’s blood).

[0015] The disadvantages of current known methods for obtaining autologous plasma include the fact that some of them show very low platelet counts in plasma after centrifugation of native blood, sometimes even a complete absence of platelets, while others show gel particles in tubes with a separating gel. Furthermore, there are many different protocols for obtaining liquid platelet-rich and platelet-rich plasma. These differences in results are due to different tube volumes, different anticoagulants in the tubes (sodium heparin, lithium heparin, sodium citrate, EDTA), the presence of a separating gel in the tubes, and different centrifuges used for blood centrifugation. Therefore, known methods for obtaining a fibrin clot in a tube are not suitable for intradermal injection, as fibrin, being dense, does not pass through a needle.

[0016] A known method (patent RU2646820C2, IPC A61K 35 / 16 A61P 17 / 00, application 2016134003 dated 08 / 19 / 2016, authors: Renat Rashitovich Akhmerov, Olga Igorevna Korotkova, patent holder OOO "Plasmolifting TM") in which test tubes with a separating gel and sodium heparin as a coagulant are used to isolate plasma and platelets. A single centrifugation of native blood is carried out. It is noted that the use of a separating gel leads to the fact that platelets are adsorbed in the separating gel, and as a result, plasma with a very low platelet content, and sometimes their complete absence, without leukocytes and erythrocytes, is obtained. Due to insufficient gel density, all cellular elements settle to the bottom of the test tube. The declared composition of the separation gel is mass.%: gelling agent - polyisobutylene - 58.5-59.5; solvent - chlorinated paraffin - 19.5-20.5; chemically and biologically inert filler - silicone dioxide - 13.0-14.0; plasticizer - propylene glycol hexanedioic acid. A disadvantage of this method is that particles of the separating gel can get into the syringe when collecting plasma from the test tube and be injected intradermally with the plasma. Another disadvantage of this method is that the gel-like form of the serum is obtained by heating the plasma in a thermostat, which leads to denaturation of the protein molecules. The resulting plasma is administered using a microneedle. In addition, it should be noted that the plasma obtained in this way cannot be considered autologous.

[0017] A known method (patent RU2682763C1, IPC A61N1 / 05, A61K35 / 16, A61P17 / 14, application 2018113386 dated 12.04.2018, author Pakhomova Elena Evgenievna, patent holders: St. Petersburg State Budgetary Healthcare Institution Skin and Venereal Diseases Dispensary No. 4, LLC "TRICHOLOGICAL CENTER FOR HAIR HEALTH AND TREATMENT", Pakhomova Elena Evgenievna), which consists in adding sodium citrate as an anticoagulant to a sterile 9 ml VACUETTE vacuum tube and collecting the patient's native blood. Double centrifugation and isolation of plasma with platelets are carried out. The advantage of the known method is the ability to administer the resulting drug using a microneedle. The disadvantage of the known method of obtaining the drug is the technological disadvantage of the known method, in particular, the fact that the use of sodium citrate results in the drug not being autologous.

[0018] A method is known for obtaining autologous plasma containing platelets and leukocytes using the Regenlab method (Internet resource

[0019] https: / / www.regenlab.com / techno1ogies / (by the Swiss company RegenLab). For the procedure, blood is drawn from the cubital vein into tubes containing Regenlab separator gel. The tubes are then placed in a centrifuge and subjected to a single centrifugation at 3100 rpm for 5 minutes. During centrifugation, the separator gel separates red blood cells and white blood cells from plasma and platelets. A disadvantage of this known method is that the platelet count in the tubes remains at physiological levels, and sometimes is lower than in native blood. Furthermore, another disadvantage of this known method is that an activator (autologous fibrin or calcium gluconate) must be added to the finished product before administration.

[0020] A known method for producing plasma gel (patent RU2644753C1, IPC A61B 17 / 00, A61K 35 / 16, application 2017100521 dated 09.01.2017, authors and patent holders: Boris Mikhailovich Belik, Aleksey Nikolaevich Kovalev, Akop Lusigenovich Khatlamadzhiyan) involves obtaining autologous plasma from 20-100 ml of the patient's peripheral blood by two-stage centrifugation. In this case, in the first stage, erythrocytes and leukocytes are removed, and in the second, platelets are concentrated. Then, the isolated fraction is converted into a gel form by adding thrombus activators (thrombin), after which the plasma gel is injected into the problem area using a microneedle. A disadvantage of this method is the risk of thrombin contamination with various viruses, since human thrombin is obtained from donor plasma. Consequently, all thrombin preparations intended for clinical use must undergo viral inactivation. For thrombin, this typically involves solvent-based detergent treatment and / or pasteurization and sterilizing filtration.Thus, this method is unsafe and non-autologous.

[0021] A known method (patent EA037413B1, IPC A61K9 / 00, A61K35 / 16, A61P 9 / 14, application 201900296 dated 05 / 16 / 2019, authors: Burumkulov Erik Rakishevich, Sultanaliev Tokai Anarbekovich, Tursynbaev Serik Erishevich, Bespaev Abdimazhit Tasibekovich, Sharipov Askhat Sagynovich, patent holder Burumkulov Erik Rakishevich) when autologous plasma gel enriched with platelets, obtained from the patient's blood using a technology, preheated in a thermostat to a temperature of 65-85 ° C for 6 minutes is introduced into problem areas using a microneedle. The disadvantage of the known method is that the test tube used for these purposes is plastic and contains the anticoagulant sodium heparin. The gel is obtained by heating the plasma in a thermostat, which causes the proteins and cellular composition of the blood to denature at this temperature.

[0022] The closest method selected as a prototype is the method (patent RU2811233C1, IPC A61K 35 / 14, A61K 35 / 16, A61K 35 / 19, A61K 38 / 36, A61P 17 / 02, A61P 19 / 02, application 2023115138 dated 06 / 08 / 2023, authors: Viktor Sergeevich Malkov, Darya Aleksandrovna Naida, Ivan Viktorovich Kurtasov, Alexander Aleksandrovich Sharshukov, patent holder ARM LLC), which consists in obtaining a platelet-rich fibrin matrix and includes the following steps: a) platelet-rich plasma is obtained in an hourglass-shaped test tube after centrifugation of the patient's blood with the anticoagulant sodium citrate place freshly collected stabilized blood from the patient;then the tube is centrifuged until the stabilized blood is separated into three fractions, the middle of which is a leukothrombocyte suspension, which is taken from the tube(s) with the addition of the upper fraction - blood plasma, which allows for the formation of platelet-rich plasma containing at least 1 million platelets in 1 μl of product; b) obtaining serum containing thrombin, for this purpose, freshly collected whole blood of the same patient is placed in a tube that does not contain filler, then the tube is centrifuged until the blood is separated into three fractions, the upper of which is plasma, after which the tube is left at room temperature until the plasma transitions from a liquid state to a fibrin clot, which is achieved by a natural coagulation cascade, after which the serum containing the formed thrombin is separated from the fibrin clot;c) forming a composition for obtaining the final product, for which purpose the following are added sequentially to a container of the selected shape and size: autologous platelet-rich plasma obtained in step a), autologous serum containing thrombin obtained in step b), and a calcium gluconate solution at a concentration of 100 mg / ml or a calcium chloride solution at a concentration of 100 mg / ml, wherein the volume ratio of the PRP components: serum containing thrombin: calcium gluconate or calcium chloride solution is 16:2:1 - 16:4:2; d) the resulting mixture is left at room temperature until a platelet-rich fibrin clot is formed;d) The resulting platelet-rich fibrin clot is separated from the serum to obtain a fibrin matrix. The advantage of this method is that it allows for the production of a platelet-rich fibrin matrix that meets clinical practice requirements—the ability to intraoperatively form a multipurpose, biodegradable, regenerative-promoting graft of the desired shape and size, resistant to surgical suture insertion. The disadvantages of this method include the technologically complex, time-consuming, and expensive process of obtaining the fibrin matrix.

[0023] The proposed method for producing a gel-like cellular substance from a patient's native blood differs from known methods in that the resulting substance contains a high concentration of platelets and a homogenized fibrin matrix in gel form (HFM), and its production does not require thermal heating or the introduction of coagulation activators. Unlike known methods, the proposed method for producing liquid plasma with platelets and a cellular fibrin substance is obtained using a single, one-step centrifugation of the patient's native blood in a test tube without the use of a coagulant. Consequently, the proposed method for producing the substance is completely autologous. This substance contains platelets, leukocytes, a small amount of erythrocytes, a large number of biologically active substances, primarily growth factors, and, most importantly, an extracellular fibrin matrix.Depending on the centrifugation protocol, the substance can be obtained in liquid or gel form. This ensures a prolonged release of biologically active components and a prolonged regeneration mechanism when the substance is injected into the scalp.

[0024] The objectives of the invention are to develop a method for producing a cellular fibrin substance, as well as a method for administering the resulting substance for the treatment of hair loss and stimulation of hair growth. The proposed method differs from methods for producing liquid plasma with platelets in that the resulting suspension is gel-like rather than liquid. The suspension contains homogenized fibrin, which is formed from the patient's native blood in a cylindrical, vacuum-sealed, sterile borosilicate glass tube without the use of anticoagulants, coagulation activators, or the need to heat the resulting plasma in a thermostat. The resulting substance is homogenized and finely dispersed, allowing for its administration using a syringe and microneedle.Injecting the substance using a syringe and microneedle will allow the substance to remain in the skin for a longer period of time, which will ensure the prolonged release of growth factors, as well as faster and more noticeable regenerative effects.

[0025] To achieve the specified result, the following set of essential features is used: a method for treating hair loss, including collecting the patient's blood in a sterile test tube, centrifuging the blood and introducing a homogenized fibrin matrix, characterized in that venous blood is collected from the patient in a vacuum cylindrical test tube made of borosilicate glass; the obtained venous blood is centrifuged once at a relative centrifugal force of 900-1200 for 5-10 minutes; the fibrin-platelet mass is separated from the erythrocytes under aseptic conditions; the fibrin clot is homogenized to a finely dispersed consistency at a temperature of 4 to 10 ° C; the finished homogenized fibrin matrix is ​​collected; subsequent intradermal or subcutaneous administration is performed into the hair loss area 1-2 times a month, a course of 8-12 procedures.

[0026] To achieve the specified result, the following set of essential features is used: a method for treating hair loss, including collecting the patient's blood in a sterile test tube, centrifuging the blood and introducing homogenized fibrin matrix, characterized in that venous blood is collected from the patient in a vacuum cylindrical test tube made of borosilicate glass; the obtained venous blood is centrifuged once at a relative centrifugal force of 900-1200 for 5-10 minutes; the fibrin-platelet mass is separated from the erythrocytes under aseptic conditions; the fibrin clot is homogenized mechanically using a homogenizer; the finished homogenized fibrin matrix is ​​collected; subsequent intradermal or subcutaneous administration is performed into the hair loss area 1-2 times a month, a course of 8-12 procedures.

[0027] In addition, hair loss treatment can be performed by injecting the resulting homogenized fibrin matrix into the scalp using a sterile syringe and microneedles.

[0028] The mechanism of action of the obtained drug using the proposed method at the level of the scalp and hair follicles is associated with the release of biologically active growth factors by platelets: PDGF (platelet-derived growth factor) stimulates cell proliferation and angiogenesis; VEGF (vascular endothelial growth factor) promotes the formation of new capillaries, improving follicle nutrition; TGF-P (transforming growth factor beta) regulates tissue repair and follicular cell function; EGF (epidermal growth factor) stimulates the regeneration of epithelial cells of the scalp. Growth factors released from platelets stimulate the transition of follicles from the telogen (resting) phase to the anagen (growth phase). This is especially important for diffuse alopecia and androgenetic alopecia.

[0029] The homogenized fibrin clot obtained by the proposed method is a biologically active cellular matrix obtained from the patient's own venous blood after homogenization of the fibrin clot. Unlike traditional forms of fibrin clots, the homogenized form is a uniform mass, convenient and suitable for injection, while preserving important cellular components—platelets, leukocytes, and mononuclear cells.

[0030] Leukocytes (leukocyte elements) provide antimicrobial protection and participate in modulating the inflammatory response during the early stages of tissue repair. Leukocytes contained in the matrix help modulate local inflammation, often present in seborrheic dermatitis, scalp psoriasis, and other conditions associated with hair loss. Mononuclear cells, including lymphocytes and monocytes, play a crucial role in the immune response and hair regeneration processes.

[0031] The technical result achieved using the proposed method consists of obtaining a completely autologous, biologically active cellular substance containing a high concentration of growth factors, platelets, leukocytes, and an extracellular fibrin matrix. Its homogenized form allows for intradermal administration via microneedles and prolonged release of biologically active substances, increasing the effectiveness of therapy for all forms of alopecia, including cicatricial alopecia. The homogenized fibrin matrix enhances tissue vascularization and improves oxygen and nutrient delivery to the hair roots, which positively impacts hair thickness and strength. The fibrin matrix provides temporary extracellular matrix support, creating a favorable microenvironment for the stem cells of the hair papilla.

[0032] The proposed method was developed through experimental and clinical studies with the aim of solving an important practical problem - creating a universal, biodegradable material capable of activating hair growth.

[0033] To confirm the effectiveness of the proposed methods, studies were conducted to visually assess the effectiveness of the proposed technique in reducing hair loss and stimulating hair growth.

[0034] Examples of implementation of the proposed method for obtaining whey.

[0035] Example 1. A 32-year-old patient diagnosed with androgenetic alopecia, grade 5 according to the Norwood-Hamilton classification. A procedure was performed to obtain a homogenized fibrin matrix. After collecting 9 ml of venous blood, a single centrifugation at 1000xg for 7 minutes was performed in a borosilicate glass test tube. The resulting fibrin clot was homogenized at +4°C. The prepared substance was injected intradermally into the balding area using a syringe and microneedle. A course of 10 procedures was performed. An increase in hair density, a decrease in hair loss, and an improvement in the overall condition of the scalp were noted. Figs. 1-4.

[0036] Example 2. A 28-year-old female patient diagnosed with androgenetic alopecia stages 1-2 according to Ludwig. A procedure was performed to obtain a homogenized fibrin matrix. After collecting 9 ml of venous blood, a single centrifugation at 1000 x g for 7 minutes was performed in a borosilicate glass test tube. The resulting fibrin clot was homogenized at +4°C. The prepared substance was injected intradermally into the balding area using a syringe and microneedle. A course of 12 procedures was performed. An increase in hair density, a decrease in hair loss, and an improvement in the overall condition of the scalp were noted. Figs. 5-10.

[0037] Example 3. A 65-year-old female patient diagnosed with chronic diffuse alopecia. A procedure was performed to obtain a homogenized fibrin matrix. After collecting 9 ml of venous blood in a borosilicate glass test tube, a single centrifugation was performed at 1000xg for 7 minutes. The resulting fibrin clot was homogenized at +4°C. The prepared substance was injected intradermally into the balding area using a syringe and microneedle. A course of 12 procedures was performed. An increase in hair density, a decrease in hair loss, and an improvement in the overall condition of the scalp were noted (Figs. 11-14).

[0038] The results obtained from the practical implementation of the proposed methods for producing a homogenized fibrin matrix and its administration demonstrate their effectiveness. The homogenized fibrin matrix is ​​characterized by a high concentration of growth factors, platelets, leukocytes, and mononuclear cells, as well as the presence of an extracellular fibrin matrix designed to provide prolonged release of biologically active substances in scalp tissue.

[0039] The proposed method for producing a homogenized fibrin clot can be used in medicine, specifically in trichology, cosmetology, and dermatology for the treatment of androgenetic, diffuse, seborrheic, and focal alopecia, as well as for the treatment of hair dystrophies, and for improving the survival rate of hair grafts during hair transplantation. Furthermore, the proposed method can be used to prevent premature hair loss and improve hair structure.

[0040] Thus, the present invention provides a simple, clinically effective, cost-effective, and promising method for producing a homogenized fibrin matrix based on a patient's own blood. The resulting material is homologous, finely dispersed, and represents a biologically active, fully autologous substrate.

[0041] The presented method was developed and tested at the Clinic of Your Health LLC (St. Petersburg, Russia) and was named Trichogel.

Claims

1. A method for treating hair loss comprising collecting the patient's blood in a sterile test tube, centrifuging the blood and introducing a homogenized fibrin matrix, characterized in that venous blood is collected from the patient in a vacuum cylindrical borosilicate glass test tube; the obtained venous blood is centrifuged once at a relative centrifugal force of 900-1200 for 5-10 minutes; the fibrin-platelet mass is separated from the erythrocytes under aseptic conditions; the fibrin clot is homogenized to a finely dispersed consistency at a temperature of 4 to 10°C; the finished homogenized fibrin matrix is ​​collected; subsequent intradermal or subcutaneous administration is performed into the area of ​​hair loss 1-2 times a month, for a course of 8-12 procedures.

2. A method for treating hair loss comprising collecting patient's blood in a sterile test tube, centrifuging the blood and introducing homogenized fibrin matrix, characterized in that venous blood is collected from the patient in a vacuum cylindrical test tube made of borosilicate glass; the obtained venous blood is centrifuged once at a relative centrifugal force of 900-1200 for 5-10 minutes; the fibrin-platelet mass is separated from the erythrocytes under aseptic conditions; the fibrin clot is homogenized mechanically using a homogenizer; the finished homogenized fibrin matrix is ​​collected; subsequent intradermal or subcutaneous administration is performed into the area of ​​hair loss 1-2 times a month, for a course of 8-12 procedures.

3. A method for treating hair loss according to any one of claims 1 or 2, characterized in that the resulting homogenized fibrin matrix is ​​introduced into the scalp using a sterile syringe and microneedles.

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