WO2021210026A1

WO2021210026A1 - A cell-free growth factor concentrate, method of preparing the same and a kit thereof

Info

Publication number: WO2021210026A1
Application number: PCT/IN2021/050383
Authority: WO
Inventors: Vasanthi Palanivel; Shrinivas RANGACHARI
Original assignee: Vasanthi Palanivel; Rangachari Shrinivas
Priority date: 2020-04-17
Filing date: 2021-04-17
Publication date: 2021-10-21

Abstract

The present invention discloses a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF 1), platelet derived growth factor - BB (PDGF-BB), and transforming growth factor beta 1 (TGF β1); formulation of cell-free growth factor concentrate; a method of preparing the cell-free growth factor concentrate and a kit thereof. The present disclosure relates to compositions for treating/ improving rejuvenation of lung tissues in patients with compromised pulmonary function due to various causes, resulting in catalysing lung tissue healing, controlling the inflammation, preventing fibrosis progression, activation of pulmonary stem cells thereby leading to improve the quality of patients suffering from respiratory distress and compromised lung function.

Description

A CELL-FREE GROWTH FACTOR CONCENTRATE. METHOD OF

PREPARING THU SAME AND A KTT THEREOF TECHNICAL FIELD

[0001] The present disclosure generally relates to compositions for treating/ improving rejuvenation of lung tissues in patients with compromised pulmonary function due to various causes, resulting in catalysing lung tissue healing, controlling the inflammation, preventing fibrosis progression, activation of pulmonary stem cells thereby leading to improve the quality of patients suffering from respiratory distress and compromised lung function. In particular, the present disclosure relates to a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF 1), platelet derived growth factor - BB (PDGF-BB), and transforming growth factor beta 1 (TGF bΐ); a formulation of cell-free growth factor concentrate, a method of preparing the cell-free growth factor concentrate and a kit thereof.

BACKGROUND ART

[0002] Acute respiratory distress syndrome (ARDS) is driven by a severe pro- inflammatory response resulting in lung damage, impaired gas exchange and severe respiratory failure.

[0003] There is no specific treatment that effectively improves outcome in ARDS. ARDS is the primary cause of fatality following COVID-19 infection, resulting in a significantly decreased oxygen uptake, but currently has limitedly effective treatments. ARDS can be caused by a number of different conditions, including a range of pathogens, and while various techniques are currently being investigated to mitigate the damage that is caused before it becomes too severe or to regenerate the damage to the lungs, many are still in the early stages of development. Other than placing patients on a ventilator, there is currently no effective treatment for ARDS.

[0004] Respiratory-related morbidity and mortality are on the rise. While some chronic lung injuries have been linked to environmental factors or lifestyle choices such as smoking, the cause of Idiopathic Pulmonary Fibrosis (IPF) remains unknown (as the name implies). IPF is actively being studied by many groups as it is a progressive and fatal form of interstitial lung disease, characterized by fibroblastic foci, alveolar honeycombing, and persistent, unremitting fibrosis that ultimately leads to the destruction of lung architecture and fulminant respiratory failure. The exact pathological mechanism(s) and cause(s) of IPF are unknown, resulting in poor prognosis.

[0005] There remain few treatment options for IPF. To date, only two Food and Drug Administration (FDA) approved therapies exist: pirfenidone and nintedanib. These drugs, however, are palliative and merely delay disease progression. They do not halt or reverse the fibrosis that is already established. As new treatment strategies continue to evolve, cell-based therapies have emerged as promising options for a number of diseases.

[0006] Rather than indiscriminate therapy with an anti-immune drug, such as an inhibitor of a cytokine, like IL-6, growth factors and anti-inflammatory cytokines present in the growth factor concentrate can potentially and specifically calm inflammation exactly where it is most active, without causing a more general ‘global’ immunosuppression which would be harmful for a virally infected patient.

[0007] However, in recent years, cell therapy has shown great promise in preclinical ARDS studies. A wide range of cells have been identified as potential candidates for use, among these are mesenchymal stromal cells (MSCs), which are adult multi-lineage cells that can modulate the immune response and enhance repair of damaged tissue. The therapeutic potential of MSC therapy for sepsis and ARDS has been demonstrated in multiple in-vivo models.

[0008] The therapeutic effect of these cells seems to be due to two different mechanisms; direct cellular interaction, and paracrine release of different soluble products such as extracellular vesicles (EVs)/exosomes. Different approaches have also been studied to enhance the therapeutic effect of these cells, such as the over-expression of anti inflammatory or pro-reparative molecules. Several clinical trials (phase I and II) have already shown safety of MSCs in ARDS and other diseases.

[0009] However, several translational issues still need to be addressed, such as the time taken for growing cells, large-scale production of cells, and their potentiality and variability, before the therapeutic potential of stem cells therapies can be realized. In a simple equivalent non-invasive preparation like Platelet rich plasma (PRP), depositing highly concentrated cells in a heavily inflamed lungs as in COVID-19 patients, tend to aggregate and aggravate the inflammation. While there have been investigations to show the recruitment of the cells to the lungs following systemic (e.g., intravenous) administration, these depend critically on many factors.

[0010] Platelet Derived Growth Factors are studied for Lung Regeneration. Platelets are vehicles for the delivery of growth factors (PDGF, TGF-B, IGF, EGF, VEGF) that induce proliferation of fibroblasts, osteoblasts and endothelial cells, promoting and accelerating healing of hard and soft tissues. Autologous Platelet Rich Plasma also contains fibrin, fibronectin and vitronectin that act as cell adhesion molecules for lung epithelial migration.

[0011] By allowing the patient to breathe aerosolized particles of growth factor concentrate, the new delivery method increases the number of platelets into the lungs' endothelial cells, which reduces inflammation and stimulates the healing process (Lung Regenerative Program of 2019). Inhalation based delivery method increases the number of platelets into the lungs' endothelial cells, which reduces inflammation and stimulates the healing process and stops the progression into fibrosis.

[0012] Angiogenesis, the growth of new blood vessels, plays a key role in organ development, homeostasis, and regeneration. The cooperation of multiple angiogenic factors, rather than a single factor, is required for physiological angiogenesis. Soluble platelet extract, which contains abundant angiopoietin-1 and multiple other angiogenic factors which is a cell free only growth factor concentrate, stimulates angiogenesis and maintains vascular integrity in vitro and in vivo and lung vascular and alveolar regeneration through Angl-LRP5-Tie2 signalling (Mammoto etal, 2016).

[0013] Scientists have studied inflammation to address lung tissue regeneration and healing. Inflammation is a complex cellular and biochemical process that occurs in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent, such as a pathogen, allergen or irritant.

[0014] The inflammatory process includes local reactions and resulting morphologic changes in tissue; the destruction or removal of the causative agent; and the responses that lead to repair and healing. An inflammatory response consists of a cascade of biochemical events, involving the local vascular system and immune system, and various cells within the injured tissue.

[0015] The process involves the release of numerous cell-derived mediators, including histamine, interferon-gamma, interleukin-8, leukotriene, nitric oxide, prostaglandins, tumor necrosis factor-alpha, and interleukin-1. In most instances, inflammation is a beneficial and transient process, which subsides as the body attacks and overcomes an infectious or other harmful agent. However, in some instances, inflammation can be acute self-perpetuating process, for example, as part of an ongoing degenerative process due to cytokine storm in the case of Corona virus infection leading to destruction of lung tissues leading to pulmonary inflammation.

[0016] Various groups worldwide are currently involved in investigating how cell- based therapy, might be of use in treating inflammatory disorders, including pulmonary inflammation. There is a need in the art to develop a cell-based therapy for pulmonary inflammation which are self-perpetuating processes, for instance as in case of degenerative process due to cytokine storm in the case of Corona virus infection leading to destruction of lung tissues.

[0017] It is known to use activated autologous platelets as a treatment in a number of medical and surgical procedures, including but not limited to oral and maxillofacial surgery, orthopedic surgery, cosmetic and reconstructive surgery, chronic tissue repair, sports medicine injuries, neurosurgery, cardiovascular surgery, podiatry, hair transplant surgery, medical research, tissue engineering, and non-surgical cellular therapy. U.S. Pat. Nos. 4,957, 742 and 6,649,072 disclose wound healing compositions that include platelet enriched plasma which prior to use is activated by thrombin to release growth factors from the alpha granules of the platelets.

[0018] Extracting therapeutic levels of platelets has been a technical challenge requiring trained resources to operate the equipment originally designed for the production of platelet rich plasma (PRP). The clinical practitioner now has access to more simplified equipment that allows him to process PRP with smaller amounts of whole blood in a shorter amount of time. Venous access, clinical expertise, and cost are still challenges that have limited the widespread use of this process throughout the world. Moreover from a commercial standpoint, wound healing compositions that include platelets must meet costly FDA guidelines applicable to blood products.

[0019] Growth factors are responsible for the regeneration process, as described above. Platelets function merely as carriers for the growth factors. Therefore, there is a need for an inexpensive and efficient process for extracting and isolating growth factors from the platelets contained in plasma for subsequent use in wound healing. The final product preferably may be free of other components that are typically found in conventional platelet enriched products, namely the platelets themselves, ghost platelets, white blood cells, red blood cells, bacteria, and other cellular debris.

[0020] It is further desirable to prepare a wound healing product that can be subjected to conventional preservations, such as lyophilization, freeze drying, and cryopreservation in a process that does not destroy the growth factors. In this manner the shelf life of the product would be prolonged. A product with prolonged stability and shelf life and ability to preserve at domestic refrigeration, and do-it-yourself mode of administration will make the product availability even at remote places and extend the benefit to people who may otherwise be dependent on healthcare systems for the preparation and administration.

[0021] Therefore, there is a need for a process for isolating and extracting growth factors in a non-destructive manner from platelets. The resulting composition may or may not be substantially free of other components, such as platelets, ghost platelets, white blood cells, red blood cells and bacteria, and can be used immediately fresh or lyophilized or freeze dried into a shelf-stable product for subsequent use.

SUMMARY

[0022] It is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solve at least the above-mentioned problem.

[0023] In view of the foregoing, an embodiment herein provides a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml- 233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

[0024] Another embodiment of the present disclosure relates to a formulation comprising a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml along with 200-1000 mg of Glutathione, 2-4 ml of 0.9% sodium chloride, and 10-1000 pg of a bronchodilator.

[0025] According to an embodiment, the formulation comprises exosomes, secretome of cells or stem cells derived from lung or any tissue, induced pluripotent stem cells, mesenchymal stem cells from blood, bone marrow, cord tissue, cord blood, menstrual blood and similar sources, and conditioned media of cells or stem cells.

[0026] The present disclosure provides a method for preparing a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml, the method comprising steps of: collecting whole blood samples from volunteers at a volume of 10 ml to 60 ml; enumerating platelets in the collected whole blood; taking aliquots of 10 ml of blood in 0.1-lml of acid citrate dextrose solution or K2 EDTA; incubating the blood samples at a temperature of 4°C to 37°C with RBC aggregators for a time period of 5 minutes to 120 minutes; centrifuging the samples post incubation at a centrifugal force of 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes at a temperature of 21°C to 24°C to obtain platelet poor plasma, huffy coat, platelet rich plasma and red blood cells in different layers; transferring the platelet rich plasma layer into a falcon tube and centrifuging at 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes; incubating the platelet rich plasma layer with platelet activators and incubating for 15 to 45 minutes; and collecting the supernatant after incubation, filtering and dispensing as aliquots of 0.5ml to 10 ml into cryovials.

[0027] According to an embodiment, the red blood cell aggregators are selected from a group comprising heparin, collagen, calcium, hyaluronic acid, polygeline, thrombin, gelatin, ethylenediaminetetra acetic acid, sodium citrate and any combination thereof.

[0028] According to another embodiment, the platelet activators are selected from a group comprising collagen, calcium, hyaluronic acid, thrombin and any combination thereof. [0029] The present disclosure relates to a kit comprising a formulation of a cell-free growth factor concentrate, a hand-held nebulization and an instructions manual, wherein the formulation comprises cell-free growth factor concentrate, 200-1000 mg of Glutathione, 2- 4 ml of 0.9% sodium chloride, and 10-1000 pg of a bronchodilator, and wherein the cell- free growth factor concentrate comprises vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml- 129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

[0030] According to an embodiment, the formulation of the cell-free growth factor concentrate is contained in one to fifty vials.

[0031] The present disclosure relates to a method of rejuvenation of lung issues in patients with lung disease or compromised pulmonary function by administering a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml- 129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

[0032] According to an embodiment, the lung disease is Acute Respiratory Distress Syndrome (ARDS), Alpha- 1 -Antitrypsin Deficiency, Asbestos-Related Lung Diseases, Asbestosis, Asthma, Bronchi- ectasis, Bronchitis, Bronchopulmonary Dysplasia (BPD), Chronic Bronchitis, Chronic Obstructive Pulmonary Disease (COPD), Collapsed Lung, Cough, Cystic Fibrosis, Emphysema, Hemothorax, Idiopathic Pulmonary Fibrosis, Infant Respiratory Distress Syndrome (Respiratory Distress Syndrome in Infants), LAM (Lymphangioleiomyomatosis), Lung Transplant, Pleural Effusion, Pleurisy and Other Pleural Disorders, Pneumonia, Pneumonoconiosis, Pneumothorax, Pulmonary Embolism, Pulmonary Arterial Hypertension, Pulmonary Fibrosis, Respiratory Distress Syndrome in Infants, Respiratory Failure, Sarcoidosis, Tracheostomy, and Ventilator/Ventilator Support. [0033] Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular component parts of the device described or steps of the methods described since such device and method may vary. It is also to be understood that the terminology used herein is for purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

[0034] The present disclosure will become apparent from the detailed description given below. These and other aspects of the embodiments and other objects and advantages of the present invention herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The accompanying drawings are incorporated for illustration of preferred embodiments of the present invention and are not intended to limit the scope thereof. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Different configuration changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0035] The detailed description is set forth with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different figures indicates similar or identical items.

[0036] FIG. 1 illustrates a method for preparing a cell-free growth factor concentrate according to an embodiment herein;

[0037] FIG. 2 illustrates shows the steps followed by a patient for self-administering the formulation comprising cell-free growth factor according to an embodiment herein; [0038] FIG. 3 illustrates the levels of VEGF and EGF in the growth factor concentrate at certain storage durations according to an embodiment herein;

[0039] FIG. 4 illustrates the levels of bFGF and IGF-1 in the growth factor concentrate at certain storage durations according to an embodiment herein; and

[0040] FIG. 5 illustrates the levels of bFGF and IGF-1 in the growth factor concentrate at certain storage durations according to an embodiment herein.

[0041] FIG. 6 illustrates the levels of PDGF-BB and TGF- bΐ in the growth factor concentrate at certain storage durations according to an embodiment herein.

PET AIT, ED DESCRIPTION OF PREFERRED EMBODIMENTS [0042] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0043] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0044] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[0045] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. [0046] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[0047] Example embodiments of the present disclosure are described with reference to the accompanying figures.

[0048] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[0049] Definitions: The following terms are used as defined below throughout this application unless otherwise indicated.

[0050] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.

[0051] The terms "subject", "patient" or "individual" generally refer to a human or mammals.

[0052] "Sample" refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.

[0053] “Body fluid” refers to, but is not limited to, plasma, serum, urine, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.

[0054] The primary objective of the present disclosure is to provide compositions and dosage forms for treating/improving rejuvenation of lung tissues in patients with compromised pulmonary function due to various causes, resulting in catalysing lung tissue healing and preventing fibrosis progression, activation of pulmonary stem cells thereby leading to improvement in the quality of patients suffering from respiratory distress and compromised lung function.

[0055] The present disclosure provides a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml -74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

[0056] An embodiment of the present disclosure relates to a composition comprising platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, epidermal growth factor, interferons, stromal cell derived factor 1, hepactocyte growth factor and antibodies against respiratory virus, wherein the composition is targeted against respiratory viruses to neutralise the virus including but not limited to COVID-19, to rejuvenate lung tissues.

[0057] In an embodiment, the present disclosure aims at addressing the challenges with regard to self-perpetuating pulmonary disorders mentioned above and the present technology provides methods for generating acellular cytokine compositions from blood, alone or in combination with similar biologicals comprising majorly anti-inflammatory molecules for use in treatment of lung inflammation this repurposing of the well-established biologicals that needs to be tested and validated which are the urgently needed studies and need of the hour.

[0058] The present disclosure provides compositions, methods and kits for rejuvenation of lung issues in patients with compromised pulmonary function.

[0059] The present disclosure relates to a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF 1), platelet derived growth factor - BB (PDGF-BB), and transforming growth factor beta 1 (TGF bΐ); a method of preparing the cell-free growth factor concentrate and a kit thereof.

[0060] Another embodiment of the present disclosure relates to a formulation comprising a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml along with along with 200-1000 mg of Glutathione, 2-4 ml of 0.9% sodium chloride, and 10-1000 pg of a bronchodilator.

[0061] In yet another embodiment of the present disclosure, the bronchodilator includes but is not limited to beta-2 agonists, long acting bronchodilators (LABA), short acting bronchodilators (SABA) and antimuscarinics.

[0062] In still another embodiment of the present disclosure, bronchodilators includes muscarinic agonists, methylxanthines, Anti-inflammatory agents (steroids, slow anti inflammatory drugs, release inhibitors), and Leukotriene antagonists (lipoxygenase inhibitors, receptor inhibitors).

[0063] Activation of a platelet by an agonist, such as thrombin, or other agonists known in the art, leads to the release of granule material from within the platelet. Such granulation activation results in the release of proteins known as growth factors, primarily concentrated in the alpha granules of platelets. Released growth factors stimulate the formation of new tissue. When used to regenerate tissues, growth factors are known to increase the rate of collagen lay-down, vascular ingrowth, fibroblast proliferation and overall healing. The release of a protein known as platelet-derived growth factor (PDGF) is a chemotactic signal for monocytes, neutrophils and fibroblasts which then move into the wound to begin the inflammatory stage of the healing process. During this time, monocytes secrete a number of factors, including PDGF and transforming growth factor-beta 1 (TGF-bI) (also found in platelets). In this manner fibroblasts are activated to begin the repair stage of the healing process. Subsequently, tissue repair continues through the process of collagen remodeling within the tissue.

[0064] In one of the embodiment, as will be appreciated by a person skilled in the art the present invention provides a variety of following uses and benefits:

- Based on scientific and clinical evidence of similar autologous preparations for the treatment of pulmonary fibrosis,

- Highly scalable protocol that can treat millions

- Easy to administer by Doctors with minimal training Takes less than 15 minutes for the preparation Also can be used as DIY-at home post discharge

[0065] In an embodiment, the protocol of the present disclosure is developed to meet the following objectives:

- Reduce inflammation in early and late stage illness patients

To improve the lung function and accelerate lung tissue healing and preventing progression of fibrosis, reduction in number of days on ventilators and hospital stay. Clinical improvement of patients in comparison to standard treatment

[0066] The present disclosure provides compositions, kits and integrated systems comprising several transport materials, components, binding agents, containers for treating/ improving rejuvenation of lung tissues in patients with compromised pulmonary function.

[0067] The rationale behind use of regenerative medicine based approach stems from the already demonstrated use of platelet-rich plasma (PRP)and its derivatives including autologous PRP (those derived from the patient) with minimal manipulation is safe and effective to promote the healing of lung tissue affected by COPD and ILD. The administration of glutathione to mobilize secretions in the lung has been determined a safe and effective addition to treatment (Jones, 2011). “Platelet rich plasma (PRP)” is defined as “autologous blood with concentrations of platelets above baseline levels, which contains at least seven growth factors.” Cell ratios in normal blood contain only 6% platelets, however in PRP there is a concentration of 94% platelets.

[0068] Autologous blood cell derivative (ABCD) also known as Growth factor concentrate (GFC) is prepared from concentrated platelets isolated from fresh peripheral blood of the patient which is collected from a peripheral vein, stored in anticoagulant and processed to separate various components of blood. From the above separations, platelet fraction alone is concentrated further and through stimulating platelets cytokines and growth factors (GFs) become bioactive and are secreted by stimulated platelets within 10 min after stimulation.

[0069] These growth factors include platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), and epidermal growth factor (EGF). They can regulate cell migration, attachment, proliferation and differentiation, and promote extracellular matrix accumulation. ABCD also contains stromal cell derived factor 1 (SDF-1) and hepatocyte growth factor (HGF) which control recruitment, proliferation, and activation of fibroblasts, neutrophils, monocytes and other cells central to wound healing.

[0070] Platelet extract increases phosphorylation levels of the Wnt coreceptor low- density lipoprotein receptor-related protein 5 (LRP5) and thereby activates angiogenic factor receptor Tie2 in endothelial cells (ECs) and accelerates EC sprouting and lung epithelial cell budding in- vitro.

[0071] Platelet extract also increases phosphorylation levels of Tie2 in the mouse lungs and accelerates compensatory lung growth and recovery of exercise capacity after unilateral pneumonectomy in mice, whereas soluble Tie2 receptor or Lrp5 knockdown attenuates the effects of PRP extract.

[0072] Because human platelet extract is generated from autologous peripheral blood and can be stored at -80°C, these strategies may lead to the development of novel therapeutic interventions for various angiogenesis-related lung diseases and to the improvement of strategies for lung regeneration.

[0073] The present disclosure is further described in two FIG. 1 and FIG. 2.

FIG.l describes a method for preparing a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml -74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml, as described in the below example.

[0074] An embodiment of the present disclosure relates to a method for preparing a cell- free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml- 129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml, wherein the method comprises steps of: collecting whole blood samples from volunteers at a volume of 10 ml to 60 ml; enumerating platelets in the collected whole blood; taking aliquots of 10 ml of blood in 0.1-lml of acid citrate dextrose solution or K2 EDTA; incubating the blood samples at a temperature of 4 °C to 37°C with RBC aggregators for a time period of 5 minutes to 120 minutes; centrifuging the samples post incubation at a centrifugal force of 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes at a temperature of 21°C to 24°C to obtain platelet poor plasma, buffy coat, platelet rich plasma and red blood cells in different layers; transferring the platelet rich plasma layer into a falcon tube and centrifuging at 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes; incubating the platelet rich plasma layer with platelet activators and incubating for 15 to 45 minutes; and collecting the supernatant after incubation, filtering and dispensing as aliquots of 0.5ml to 10 ml into cryovials.

[0075] Another embodiment of the present disclosure relates to a kit comprising a formulation of a cell-free growth factor concentrate, a hand-held nebulization and an instructions manual, wherein the formulation comprises cell-free growth factor concentrate, 200-1000 mg of Glutathione, 2-4 ml of 0.9% sodium chloride, and 10-1000 pg of a bronchodilator, and wherein the cell-free growth factor concentrate comprises vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF- BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

[0076] EXAMPLE 1:

Preparation of Cell free-Growth factor concentrate

10-60 ml of blood was drawn and dispensed as aliquots of 10 ml each in acid citrate dextrose (ACD-A) solution gel tube / K2 EDTA tube. Samples were incubated (15-45 minutes) with RBC aggregators (One or more of the following: Heparin, Collagen, Calcium, Hyaluronic acid, Polygeline, Thrombin, Gelatin, EDTA, Sodium citrate) Samples post incubation were centrifuged at 600 rpm for 2 minutes. The recommended temperature during processing was 21°C - 24°C to prevent platelet activation during centrifugation. Post centrifugation, platelet poor plasma (PPP), buffy coat / Platelet rich plasma (PRP) and RBCs were obtained from top to bottom layer of test tube respectively. Platelet poor plasma (PPP) was kept aside. The middle portion, Platelet rich plasma (PRP) was transferred into a falcon tube and again centrifuged at 3000 rpm for 12 minutes. The supernatant-PPP was saved for future use and platelet pellet was collected into a falcon tube to which PPP was added (approximately 3ml). Samples were incubated (15-45 minutes) with Platelet activators (One or more of the following: Collagen, Calcium, Hyaluronic acid, Thrombin, freeze-thaw cycles). The supernatant collected after incubation was filtered and dispensed as aliquotes into cryovials, which were then well capped and tightened and ready to be administered or preserved for future use.

[0077] MODE OF ADMINITRATION - Inhalation/ Aerosol based delivery method increases the proposed combination of biomolecules whole/in part as per the indication and severity directly into the lungs' endothelial cells, which reduces inflammation and stimulates the healing process and stops the progression into fibrosis.

[0078] FIG. 2 shows the steps followed by a patient for self-administering the formulation comprising cell-free growth factor concentrate of the present disclosure. Approximately 50 cc of autologous/allogenic venous blood is drawn or platelet concentrate (infectious disease screened) from blood banks is procured and growth factor concentrate (GFC), is produced by purifying and enriching the patients baseline platelet levels (~4-6x baseline) in a clean room setting using the proprietary lab protocols (ABCD-Autologous Blood Cell Derivative) utilizing a rapid stimulation technique. From that sample, a high growth factor concentrate is created using a filtration based protein concentrate technique, and a sample is retained to quantify the protein profile of the GFC by ELISA. The GFC is aliquoted into 2 ml ampules using sterile technique which is then frozen at -20°C. To use the GFC, the patient is required to unfreeze each ampule and place it into a handheld mesh nebulizer and inhale the GFC following the nebulizer manufacture's protocol until the treatment is completed. The treatment is to be applied once a day for two weeks and extension of the treatment is progress dependent.

[0079] The protocol to be followed to use the nebulizer is as follows:

- Ensuring the device is fully charged.

Carefully removing the mesh protector.

Inserting the chosen nuts by pushing firmly onto the body with clean hands. - Unblock the lid of the atomizer cap and pulling the medication ends of the device - sealing.

Shutting the cup can hold upto eight milliliters of liquid medication.

- Press the power button and placing the mask over nose and mouth and breathing deeply holding the device upright.

[0080] The high quality restoration technology converts your liquid medicine into a fine mist that can be inhaled evenly into the lungs for optimum results and faster more effectively. If preferred, a patient can even use the nozzle for an oral route delivery also. It is important to ensure that all of the medications has been inhaled before removing the mask. Also, it’s important to thoroughly clean the device after use to prevent infection, remove the cup by pulling sharply upwards open the lid and pour away any remnants of medicine then clean and disinfect allowing the device to air dry in a clean environment.

[0081] For a deeper clean, the cup is filled with hot pure water, the cap is closed and the nebulizer is turned on allowing the hot water to run through as a mist for three to five minutes. Thereafter, the nebulizer is gently wiped away so that any remaining water is removed and left to air dry.

[0082] Proper maintenance is extremely important as not doing this may hamper the performance of the nebulizer for reliable handheld medicine delivery for patient’ s respiratory needs.

[0083] An embodiment of the present disclosure relates to formulations specific to COVID -19, a treatment mechanism that allows treatment of the inflamed respiratory tract using activated growth factors from autologous, allogenic plasma (Healthy volunteers and living related donors) or from platelet concentrates procured from blood banks. This is based on scientific and clinical evidence of similar autologous preparations for the treatment of pulmonary fibrosis.

[0084] The composition of the present disclosure improves the lung function and accelerates lung tissue healing, preventing progression of fibrosis, thereby leading to reduction in number of days on ventilators and hospital stay. There is clinical improvement of patients administered with the present composition in comparison to standard treatment.

[0085] EXAMPLE 2: Comparison of concentration of growth factors in cell-free growth factor concentrate and Conventional platelet rich plasma

The concentration of growth factors namely VEGF, EGF, bFGF, IGF-1, PDGF-BB and TGF- bΐ in the present cell-free growth factor concentrate was compared with that of conventional platelet rich plasma, wherein a relatively significant increase in the concentration of the said growth factors in cell-free growth factor concentrate was observed as indicated in the below table.

[0086] EXAMPLE 3: Stability Studies

To compare the stability of the growth factors in the cell-free growth factor concentrate with that of platelet poor plasma, stability studies were conducted by performing ELISA assays after platelets activation. Three samples in triplicates were used to evaluate the growth factor levels immediately after preparation and after storage duration of 3, 6, 9, 12 and 24 h at 20°C. As the reduction of growth factors were found in 9h and 12 h, 24 h samples were not considered for analysis.

The following observations were made:

• Mean levels of VEGF, PDGF-BB, and TGF-bl were significantly higher in the cell-free growth factor concentrate compared to PPP. • No significant difference was observed in IGF-1 levels between cell-free growth factor concentrate and PPP.

• PDGF- AB release by the cell-free growth concentrate was found to be significantly correlated to the platelet count. · Such relationship was not significant for TGF-bl and VEGF levels.

[0087] FIG. 3 shows the levels of VEGF and EGF in the growth factor concentrate at storage durations of 1, 3, 6, 9 and 12 hours at 20°C. FIG. 4 shows the levels of bFGF and IGF-1 in the growth factor concentrate at storage durations of 1, 4, 8, 12 and 24 weeks at frozen conditions. FIG. 5 shows the levels of bFGF and IGF-1 in the growth factor concentrate at storage durations of 1, 3, 6, 9 and 12 hours at 20°C. FIG. 6 shows the levels of PDGF-BB and TGF- bΐ in the growth factor concentrate at storage durations of 1, 4, 8, 12 and 24 weeks at frozen conditions. The recorded levels of each growth factor over the aforementioned intervals of time are indicated in the below tables.

[0088] Taken together, stability experiments demonstrated that a storage period of 6 h at 20 degrees may be compatible with preservation of platelet functionality and growth factor release capacity, whereas longer storage duration causes growth factors within platelet granules to decrease necessities the need for better cold storage of the cell-free growth factor concentrate to preserve the growth factors.

[0089] Three samples in triplicates were used to evaluate the growth factor levels immediately after preparation and after storage duration of lweek, 4 weeks, 8weeks, 12 weeks and 24 weeks at -10°C (frozen in the freezer of the house hold refrigerator). [0090] An increase of growth factors concentration was noted within lweek of storage and decrease within the following 6 months.

[0091] This present study discloses a standardized method for PRP derived growth factor concentrate preparation and home based storage based on the formulation of the obtained biotherapy product. It is evidenced that a simple protocol that can be adopted for do- it-yourself mode of applications especially for rapid rejuvenation and home based application for regeneration and healing where periodical, multiple applications are crucial for pro-longed clinical outcome.

[0092] ADVANTAGES OF THE CELL-FREE GF CONCENTRATE AND FORMULATION COMPRISING THE SAME:

Therapies known in the art may be directed to removal of the underlying irritant or agent causing the inflammatory reaction, or by mediating one or more aspects of the inflammatory response. Examples include glucocorticoid steroids (such as hydrocortisone, cortisone, prednisone, and beclomethasone), non-steroidal anti-inflammatory drugs (such as aspirin, ibuprofen and naproxen), and immune selective anti-inflammatories. However, many such treatments present side effects, particularly during chronic administration, or have pharmacologic characteristics that limit their use. Moreover, many treatments do nothing to address the underlying causes of the inflammatory process. Accordingly, improved methods of treating inflammation are needed, offering one or more of improved efficacy, reduced side effects, and improved dosing characteristics.

[0093] The process of preparation is simplified enough to carry out the preparation at home, at any healthcare centre even at primary health care centre and also patients can continue the treatment at home as it is designed to be do-it-yourself way.

[0094] CLINICAL APPLICATIONS

Any lung pathology where regeneration can provide the required therapeutic out- come, this process innovation can be used to treat any lung disease. Examples of lung disease include, but are not limited to: Acute Respiratory Distress Syndrome (ARDS), Alpha- 1 -Antitrypsin Deficiency, Asbestos-Related Lung Diseases, Asbestosis, Asthma, Bronchi- ectasis, Bronchitis, Bronchopulmonary Dysplasia (BPD), Chronic Bronchitis, Chronic Obstructive Pulmonary Disease (COPD), Collapsed Lung, Cough, Cystic Fibrosis, Emphysema, Hemothorax, Idiopathic Pulmonary Fibrosis, Infant Respiratory Distress Syndrome (Respiratory Distress Syndrome in Infants), LAM (Lymphangioleiomyomatosis), Lung Transplant, Pleural Effusion, Pleurisy and Other Pleural Disorders, Pneumonia, Pneumonoconiosis, Pneumothorax, Pulmonary Embolism, Pulmonary Arterial Hypertension, Pulmonary Fibrosis, Respiratory Distress Syndrome in Infants, Respiratory Failure, Sarcoidosis, Tracheostomy, and Ventilator/Ventilator Support. In some embodiments, proposed compositions are delivered directly to the lung via bronchoscopy or delivering indirectly to the lung via the heart or blood vessel. Measurements of tissue perfusion or function may be done to evaluate the efficacy of the treatment.

[0095] BEST MODE OF PRACTICE:

The present disclosure provides compositions, kits and integrated systems for transportation of biological samples using biopolymers. In addition, the kits may include several transport materials, components, binding agents, containers for treating patients.

[0096] The present disclosure provides compositions, kits and integrated systems for practicing the invention. In addition, the kits may include several transport materials, components, binding agents, containers for the transport of biological samples as part of the embodiments of the invention. [0097] Those of skill in the art will appreciate that for use in the disclosed methods, the compositions and constructs disclosed herein may be present in compositions including one or more physiologically acceptable carriers or diluents, such as water or saline. Such compositions may additionally contain other components, such as preservatives, stabilizers, buffers and the like. Carriers, diluents and other components suitable for use in the present compositions.

[0098] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[0099] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0100] [0100] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0101] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[0102] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that a person skilled in the art can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein.

Claims

CLAIMS WE CLAIM:

1. A cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

2. A formulation comprising the cell-free growth factor concentrate as claimed in claim 1 along with 200-1000 mg of Glutathione, 2-4 ml of 0.9% sodium chloride, and 10- 1000 pg of a bronchodilator.

3. The formulation as claimed in claim 2, wherein the formulation comprises exosomes, secretome of cells or stem cells derived from lung or any tissue, induced pluripotent stem cells, mesenchymal stem cells from blood, bone marrow, cord tissue, cord blood, menstrual blood and similar sources, and conditioned media of cells or stem cells.

4. A method for preparing a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml- 339.2 ng/ml, the method comprising steps of: collecting whole blood samples from volunteers at a volume of 10 ml to 60 ml; enumerating platelets in the collected whole blood; taking aliquots of 10 ml of blood in 0.1-lml of acid citrate dextrose solution or K2 EDTA; incubating the blood samples at a temperature of 4 °C to 37°C with RBC aggregators for a time period of 5 minutes to 120 minutes; centrifuging the samples post incubation at a centrifugal force of 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes at a temperature of 21°C to 24°C to obtain platelet poor plasma, buffy coat, platelet rich plasma and red blood cells in different layers; transferring the platelet rich plasma layer into a falcon tube and centrifuging at 200 rpm to 4000 rpm for a time period of 2 minutes to 30 minutes; incubating the platelet rich plasma layer with platelet activators and incubating for 15 to 45 minutes; and collecting the supernatant after incubation, filtering and dispensing as aliquots of 0.5ml to 10 ml into cryovials.

5. The method as claimed in claim 4, wherein the red blood cell aggregators are selected from a group comprising heparin, collagen, calcium, hyaluronic acid, polygeline, thrombin, gelatin, ethylenediaminetetra acetic acid, sodium citrate and any combination thereof.

6. The method as claimed in claim 4, wherein the platelet activators are selected from a group comprising collagen, calcium, hyaluronic acid, thrombin and any combination thereof.

7. A kit comprising a formulation of a cell-free growth factor concentrate, a hand-held nebulization and an instructions manual, wherein the formulation comprises cell-free growth factor concentrate, 200-1000 mg of Glutathione, 2-4 ml of 0.9% sodium chloride, and 10-1000 pg of a bronchodilator, and wherein the cell-free growth factor concentrate comprises vascular endothelial growth factor (VEGF) at a concentration of 514 pg/ml-1314 pg/ml, epidermal growth factor (EGF) at a concentration of 133 pg/ml-233 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 26.2 pg/ml-74.2 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 76.2 ng/ml-129.2 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 20.9 ng/ml-85.5 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 248.8 ng/ml-339.2 ng/ml.

8. The kit as claimed in claim 7, wherein the formulation of the cell-free growth factor concentrate is contained in one to fifty vials.

9. A method of rejuvenation of lung issues in patients with lung disease or compromised pulmonary function by administering a cell-free growth factor concentrate comprising vascular endothelial growth factor (VEGF) at a concentration of 914±400 pg/ml, epidermal growth factor (EGF) at a concentration of 183±50 pg/ml, basic fibroblast growth factor (bFGF) at a concentration of 50.2 ± 24.0 pg/ml, insulin-like growth factor 1 (IGF 1) at a concentration of 102.7±26.5 ng/ml, platelet derived growth factor - BB (PDGF-BB) at a concentration of 53.2±32.3 ng/ml, and transforming growth factor beta 1 (TGF bΐ) at a concentration of 294±45.2 ng/ml.

10. The method as claimed in claim 9, wherein the lung disease is Acute Respiratory Distress Syndrome (ARDS), Alpha- 1 -Antitrypsin Deficiency, Asbestos-Related Lung Diseases, Asbestosis, Asthma, Bronchi- ectasis, Bronchitis, Bronchopulmonary Dysplasia (BPD), Chronic Bronchitis, Chronic Obstructive Pulmonary Disease (COPD), Collapsed Lung, Cough, Cystic Fibrosis, Emphysema, Hemothorax, Idiopathic Pulmonary Fibrosis, Infant Respiratory Distress Syndrome (Respiratory Distress Syndrome in Infants), LAM (Lymphangioleiomyomatosis), Lung Transplant, Pleural Effusion, Pleurisy and Other Pleural Disorders, Pneumonia, Pneumonoconiosis, Pneumothorax, Pulmonary Embolism, Pulmonary Arterial Hypertension, Pulmonary Fibrosis, Respiratory Distress Syndrome in Infants, Respiratory Failure, Sarcoidosis, Tracheostomy, and Ventilator/Ventilator Support.