Use of plasma and plasma components for ameliorating pain

Abstract

The use of a plasma component for the preparation of a medicament for treating a subject diagnosed with pain is described, wherein an effective amount of the plasma component is administered to the subject.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of October 21, 2019, application number 201980070839.3, and invention name "Use of plasma and plasma components for improving pain, wound healing and postoperative recovery". Technical Field

[0002] The present invention relates to the prevention and treatment of diseases and aging-related diseases. The present invention relates to the use of blood products, such as plasma and plasma fractions, to improve and accelerate recovery from surgery, including recovery from surgery-related conditions and indications. The present invention also relates to the use of blood products, such as plasma and plasma fractions, to relieve chronic pain or neuropathy and treat indications related to wound healing. Background Art

[0003] The following is provided as background information only and is not admitted to be prior art to the present invention.

[0004] Surgery is often accompanied by complications such as pain, cardiopulmonary problems, infection, thromboembolic problems, and postoperative wound healing. In addition, wound healing takes time, whether it is a wound caused by the surgery itself (e.g., an incision), or caused by an accident, violence, or disease and subsequently treated by surgery. This complication usually worsens with age. Surgical stress and the subsequent demand for organ function may cause other complications, which are usually mediated by trauma-induced endocrine metabolic changes and cascade reactions (cytokines, complement, arachidonic acid metabolites, nitric oxide, and free oxygen radicals) activation. (Kehlet H. et al., Br. J. Anaesthesia, 78:606-17 (1997)). In the surgical stress response, the sympathetic nervous system is activated. (Starkweather A et al., Topics in Pain Management, 32(8):1-11 (2017)). Pituitary hormone secretion increases, leading to the mobilization of energy through catabolism. This in turn leads to salt and water retention. Increased secretion of adrenocorticotropic hormone (ACTH) leads to increased norepinephrine and sympathetic nerve activity. This results in cardiovascular responses such as tachycardia and hypertension, and glucagon release leading to hyperglycemia. Increased growth hormone and cortisol also inhibit differentiation of monocytes into macrophages. This, in turn, interferes with T cell signaling / histamine production and reduces migration of immune cells. (Same)

[0005] Current treatments for postoperative recovery include reducing postoperative pain and multimodal interventions. (Same) Pain management is important in recovery from many types of surgery, and acute pain is expected. (Pinto PR, J Pain Res, 10:1087-98 (2017)). Patients undergoing common surgery experience postoperative pain to a large extent. (Couceiro TC, Rev Bras Anestesiol, 59(3):314-20 (2009)). Pain also plays a negative role in clinical outcomes because it impairs healing and recovery. Same. Hip and knee replacements in particular cause chronic pain (e.g., osteoarthritis) and acute pain. Same. Therefore, analgesics are often used during postoperative recovery, both during hospitalization and during home rehabilitation.

[0006] Enhanced Recovery After Surgery (ERAS) is a multimodal intervention. (Starkweather A, supra). ERAS addresses a variety of surgical procedures, such as colorectal surgery, orthopedics, gynecology, urology, head and neck cancer, bladder cancer, liver disease, rectal / pelvic disease, colon pathology, pancreaticoduodenectomy, gastrectomy, and bariatric and gynecologic surgical oncology. Same. As a multimodal intervention, it emphasizes: preoperative techniques (counseling services, fluid / carbohydrate loading; shortened fasting period); perioperative techniques (short-acting anesthetics; normothermia; antibiotic prophylaxis; thromboembolism prevention; prevention of salt / water overload; vomiting prevention); and postoperative techniques (early oral diet; exercise; non-opioid analgesia; and post-discharge support). Same.

[0007] However, current treatments have failed to eliminate postoperative morbidity and mortality. The nature of multimodal techniques is time and resource intensive. At the same time, no single technique or medical therapy can match this multimodal approach. Due to these deficiencies, new treatments are needed to improve postoperative recovery. Summary of the invention

[0008] The present invention is based on the production and use of blood products for treating symptoms and conditions that affect surgical recovery, including, for example, pain and wound healing. The present invention recognizes, among other things, the need for new treatments to treat adverse conditions associated with postoperative recovery and improve postoperative recovery. The compositions of the present invention are derived from blood and plasma and involve solutions to address the failures and inadequacies of current treatments by using plasma components that have efficacy in treating adverse conditions associated with postoperative recovery and for improving postoperative recovery.

[0009] The present invention is also based on the production and use of blood products for treating symptoms and conditions associated with acute or chronic pain. Among other things, the present invention recognizes the need for new treatments to relieve pain. While there are treatments for acute and chronic pain, many of these therapies, such as opioid analgesics, are subject to high levels of addiction, abuse, and associated morbidity and mortality.

[0010] Incorporation by reference

[0011] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Chronic constriction injury (CCI) experiments are described. Wild-type mice aged 23 months were given CCI or sham surgery via ligation 24 hours before 7 consecutive days of pulse dosing in PPF1, gabapentin, recombinant human albumin (rhAlb) or vehicle control groups. Behavioral assessments were performed from the second to fifth week, and tissues were collected for histological examination during the fifth week.

[0013] Figure 2 Is a diagram of the location of CCI administration to wild-type mice at twenty-two months of age. As shown, a ligature was made on the sciatic nerve. This diagram is adapted from Suter MR et al., Anesthesiology Res and Practice, (2011), the entire contents of which are incorporated herein by reference.

[0014] Figure 3 Shows the Figure 1 Mechanical von Frey allodynia test data for wild-type mice treated with CCI or sham surgery are shown. Von Frey filament stimulation applied to the paralyzed hind paw of the subject's sciatic nerve is useful for analysis of pain behavior. Figure 3 The pressure when the mice withdrew their hind paws was measured and plotted in the Figure 1. The graph shows that mice treated with PPF1 after CCI had significantly less pain (could tolerate more pressure) than control mice treated with vehicle after CCI. Animals treated with vehicle after sham surgery also had significantly less pain relative to animals treated with vehicle control after CCI. This suggests that PPF1 has a positive effect on mechanical nociception deficits.

[0015] Figure 4 Shows Figure 1Hippocampal histology data of wild-type mice shown. Neurogenesis was measured using the doublecortin (DCX) marker. Mice that underwent CCI surgery and were treated with PPF1 had a significant increase in hippocampal neurogenesis compared to mice that received vehicle. Mice that received sham surgery with vehicle had a trend toward greater neurogenesis than mice that underwent CCI and received vehicle after surgery. Therefore, PPF1 has the ability to restore neurogenesis after chronic nerve injury.

[0016] Figure 5 Shows Figure 1 Hippocampal histology data of wild-type mice are shown. CD68 expression was quantified, and the number of CD68-positive cells in the hippocampus of mice given CCI-loaded was significantly greater than that of mice given CCI plus PPF1. A similar degree of difference was observed between CCI-loaded and sham-loaded mice. This suggests that PPF1 contributes to the suppression of neuroinflammation induced by chronic nerve injury.

[0017] Figure 6 Data are shown for mechanical von Frey allodynia in 22-month-old C57BL / 6J mice that underwent CCI or sham surgery and were followed by Figure 1 Weeks after CCI or sham surgery, mice were assessed for hind paw withdrawal pressure and are shown in Figure 6 In. This figure illustrates that mice administered PPF1 after CCI surgery had significantly higher tolerance to mechanical nociception at all evaluated time points than mice treated with vehicle after CCI. In contrast, mice administered gabapentin had significantly improved mechanical nociception only 2 weeks after CCI surgery, and mechanical nociception at all other time points was similar to that of mice treated with vehicle. Sham-operated mice had significantly increased responses to mechanical nociception at 3 and 5 weeks after surgical treatment. In summary, these data suggest that PPF1 improves peripheral pain for a longer period of time compared to the standard of care treatment (gabapentin).

[0018] Figure 7 Shown are data from the hot plate test of 22-month-old wild-type mice that received CCI or sham surgery and were followed by Figure 1The test was performed on the time axis shown in Figure 1. This test was performed according to the method described by Woolfe and Macdonald. (Woolfe G. and Macdonald AD, J. Pharmacol. Exp. Ther. 80:300-07 (1944), the entire contents of which are incorporated herein by reference). The temperature of the hot plate was set to 55°C. The mice were placed in a transparent cylinder for 30 minutes to adapt. The cylinder was placed on the hot plate and a timer was started. When the first pain response behavior (e.g., licking the hind paw or hind paw jumping) was observed, the time was recorded as the latency. Figure 7 Hot plate pain response latency 5 weeks after CCI or sham surgery is shown. PPF1-treated groups showed significantly reduced sensitivity to hot plate stimulation compared to controls treated with CCI vehicle, indicating a rescue effect of PPF1. The baseline of care effect (gabapentin) was similar to that of vehicle.

[0019] Figure 8 Shown are data from the hot plate test for wild-type mice that received CCI or sham surgery and were followed Figure 1 The timeline shown is for testing. Figure 8 Shown are the latency to hot plate pain responses 5 weeks after CCI or sham surgery. PPF1-treated mice and rhALB showed significantly reduced sensitivity to hot plate stimulation compared to controls that received CCI loading agent treatment.

[0020] Fig. 9 Data are shown for mechanical von Frey allodynia in C57BL / 6J mice that underwent CCI or sham surgery and were followed by Figure 1 The timeline shown is for testing. Fig. 9 This indicates that mice administered PPF1 after CCI surgery were significantly more tolerant to mechanical nociception at all time points assessed than mice treated with vehicle after CCI. In contrast, mice administered rhALB showed mechanical allodynia responses similar to those of vehicle-treated mice at all time points.

[0021] Fig.10 Data from histological analysis of sciatic nerves (approximately 1000 μm from the last ligature) for myelin basic protein (MBP) expression in C57BL / 6J mice that underwent CCI or sham surgery are shown and are shown in Figure 4. Figure 1 Tissues were collected and analyzed on day 35 as indicated. Fig.10 Mice that were given PPF1 after CCI surgery had a significant increase in MBP intensity, indicating increased myelin expression compared to animals treated with vehicle. Mice that were sham-operated also expressed more MBP compared to mice that were treated with vehicle after CCI.

[0022] Fig.11 Data from histological analysis of sciatic nerves (approximately 1000 μm from the last ligature) of S-100 protein (expressed by Schwann cells) in C57BL / 6J mice that underwent CCI or sham surgery are shown and are shown in Figure 4. Figure 1 Tissues were collected and analyzed on day 35 as indicated. Fig.11 The results showed that mice given PPF1 after CCI surgery had a significant increase in S-100 intensity, indicating an increase in Schwann cells (cells that produce myelin in peripheral nerves) compared to animals treated with vehicle. Mice treated with sham surgery also expressed more S-100 than mice treated with vehicle after CCI.

[0023] Fig.12 are selected images from histological analysis of sciatic nerves, which determined Fig.10 and 11 The position used for quantification in Figure 5 (approximately 1000 μm from the last ligature) and the typical intensity of S-100 protein (expressed by Schwann cells) and myelin basic protein in C57BL / 6J mice that underwent CCI surgery and were treated with vehicle or PPF1 and were Figure 1 The indicated days 35 were used for qualitative analysis of sciatic nerve tissue.

[0024] Fig.13 Data from spinal cord histological analysis of C57BL / 6J mice that underwent CCI or sham surgery (analysis was performed on spinal cord tissues collected from L4-L6 lumbar segments) are shown and are presented in Figure 4. Figure 1 Tissues were collected for analysis after day 35 as indicated. Fig.13 The researchers showed that BDNF intensity within the dorsal horn of the spinal cord was significantly reduced in mice that were administered PPF1 after CCI surgery, indicating reduced activity of microglia within the spinal cord. Since BDNF is a pro-inflammatory cytokine released by activated microglia, these findings suggest that PPF1 reduces essential regulators of pain states within the spinal cord, restoring levels to those of sham-operated (no CCI injury) mice.

[0025] Fig.14 Data from spinal cord histological analysis of C57BL / 6J mice that underwent CCI or sham surgery (analysis was performed on spinal cord tissues collected from L4-L6 lumbar segments) are shown and are presented in Figure 4. Figure 1 Tissues were collected for analysis after day 35 as indicated. Fig.14The results showed that the intensity of CD68 in the dorsal horn of the spinal cord of mice administered PPF1 after CCI surgery was significantly reduced, indicating a decrease in the activity of microglia in the spinal cord. Since the CD68 protein is expressed by activated microglia, this suggests that PPF1 reduced the activation of essential cell types in the spinal cord that induce pain states, restoring their levels to those of sham-operated (no CCI injury) mice. Fig.13 and Fig.14 The data presented indicate that PPF1 centrally modulates the pain state caused by sciatic nerve injury, alleviating or preventing the establishment of pain signals between peripheral nerves and the brain, also known as central sensitization.

[0026] Fig.15 are selected from images of spinal cord histological analysis that determined Fig.14 The dorsal horn locations used for quantification in (analysis was performed on spinal cord tissue collected from L4-L6 lumbar segments) and the representative intensity of CD68 protein (expressed by activated microglia) in C57BL / 6J mice that underwent CCI surgery and were treated with vehicle or PPF1 and in Figure 1 The indicated days 35 were used for qualitative analysis of spinal cord tissue.

[0027] Fig.16 are selected images from spinal cord histology analysis that identified Fig.13 Figure 3 Dorsal horn location quantified in (analysis of spinal cord tissue collected from L4-L6 lumbar segments) and representative intensity of BDNF protein (a cytokine released by activated microglia) in C57BL / 6J mice that underwent CCI and were treated with vehicle or PPF1 and were placed in the same Figure 1 The indicated days 35 were used for qualitative analysis of spinal cord tissue.

[0028] Fig.17 Chronic constriction injury (CCI) experiments are described. Wild-type mice aged 22 months were given CCI or sham surgery via ligation 2 weeks before a 7-day pulsed administration in PPF1, rhAlb or vehicle control groups. Behavioral assessments were performed weekly from the second to seventh week, and tissues were collected for histological examination in the seventh week.

[0029] Fig.18 Data are shown for mechanical von Frey allodynia in C57BL / 6J mice that underwent CCI or sham surgery and were followed by Fig.17 The timeline shown is for testing. Fig.18It was demonstrated that mice administered PPF1 two weeks after CCI surgery developed a significant increase in tolerance to mechanical nociception starting at one week after cessation of PPF1 treatment, which remained unchanged throughout the study. Fig.18 The findings in the study suggest that PPF1 treatment initiates a process that reduces sensitivity to mechanical allodynia in a longitudinal manner, as improvements in tolerance were not demonstrated until one week after treatment (in contrast to treatments that provide benefits only during treatment, such as opioid analgesics) and persisted for at least 28 days. In contrast, the responses to mechanical allodynia in mice administered rhALB were similar to those of vehicle-treated mice at all time points.

[0030] Fig.19 Shown are data from the hot plate test for wild-type mice that received CCI or sham surgery and were followed Fig.17 The timeline shown is for testing. Fig.19 Shown are the latency to hot plate pain responses 5 weeks after CCI or sham surgery. PPF1-treated mice showed a significant decrease in sensitivity to hot plate stimulation compared to controls that received CCI loading agents.

[0031] Fig. 20 Shown are data from the hot plate test for wild-type mice that received CCI or sham surgery and were followed Fig.17 The timeline shown is for testing. Fig. 20 Shown are the latency to hot plate pain responses 7 weeks after CCI or sham surgery. PPF1-treated mice showed a significant decrease in sensitivity to hot plate stimulation compared to controls that received CCI loading agents.

[0032] Fig.21A and Fig.21B is untreated ( Fig.21A ) and PPF1 treated ( Fig.21B ) of diabetic wounds (B6.BKS(D)-Lepr db Histological comparison of PPF1 in diabetic mouse model (Figure 2A). The black bar indicates the wound bed thickness (epidermis plus granular layer). The arrow indicates the wound boundary. As determined by the wound bed thickness, the wound bed thickness of PPF1-treated mice increased. Therefore, it is demonstrated that PPF1 improves wound healing.

[0033] Fig.22A and Fig. 22B is untreated ( Fig.22A ) and PPF1 treated ( Fig. 22B ) of diabetic wounds (B6.BKS(D)-Lepr dbHistological comparison of PPF1-treated and untreated wounds (Figure 2A). Black bars indicate the granular layer. Blue bars indicate the epidermis. PPF1-treated wounds showed a thicker epidermis than untreated wounds, while the granular layer of PPF1-treated wounds and untreated wounds showed a more obvious trend of difference (i.e., the granular layer of PPF1-treated wounds was thicker than that of untreated wounds).

[0034] Fig.23 yes Figures 24 to 28 Overall design of the diabetic wound healing experiment. When blood was collected, the fasting blood glucose level was measured by blood drop. Skin wounds were made on the second day, and intravenous (iv) injections were performed from the first day to the seventh day. Histological examinations (microscope display) were performed after sacrifice.

[0035] Fig.24 Shown is the percentage of wounds that remained open at several time points after wounding in the first study (Study 1). Mice were treated with PPF1 (150 µL) or saline control for 7 days. After 10 days, open wound size was significantly reduced in animals treated with PPF1 compared to saline controls. (** p< 0.006, unpaired T test).

[0036] Fig.25 Shown is the percentage of wounds that remained open at several time points after wounding in a second similar study (Study 2). Mice were treated with PPF1 (150 µL) or saline controls for 7 days. After 8 days, open wound size was significantly reduced in animals treated with PPF1 compared to saline controls. (** p< 0.0018, unpaired T test).

[0037] Fig.26 Data from Study 1 and Study 2 are combined to show the percentage of wounds that remained open 11 days after injury. PPF1-treated mice had a statistically significant reduction in the percentage of wounds that remained open after 11 days. (** p<0.006, unpaired samples T-test). The difference between PPF1-treated and vehicle-treated animals was also significant at day 10 (** p<0.006, unpaired samples T-test).

[0038] Fig. 27 Shown are the results of a study in which PPF1 or vehicle was topically applied to wounds of B6 ob / ob (B6.Cg-Lepob / J mice). Fig. 27 A study paradigm of topical application of 30 μL of PPF1 or vehicle control to the wound daily is shown. Fig.10 Treat injuries as indicated.

[0039] Fig.28Results of a local study are shown as the percentage of the initial wound area remaining after 10 days of treatment. Fig.15 It was shown that PPF1 significantly reduced the percentage of open wounds remaining after 10 days compared to vehicle controls. DETAILED DESCRIPTION

[0040] A. Introduction

[0041] The present invention relates to methods and compositions for treating adverse conditions associated with postoperative recovery and for improving postoperative recovery that have been identified and discovered. The term "improving postoperative recovery" refers to accelerating the postoperative recovery of a subject, i.e., the subject may be mobile or discharged from inpatient care in a shorter period of time than without the intervention of the embodiments of the present invention. The term "adverse condition" refers to conditions or symptoms caused by, for example, but not limited to, pain, cardiopulmonary problems, infection, thromboembolic problems, inflammation, and delayed wound healing. Aspects of the present invention are methods and compositions described herein for treating subjects suffering from adverse conditions associated with postoperative recovery, and for improving postoperative treatment. Also described herein is a dosing regimen that causes improvement in subjects suffering from adverse conditions associated with postoperative recovery, and for improving postoperative treatment. The methods and compositions described herein can be used to: prevent postoperative recovery complications; relieve symptoms of postoperative recovery or prevent postoperative recovery complications; accelerate postoperative recovery. The methods and compositions of the present invention can be used or administered preoperatively (before surgery); perioperatively (during surgery); or postoperatively (after surgery).

[0042] Another aspect of the present invention is to treat a wider range of chronic pain / neuropathy, not just chronic pain / neuropathy associated with postoperative recovery. The methods and compositions of the present invention described herein can be used to treat chronic pain and neuropathy. "Chronic pain and neuropathy" means that the degree of chronic pain felt by the subject to whom the composition of the present invention is applied is slightly, moderately or significantly reduced, as assessed by subjective or objective means. The means may include self-administered tests or tests administered by medical technicians, such as, for example and without limitation: X-rays; MRI, CT scans; assessment or description of pain by the subject; activity; reaction, muscle strength; sensitivity (e.g., how long it takes for the subject to remove a limb subjected to pressure or other stimulation); blood tests for inflammatory markers; electromyography (EMG); and nerve conduction velocity.

[0043] Embodiments of the present invention include using plasma components as a treatment method, such as one or more than one fraction or effluent obtained from a blood fractionation process, such as Cohen fractionation as described below. Embodiments of the present invention include using plasma components (a solution containing normal human albumin, alpha globulin and beta globulin, gamma globulin and other proteins, hereinafter referred to as "plasma components" alone or in combination). Other embodiments of the present invention include using plasma protein components (PPF) for treatment. Other embodiments of the present invention include using human albumin solution (HAS) component treatment. Other embodiments include using effluent in a blood fractionation process, such as effluent I or effluent II / III as described below. Additional embodiments include plasma components that have substantially removed all coagulation factors to retain efficacy while reducing the risk of thrombosis (e.g., see U.S. Patent Application Nos. 62 / 236,710 and 63 / 376,529, the entire contents of which are incorporated herein by reference).

[0044] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods or compositions described, which, of course, can be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit specific embodiments, because the scope of the present invention will be limited only by the appended claims.

[0045] The publications discussed herein are provided here only because they are disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to predate the publications by virtue of prior invention. In addition, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0046] Where a numerical range is provided, it is to be understood that, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of the range, up to one tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any specified value or intermediate value in a specified range and any other specified value or intermediate value in a specified range is included in the present invention. The upper and lower limits of the smaller range may be independently included in or excluded from the smaller range, and one, neither, or both of the two limits of the range included in the smaller range are also included in the present invention, subject to any explicitly excluded limits in the specified range. If a specified range includes one or both of the limits, a range that does not include one or both of the included limits is also included in the present invention.

[0047] It should be noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as a basis for incorporating exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0048] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete parts and features that can be easily separated or combined with features in any other several embodiments without departing from the scope or spirit of the invention. Any method described can be performed in the order of events recited or in any other order that is logically possible.

[0049] B. Definitions

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar to or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. It should be understood that in the event of a conflict, the present disclosure supersedes any disclosure incorporated into the publication.

[0051] It must be noted that as used herein and in the appended claims, indefinite quantifiers include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to a "peptide" includes reference to one or more than one peptide and equivalents thereof known to those skilled in the art, such as polypeptides, and so forth.

[0052] When describing the methods of the present invention, the terms "host", "subject", "individual" and "patient" are used interchangeably and refer to any mammal that needs such treatment according to the disclosed methods. The mammals include, for example, humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice and rats. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a livestock. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs and rats, such as in animal models of diseases) and non-human primates (e.g., chimpanzees and monkeys). Therefore, the objects of the present invention include, but are not limited to, mammals such as humans and other primates such as chimpanzees and other apes and monkeys; and the like, wherein the object is a human in a specific embodiment. The term "subject" is also intended to include a person or organism of any age, weight or other physical characteristics, wherein the object may be an adult, a child, an infant or a newborn.

[0053] "Young people" or "young individuals" refer to individuals whose chronological age is 40 years old or less than 40 years old, such as 35 years old or less than 35 years old, including 30 years old or less than 30 years old, such as 25 years old or less than 25 years old, or 22 years old or less than 22 years old. In some cases, the source individual of the blood product containing young plasma is an individual of 10 years old or less than 10 years old, such as 5 years old or less than 5 years old, including 1 year old or less than 1 year old. In some cases, the object is a newborn and the source of the plasma product is the umbilical cord, wherein the plasma product is obtained from the umbilical cord of the newborn. Therefore, "young" or "young individuals" can refer to objects whose age is 0 to 40 years old, such as 0 years old, 1 year old, 5 years old, 10 years old, 15 years old, 20 years old, 25 years old, 30 years old, 35 years old or 40 years old. In other cases, "young" or "young individuals" can refer to biological age (rather than chronological age), such as individuals whose plasma does not show plasma inflammatory cytokine levels shown by older people. In contrast, "young" or "young individual" can refer to biological age (rather than chronological age), such as an individual who exhibits higher plasma inflammatory cytokine levels than those exhibited by older individuals. By way of example and not limitation, the inflammatory cytokine is eotaxin, and the multiple difference between the young subject or young individual and the elderly individual is at least 1.5 times. Similarly, the multiple difference between elderly individuals and young individuals in other inflammatory cytokines can be used to reference biological age. (See U.S. Patent Application No. 13 / 575,437, which is incorporated herein by reference). Generally, the individual is healthy, for example, the individual does not have a hematological malignancy or autoimmune disease at the time of collection.

[0054] As used herein, "treatment" refers to any of the following: (i) preventing a disease or condition, or (ii) alleviating or eliminating the symptoms of a disease or condition. Treatment can act prophylactically (before the onset of disease) or therapeutically (after the onset of disease). This effect can be a prophylactic effect that completely or partially prevents the disease or its symptoms, and / or can be a therapeutic effect that partially or completely cures the disease and / or adverse reactions caused by the disease. Therefore, the term "treatment" as used herein covers the treatment of any condition related to postoperative recovery in mammals, including: (a) preventing the subject from developing the condition; (b) inhibiting the condition, i.e., preventing it from occurring; or (c) alleviating the condition, i.e., causing the condition to subside. Treatment can result in a variety of different physical clinical manifestations, for example, regulation of gene expression, tissue or organ rejuvenation, reduced inflammation, etc. Therapeutic drugs can be administered before, during, or after the onset of disease. Treatment of a subject can be administered during the symptomatic stage of the condition and in some cases after the symptomatic stage of the condition.

[0055] Blood products containing plasma components.In the practice of the object method, the blood product comprising the plasma component is administered to the individual in need, such as, for example, an individual suffering from a postoperative condition. Therefore, the method according to an embodiment of the present invention includes administering a blood product comprising the plasma component from an individual ("donor individual", or "donor") to an individual suffering from a postoperative condition ("recipient individual" or "recipient"). "Blood product comprising plasma component" refers to any product derived from blood (such as whole blood, plasma or its components) comprising plasma. The term "plasma" is used in its conventional sense to refer to a pale yellow / light yellow liquid component in the blood, which is composed of about 92% water, 7% protein such as albumin, gamma globulin, antihemophilic factor and other coagulation factors, and 1% mineral salts, sugars, fats, hormones and vitamins. Non-limiting examples of blood products containing plasma suitable for use in the subject methods include: whole blood treated with an anticoagulant (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products produced by filtering whole blood to remove leukocytes ("leukopenia"), blood products composed of plasma derived from plasma removal or apheresis, fresh frozen plasma, blood products consisting essentially of purified plasma, and blood products consisting essentially of plasma components. In some cases, the plasma product used is a non-whole blood plasma product, i.e., the product is not whole blood, i.e., the product lacks one or more components of whole blood such as red blood cells, white blood cells, etc., to the extent that at least these components are present in whole blood. In some cases, the plasma product is substantially acellular if not completely acellular, wherein in this case, the cell content may be 5% by volume or less than 5% by volume, such as 1% by volume or less than 1% by volume, including 0.5% by volume or less than 0.5% by volume, wherein in some cases, the acellular plasma component is a composition that is completely free of cells, i.e., it does not contain cells.

[0056] Collection of blood products containing plasma components. Embodiments of the methods described herein include the administration of blood products comprising plasma components, which may be derived from donors, including human volunteers. The term "derived from humans" may be used to refer to such products. Methods for collecting blood products comprising plasma derived from donors are well known in the art. (See, e.g., AABB TECHNICAL MANUAL, (Mark A. Fung et al., eds., 18th ed. 2014), which is incorporated herein by reference).

[0057] In one embodiment, the donation is obtained by venipuncture. In other embodiments, the venipuncture is only a single venipuncture. In other embodiments, saline volume replacement is not used. In a preferred embodiment, a blood product comprising plasma is obtained using plasmapheresis. Plasmapheresis can include removing a volume of plasma corrected for weight while returning cellular components to the donor. In a preferred embodiment, sodium citrate is used during plasmapheresis to prevent cell coagulation. After the use of citrate, the volume of plasma collected from the recipient is preferably 690 mL to 880 mL, and is preferably coordinated with the recipient's body weight.

[0058] C. Plasma components

[0059] During World War II, there was a need for a stable plasma expander that could be used when soldiers were losing a lot of blood on the battlefield. Therefore, a method for preparing freeze-dried plasma was developed. However, since sterile water is required for the reconstitution of freeze-dried plasma, it is difficult to use on the battlefield. As an alternative, Dr. EJ Cohn suggested that albumin could be used and prepared a ready-to-use stable solution that could be immediately introduced to treat shock. (See Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., first edition 1991)). Dr. Cohn's method for purifying plasma components uses pre-cooled ethanol, takes advantage of its denaturing effect, and achieves separation by changing the pH value and temperature.

[0060] Embodiments of the methods of the present invention include administering plasma fractions to a subject. Fractionation refers to the process of separating specific protein subgroups from plasma. Fractionation techniques are known in the art and rely on procedures developed by Cohn et al. in the 1940s. (E. Cohn, Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. 68 J Am Chem Soc 459 (1946), which is incorporated herein by reference). The process involves multiple steps, each of which includes a specific ethanol concentration and pH, temperature and osmotic changes, resulting in selective precipitation of proteins. The precipitate can also be separated by centrifugation or precipitation. The original "Cohn fractionation process" involves separating proteins into five components by precipitation, namely component I, component II+III, component IV-1, component IV-4 and component V. Albumin is the endpoint (component V) product initially determined in the process. According to embodiments of the present invention, each component (or the effluent of the aforementioned separation step) contains or may contain a protein component that can be used for treatment. (See Thierry Burnouf, Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101 (2007); Adil Denizli, Plasma fractionation: conventional and chromatographic methods for albumin purification, 4 J. Biol. & Chem. 315, (2011); and T. Brodniewicz-Proba, Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products, 5 Blood Reviews 245 (1991), and U.S. Patents Nos. 3869431, 5110907, 5219995, 7531513, and 8772461, all of which are incorporated herein by reference).The experimental parameters described above can be adjusted to obtain specific protein fractions.

[0061] Recently, fractionation has become more complex and therefore constitutes other embodiments of the present invention. The recent increase in complexity is through: the introduction of chromatography leads to the isolation of new proteins from existing components such as cold precipitate, cold precipitate-reduced plasma and Cohn components; by combining chromatography and ethanol fractionation processes to improve the recovery of IgG; and virus reduction / inactivation / removal. (Similarly) anion exchange chromatography can be used to capture proteins at physiological pH and ionic strength. This preserves the functional activity of proteins and / or protein components. Heparin and monoclonal antibodies are also used for affinity chromatography. In addition, gel filtration fractionation, salt fractionation and polyethylene glycol fractionation are used. (Hosseini M Iran J Biotech, 14 (4): 213-20 (2016), which is incorporated herein by reference). It should be recognized by those skilled in the art that the above parameters and techniques can be adjusted to obtain specific required components containing plasma proteins.

[0062] Plasma fractionation can also be performed based on ammonium sulfate. (See, e.g., Odunuga OO, Biochem Compounds, 1:3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), which are incorporated herein by reference). In addition to obtaining specific blood components, ammonium sulfate-based fractionation has also been used to reduce the abundance of proteins in plasma. (Saha S et al., J. Proteomics Bioinform, 5(8) (2012), which is incorporated herein by reference).

[0063] In an embodiment of the present invention, plasma is fractionated in an industrial environment. Frozen plasma is thawed at 1°C to 4°C. Continuous refrigerated centrifugation is performed on the thawed plasma and the separated cryoprecipitate. The recovered cryoprecipitate is frozen and stored at a temperature of -30°C or below -30°C. The plasma reduced by the cryoprecipitate is immediately processed to capture (e.g., by primary chromatography) unstable coagulation factors, such as factor IX complex and its components and protease inhibitors such as antithrombin and C1 esterase inhibitors. Continuous centrifugation and precipitation separation can be used in subsequent steps. These technologies are known to those of ordinary skill in the art, such as described in U.S. Patent No. 4624780, No. 5219995, No. 5288853 and U.S. Patent Application No. 20140343255 and No. 20150343025, all of which are incorporated herein by reference.

[0064] In an embodiment of the present invention, the plasma component may include a plasma component containing a high concentration of albumin. In another embodiment of the present invention, the plasma component may include a plasma component (e.g., Gamunex-C®) containing a high concentration of IgG or intravenous immunoglobulin (IGIV). In another embodiment of the present invention, the plasma component may include an IGIV plasma component, such as Gamunex-C®, wherein immunoglobulins (IgG) have been fully removed by methods known to those of ordinary skill in the art, such as protein A-mediated removal. (See Keshishian, H. et al., Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury, Molecular & Cellular Proteomics, 14 at 2375-93 (2015)). In another embodiment, the plasma component may be a plasma component in which substantially all coagulation factors are removed, to retain the efficacy of the component while reducing the risk of thrombosis. For example, the plasma component can be a plasma component as described in U.S. Patent No. 62 / 376,529, filed on August 18, 2016; the entire disclosure of which is incorporated herein by reference.

[0065] D. Albumin products

[0066] For those skilled in the art, albumin plasma products (APP) are divided into two categories: plasma protein fraction (PPF) and human albumin solution (HAS). PPF is derived from a process having a higher yield than HAS but a lower minimum albumin purity than HAS (PPF is greater than 83%, HAS is greater than 95%). (Production of human albumin solution: a continually developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85 (6): 887-95, at 888 (2000)). In some cases, the albumin concentration of PPF is 83% to 95%, or 83% and 96%. Albumin purity can be determined by electrophoresis or other quantitative analysis methods such as mass spectrometry. In addition, it has been pointed out that PPF has a disadvantage because of the presence of protein "contaminants", such as PKA. The same. Therefore, PPF preparations have been unpopular as albumin plasma products and have even been removed from the pharmacopoeia of some countries. The same. In contrast to these problems, the present invention beneficially utilizes these "contaminants". In addition to alpha-, beta- and gamma-globulins and the above-mentioned PKA, the methods of the present invention utilize other proteins or other factors within "foci" to promote processes such as neurogenesis, neuronal cell survival, improved cognitive or motor function, and reduced neuroinflammation.

[0067] Those skilled in the art will recognize that there are or have been several commercial sources of PPF ("commercial PPF preparations"). Among them are Plasma-Plex™ PPF (Armour Pharmaceutical Co., Tarrytown, New York), Plasmanate™ PPF (Grifols, Clayton, North Carolina), Plasmatein™ (AlphaTherapeutics, Los Angeles, California), and Protenate™ PPF (Baxter Labs, Inc. Deerfield, Illinois). Those skilled in the art will also recognize that there are or have been several commercial sources of HSA ("commercial HAS preparations"). These include Albuminar™ (CSL Behring), AlbuRx™ (CSL Behring), Albutein™ (Grifols, Clayton, NC), Buminate™ (Baxatla, Inc., Bannockburn, IL), Flexbumin™ (Baxalta, Inc., Bannockburn, IL), and Plasbumin™ (Grifols, Clayton, NC).

[0068] 1. Plasma Protein Fraction (Human) (PPF)

[0069] According to the U.S. Food and Drug Administration ("FDA"), "Plasma Protein Fraction (Human)" or PPF is the proprietary name of the product, defined as "a sterile protein solution containing albumin and globulins derived from human plasma." (U.S. Code of Federal Regulations "CFR" 21 CFR 640.90, which is incorporated herein by reference). The raw material for PPF is plasma recovered from whole blood prepared in accordance with the provisions of 21 CFR 640.1-640.5 (incorporated herein by reference), or raw plasma prepared in accordance with the provisions of 21 CFR 640.60-640.76 (incorporated herein by reference).

[0070] The PPF was tested to determine compliance with the following standards of 21 CFR 640.92 (incorporated herein by reference):

[0071] (a) the final product should be a 5.0 + / - 0.30 percent protein solution; and

[0072] (b) The total protein of the finished product shall consist of at least 83 percent albumin and not more than 17 percent globulins. Gamma globulin shall not exceed 1 percent of the total protein. The method for determining protein composition shall be approved for each manufacturer by the Director of the Center for Biologics Evaluation and Research of the Food and Drug Administration.

[0073] As used herein, "plasma protein fraction" or "PPF" refers to a sterile protein solution containing albumin and globulin, which is derived from human plasma, wherein the albumin content is at least 83%, the content of globulin (including α1 globulin, α2 globulin, β globulin and γ globulin) and other plasma proteins does not exceed 17%, and the content of γ globulin does not exceed 1% by electrophoresis. (Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957)). PPF can also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin fraction can be determined by extracting albumin from the total protein. (Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY, PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker HK, Hall WD, Hurst JD ed. (1990)).

[0074] 2. Albumin (human) (HAS)

[0075] According to the FDA, "Albumin (Human)" (also referred to herein as "HAS") is the proprietary name of a product defined as "a sterile solution of albumin derived from human plasma" (US Code of Federal Regulations "CFR" 21 CFR 640.80, which is incorporated herein by reference). The raw material for Albumin (Human) is plasma recovered from whole blood prepared in accordance with the provisions of 21 CFR 640.1-640.5 (incorporated herein by reference), or raw plasma prepared in accordance with the provisions of 21 CFR 640.60-640.76 (incorporated herein by reference). Other requirements for Albumin (Human) are listed in 21 CFR 640.80-640.84 (incorporated herein by reference).

[0076] Albumin (human) was tested to determine compliance with the following standards of 21 CFR 640.82 (incorporated herein by reference):

[0077] (a) Protein concentration. The final product should meet one of the following concentrations: 4.0 + / - 0.25%; 5.0 + / - 0.30%; 20.0 + / - 1.2%; 25.0 + / - 1.5% protein solution.

[0078] (b) Protein composition.—At least 96 percent of the total protein in the final product shall be globulins, as determined by a method approved for each manufacturer by the Director, Center for Biologics Evaluation and Research, Food and Drug Administration.

[0079] As used herein, "albumin (human)" or "HAS" refers to a sterile protein solution comprising albumin and globulin, which is derived from human plasma, wherein the albumin content is at least 95%, and the content of globulin (including α1 globulin, α2 globulin, β globulin and γ globulin) and other plasma proteins does not exceed 5%. HAS may also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin composition can be determined by extracting albumin from the total protein.

[0080] Those skilled in the art will recognize that the PPF and HAS components may also be freeze-dried or in other solid forms. For example, these formulations may be used to make tablets, powders, granules or capsules with appropriate additives. The solid forms may be formulated into injection formulations by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol; and if necessary, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives may also be used.

[0081] E. Reduced components of coagulation factors

[0082] In other embodiments of the present invention, plasma components in which substantially all thrombin factors are removed are used to retain the effect of plasma components while reducing the risk of thrombosis. Conveniently, blood products can be obtained from young donors or young donor pools, and can be made to be free of IgM, so that ABO compatible young blood products are provided. At present, the plasma of transfusion is ABO blood type matching, because naturally occurring A and B antigen antibodies can cause transfusion reactions. When patients receive the mismatched plasma of ABO blood type, IgM seems to be the reason causing transfusion reactions. Removing IgM from blood products or blood components helps to eliminate the transfusion reactions of the object of using blood products of the present invention and plasma products.

[0083] Therefore, in one embodiment, the present invention relates to a method for treating an object suffering from an adverse condition associated with postoperative recovery. The method comprises: administering to the object a blood product or blood component derived from whole blood of an individual or a pool of individuals, wherein the blood product or blood component is substantially free of (a) at least one coagulation factor and / or (b) IgM. In some embodiments, the individual from which the blood product or blood component is derived is a young individual. In some embodiments, the blood product is substantially free of at least one coagulation factor and IgM. In a specific embodiment, the blood product is substantially free of fibrinogen (component I). In other embodiments, the blood product is substantially free of red blood cells and / or white blood cells. In other embodiments, the blood product is substantially acellular. In other embodiments, the blood product is derived from plasma. These embodiments of the present invention are also supported by U.S. Patent Application No. 62 / 376529, filed on August 18, 2016, the entire contents of which are incorporated herein by reference.

[0084] F. Handling of Protein-Rich Plasma Protein Products

[0085] Other embodiments of the present invention use plasma components, compared with PPF, in which the albumin concentration is reduced, but the content of globulin and other plasma proteins (called "contaminants" by some people) is increased. The embodiments using PPF, HAS, effluent I and effluent II / III all effectively remove coagulation factors. This plasma component is referred to as "protein-rich plasma protein products" in this article. For example, protein-rich plasma protein products containing 82% albumin and 18% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used in embodiments of the present invention. Protein-rich plasma protein products containing 81% albumin and 19% alpha globulin, beta globulin and gamma globulin and / or other plasma proteins can be used in other embodiments of the present invention. Protein-rich plasma protein products containing 80% albumin and 20% alpha globulin, beta globulin and gamma globulin and / or other plasma proteins can be used in other embodiments of the present invention. Protein-rich plasma protein products containing 70% to 79% albumin and correspondingly 21% to 30% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used in other embodiments of the present invention. In other embodiments of the present invention, protein-rich plasma protein preparations containing 60% to 69% albumin and correspondingly 31% to 40% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 50% to 59% albumin and correspondingly 41% to 50% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 40% to 49% albumin and correspondingly 51% to 60% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 30% to 39% albumin and correspondingly 61% to 70% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 20% ​​to 29% albumin and correspondingly 71% to 80% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 10% to 19% albumin and correspondingly 81% to 90% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 1% to 9% albumin and correspondingly 91% to 99% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used. In other embodiments of the present invention, protein-rich plasma protein preparations containing 0% albumin and 100% alpha globulin, beta globulin and gamma globulin and other plasma proteins can be used.

[0086] The above embodiments of the present invention may also contain a total gamma globulin concentration of 1% to 5%.

[0087] The specific concentration of a protein in a plasma fraction can be determined by techniques known to those of ordinary skill in the relevant art, including, by way of example and not limitation, electrophoresis, mass spectrometry, ELISA analysis, and Western blot analysis.

[0088] G. Preparation of Plasma Fractions

[0089] The preparation methods of PPF and other plasma fractions are well known to those skilled in the art. One embodiment of the present invention allows the collection of blood for the preparation of human plasma protein fractions in a flask using citrate or anticoagulant citrate dextrose solution (or other anticoagulants) for inhibiting coagulation, and further separation of fractions I, II+III, IV and PPF according to the method disclosed by Hink et al. (see Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2 (174) (1957), which is incorporated herein by reference). According to the method, the mixture can be collected at 2°C to 8°C. The plasma can then be centrifuged at 7°C, removed and stored at -20°C. The plasma can then be thawed and fractionated at 37°C, preferably within 8 hours after being removed from the -20°C storage state.

[0090] Plasma can be separated from fraction I using 8% ethanol at pH 7.2, temperature -2°C to -2.5°C, protein concentration 5.1% to 5.6%. Cold 53.3% ethanol (176 mL / L of plasma) and acetate buffer (200 mL of 4M sodium acetate, 230 mL of glacial acetic acid, plus enough H2O to 1 L) can be added at a rate of, for example, 450 mL / min using a sparger during the process of lowering the temperature of the plasma to -2°C. Fraction I can be separated and removed from the effluent (effluent I) by ultracentrifugation. Fibrinogen can be obtained from fraction I according to methods known to those of ordinary skill in the art.

[0091] Fraction II+III can be separated from effluent I by adjusting the effluent to 21% ethanol at pH=6.8, temperature -6°C, and protein concentration of 4.3%. Cold 95% ethanol (176 mL / L of effluent I) and 10M acetic acid for pH adjustment can be added at a rate of, for example, 500 mL / min using a sparger during the process of lowering the plasma temperature to -6°C. The resulting precipitate (fraction II+III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from fraction II+III according to methods known to those skilled in the art.

[0092] Fraction IV-1 can be separated from effluent II+III ("effluent II / III") by adjusting the effluent to 19 percent ethanol at pH = 5.2, temperature -6°C, and protein concentration of 3 percent. While effluent II+III is incubated at -6°C for 6 hours, water and 10 M acetic acid for pH adjustment can be added using a sparger. Precipitated fraction VI-1 can be allowed to stand at -6°C for 6 hours and then separated from the effluent by centrifugation at the same temperature. A stable plasma protein fraction can be recovered from effluent IV-1 by adjusting the ethanol concentration to 30 percent at pH = 4.65, temperature -7°C, and protein concentration of 2.5 percent. This can be achieved by adjusting the pH of effluent IV-1 using cold acid-alcohol (2 parts 2 M acetic acid and 1 part 95 percent ethanol). While maintaining the temperature at -7°C, 170 mL of cold ethanol (95%) is added per liter of conditioned effluent IV-1. The precipitated protein was allowed to stand for 36 hours and then removed by centrifugation at -7°C.

[0093] The recovered protein (stable plasma protein fraction) can be dried (e.g., freeze-dried) to remove ethanol and H2O. The obtained dry powder can be dissolved in sterile distilled water, e.g., 15 liters of water per kg of powder, and the pH of the solution is adjusted to 7.0 using 1 M NaOH. A final protein concentration of 5 percent can be obtained by adding sterile distilled water containing sodium acetyltryptophanate, sodium octanoate, and NaCl to a final concentration of 0.004 M sodium acetyltryptophanate, 0.004 M sodium octanoate, and 0.112 M sodium. Finally, the solution is filtered at 10°C to obtain a clear solution, which is then heat-treated at 60°C for at least 10 hours to inactivate pathogens.

[0094] Those of ordinary skill in the art will recognize that any of the above-described different components and effluents can be used in the methods of the present invention to treat conditions associated with postoperative recovery. For example, but not by way of limitation, effluent I or effluent II / III can be used to treat conditions associated with postoperative recovery or to accelerate postoperative recovery and are embodiments of the present invention.

[0095] The above-mentioned method for preparing plasma fraction and plasma protein fraction (PPF) is only exemplary and only relates to embodiments of the present invention. It will be appreciated by those skilled in the art that these methods can be changed. For example, in different embodiments and methods of the present invention, pH, temperature and ethanol concentration, etc. can be adjusted, among others, to produce different changes of plasma fraction and plasma protein fraction. In another embodiment, other embodiments of the present invention contemplate the use of nanofiltration to remove / inactivate pathogens from plasma fraction and plasma protein fraction.

[0096] Additional embodiments of the present invention contemplate methods and compositions using and / or comprising other plasma fractions. For example, the present invention contemplates, among other things, that a particular concentration of albumin is not critical for treating conditions associated with or accelerating postoperative recovery. Thus, the present invention contemplates the use of fractions having reduced albumin concentrations, such as fractions having an albumin content of less than 83%.

[0097] H. Treatment

[0098] Aspects of the methods of the present invention described herein include treating a subject with a blood product comprising plasma, wherein the blood product is, for example, a plasma component, wherein the plasma component is, for example, a plasma component as described above. Embodiments include treating a human subject with a blood product comprising plasma. Those skilled in the art will recognize that methods for treating a subject with a blood product comprising plasma are recognized in the art. By way of example and not limitation, one embodiment of the methods of the present invention includes administering fresh frozen plasma to a subject to treat a condition associated with postoperative recovery. In one embodiment, the blood product comprising plasma is administered immediately to a patient with a condition associated with postoperative recovery, for example, within about 12 hours to 48 hours after collection from a donor. In this case, the product can be stored refrigerated, for example, at 0°C to 10°C. In another embodiment, fresh frozen plasma is plasma frozen at or below -18°C (cryopreserved). Prior to administration, fresh frozen plasma is thawed, and after thawing, administered to the subject within 60 minutes to 75 minutes after the start of the thawing process. Each object preferably receives a single unit of fresh frozen plasma (200 mL to 250 mL), and fresh frozen plasma is preferably derived from a donor of a predetermined age range. In one embodiment of the invention, the fresh frozen plasma is donated by a young individual (derived from a young individual). In another embodiment of the invention, the fresh frozen plasma is donated by a donor of the same sex (derived from a donor of the same sex). In another embodiment of the invention, the fresh frozen plasma is donated by a donor of 18 to 22 years old (derived from a donor of 18 to 22 years old).

[0099] In an embodiment of the present invention, the composition of the present invention (e.g., a blood product comprising plasma, wherein the blood product is, for example, a plasma component) is administered by intravenous injection. The composition of the present invention can also be delivered intraperitoneally. In another embodiment of the present invention, the composition of the present invention can be delivered orally, subcutaneously or topically. As known in the art, typical preparations for treating wounds and promoting wound healing are gels, creams, ointments, gauze, patches, etc., and the composition of the present invention can be formulated in this way. (See, e.g., Kahn AW et al., Pharmacogn Mag, 9 (Suppl 1): S6-S10 (2013); U.S. Patent Application No. 5641483; U.S. Patent Application No. 4885163; U.S. Patent Application No. 8313764, all of which are incorporated herein by reference).

[0100] In an embodiment of the present invention, blood products comprising plasma are screened by blood type after donation. In another embodiment of the present invention, blood products comprising plasma are screened for infectious pathogens, such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc, according to the requirements of 21 CFR 640.33 and the recommendations contained in the FDA guidance document.

[0101] In another embodiment of the present invention, the subject receives plasma component treatment. In an embodiment of the present invention, the plasma component is PPF or HAS. In another embodiment of the present invention, the plasma component is one of a commercial PPF preparation or a commercial HAS preparation. In another embodiment of the present invention, the plasma component is derived from a specific age range individual library, for example, PPF or HAS derived from a young individual, or is a modified PPF or HAS component (for example, PPF or HAS in which one or more specific proteins are partially or substantially removed) after additional fractionation or treatment. In another embodiment of the present invention, the plasma component is an IGIV plasma component that has been substantially removed from immunoglobulins (IgG). A blood component that is "substantially removed" or its specific proteins such as IgG are "substantially removed" refers to a blood component containing less than about 50% of the content present in a reference product or whole blood plasma, for example, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, .25%, .1%, undetectable levels, or any integer between these values, and the percentage is measured using standard measurement methods known in the art.

[0102] I. Application

[0103] Various aspects of the methods of the present invention described herein include treating a subject with a blood product containing plasma, wherein the blood product is, for example, plasma or a plasma component, wherein the plasma or plasma component is, for example, plasma or a plasma component as described above. Embodiments include treating human subjects with a blood product containing plasma. Those skilled in the art will recognize that methods for treating subjects with blood products containing plasma are recognized in the art. By way of example and not limitation, one embodiment of the method of the present invention includes administering fresh frozen plasma to a subject to treat a condition associated with postoperative recovery. In one embodiment, the blood product containing plasma is administered immediately to a patient suffering from an adverse condition associated with postoperative recovery, for example, within about 12 hours to 48 hours after collection from a donor. In this case, the product can be stored refrigerated, for example, at 0°C to 10°C. In another embodiment, fresh frozen plasma is plasma frozen at or below -18°C (cryopreserved). Prior to administration, fresh frozen plasma is thawed, and after thawing, administered to the subject within 60 minutes to 75 minutes after the start of the thawing process. Each subject preferably receives a single unit of fresh frozen plasma (200 mL to 250 mL), and fresh frozen plasma is preferably derived from a donor of a predetermined age range. In one embodiment of the invention, the fresh frozen plasma is donated by a young individual (derived from a young individual). In another embodiment of the invention, the fresh frozen plasma is donated by a donor of the same sex (derived from a donor of the same sex). In another embodiment of the invention, the fresh frozen plasma is donated by a donor aged 18 to 22 years old (derived from a donor aged 18 to 22 years old).

[0104] In an embodiment of the present invention, blood products comprising plasma are screened by blood type after donation. In another embodiment of the present invention, blood products comprising plasma are screened for infectious pathogens, such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc, according to the requirements of 21 CFR 640.33 and the recommendations contained in the FDA guidance document.

[0105] In another embodiment of the present invention, the subject receives plasma component treatment. In an embodiment of the present invention, the plasma component is PPF or HAS. In another embodiment of the present invention, the plasma component is one of a commercial PPF preparation or a commercial HAS preparation. In another embodiment of the present invention, the plasma component is derived from a specific age range individual library, for example, PPF or HAS derived from a young individual, or a modified PPF or HAS component (for example, PPF or HAS in which one or more than one specific protein is partially or substantially removed) after additional fractionation or treatment. In another embodiment of the present invention, the plasma component is an IGIV plasma component that has been substantially removed from immunoglobulins (IgG). A blood component that is "substantially removed" or its specific protein, such as IgG, is "substantially removed" and refers to a blood component containing less than about 50% of the content present in a reference product or whole blood plasma, for example, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, .25%, .1%, undetectable levels, or any integer between these values, and the value is determined using standard measurement methods known in the art.

[0106] Embodiments of the present invention include treating a subject with a postoperative recovery-related condition by administering an effective amount of plasma or plasma fractions to the subject. Another embodiment of the present invention includes administering an effective amount of plasma or plasma fractions, followed by monitoring the subject for functional improvement, wound healing, marker presence, pain relief, or inflammation relief. Another embodiment of the present invention includes treating a subject with a postoperative recovery-related condition by administering an effective amount of plasma or plasma fractions to the subject, wherein after reaching the mean half-life or median half-life of the plasma protein or plasma fraction protein, the plasma or plasma fraction is administered in a manner that achieves functional improvement, wound healing, marker presence, pain relief, or inflammation relief relative to the most recent dose (referred to herein as "pulse administration" or "pulse administration") (see U.S. Patent No. 10357513, U.S. Patent Application Nos. 15 / 961618 and 62 / 701411, the entire contents of which are incorporated herein by reference). Another embodiment of the present invention includes administering the plasma or plasma fractions by a dosing regimen of at least two consecutive days, and monitoring the subject for functional improvement or HSC marker levels for at least 3 days after the last administration date. Other embodiments of the present invention include administering the plasma or plasma components by a dosing regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, and monitoring the functional improvement, wound healing, marker presence, pain relief, or inflammation relief of the subject for at least 3 days after the last administration date. Other embodiments of the present invention include administering the plasma or plasma components by a dosing regimen of at least 2 consecutive days, and after the last administration date, when the plasma or plasma components reach the average half-life, monitoring the functional improvement, wound healing, marker presence, pain relief, or inflammation relief of the subject. Other embodiments of the present invention include administering the plasma or plasma components by a dosing regimen of 2 to 14 consecutive days, wherein the interval between each administration can be 0 to 3 days.

[0107] In some cases, the pulse administration according to the present invention includes the administration of a first group of doses, such as the first group of doses as described above, followed by a period of non-administration, such as a "non-administration" period, followed by another dose or another group of doses. In some embodiments, the duration of the "non-administration" period can be changed to 7 days or more than 7 days, such as 10 days or more than 10 days, including 14 days or more than 14 days, wherein the non-administration period in some cases is 15 days to 365 days, such as 30 days to 90 days, including 30 days to 60 days. Therefore, the embodiment of the method includes non-long-term (i.e. non-continuous) administration, for example, non-long-term administration of plasma products. In some embodiments, the pattern of non-administration period followed by pulse administration is repeated multiple times as needed, wherein in some cases, this pattern lasts for 1 year or longer than 1 year, such as 2 years or longer than two years, until and including the life of the object. Other embodiments of the present invention include administering the plasma or plasma component by a dosing regimen of at least 5 consecutive days, followed by a non-administration period of 2 to 3 days, and then administering for 2 to 14 consecutive days.

[0108] Biochemically, an "effective amount" or "effective dose" of an active ingredient refers to a dose of the active ingredient that inhibits, antagonizes, reduces, decreases or suppresses about 20% or more, such as 30% or more, 40% or more, or 50% or more, in some cases 60% or more, 70% or more, 80% or more, or 90% or more, in some cases about 100% of the adverse conditions associated with postoperative recovery, even if they are negligible, and in some cases reverses the adverse conditions associated with postoperative recovery.

[0109] J. Plasma protein components

[0110] In the implementation method of the present invention, a plasma fraction is administered to a subject. In an embodiment, the plasma fraction is a plasma protein fraction (PPF). In another embodiment, the PPF is selected from a commercial PPF preparation.

[0111] In another embodiment, the PPF comprises 88% normal human albumin, 12% alpha globulin and beta globulin, and no more than 1% gamma globulin as detected by electrophoresis. Further embodiments of this embodiment for use in the embodiments of the present invention include, for example, a 5% PPF solution buffered with sodium carbonate and stabilized with 0.004M sodium octanoate and 0.004M acetyltryptophan. Other formulations may be used in the embodiments of the present invention, including adjusting the percentage of PPF in the solution (e.g., from about 1% to about 10%, from about 10% to about 20%, from about 20% to about 25%, from about 25% to about 30%) and the concentrations of solvents and stabilizers.

[0112] K. Plasma components by age of donor

[0113] Other embodiments of the present invention include administering a plasma protein fraction derived from the plasma of an individual of a specific age range. Embodiments include administering a PPS or HAS derived from the plasma of a young individual. In other embodiments of the present invention, the young individual is a single specific age or a specific age range. In other embodiments of the present invention, the average age of the donor is less than the average age of the subject or less than the average age of the subject being treated.

[0114] A specific embodiment of the present invention comprises pooling blood or plasma from individuals within a specific age range, and fractionating the plasma as described above to obtain a plasma protein fraction preparation, such as PPF or HAS. In an alternative embodiment of the present invention, the plasma protein fraction or a specific plasma protein fraction is obtained from a specific individual that meets a specific age range.

[0115] L. Indications

[0116] The methods of the present invention and blood products and plasma fractions containing plasma can be used to treat adverse conditions associated with postoperative recovery, and even accelerate postoperative recovery. By way of example and not limitation, the conditions and indications include pain and wound healing. The methods and compositions of the present invention can also be used to treat acute and chronic pain in diseases or conditions that are not necessarily related to postoperative recovery. The methods and compositions of the present invention can also be used to treat wound healing that is not necessarily related to postoperative recovery. The methods and compositions of the present invention can also be used to promote or stimulate myelin regeneration and treat diseases associated with myelination such as multiple sclerosis.

[0117] The methods of the present invention and the blood products and plasma fractions containing plasma can also be used to treat indications related to the nervous system. By way of example and not limitation, such conditions include central nervous system conditions such as central neuropathic pain, spinal cord injury, myelopathy, and central neuropathic pain associated with postoperative recovery. There are 17,000 new cases of spinal cord injury each year, affecting approximately 300,000 people, of which 40% to 75% of patients with spinal cord injury suffer from central neuropathic pain. (Jadad A et al., AHRQ Evidence Report Summaries, Agency for Healthcare Research and Quality; (1998-2005); https: / / www.nscisc.uab.edu / Public / Facts%202016.pdf ; and https: / / www.nscisc.uab.edu / PublicDocuments / fact_figures_docs / Facts%202012%20Feb% 20Final.pdf). One-third of patients experience severe pain, and only one-third of these patients experience a 50% or greater pain relief after treatment. (Charbonneau R, CMAJ, 189(2):E48-E49 (2017); and Hadjipavlou G et al., BJA Education, 16(8):264-68 (2016)). The incidence of myelopathy is 605 cases per 1,000,000 population, and there are surgical options but no medical treatments, indicating an unmet need in this area. (Nouri A et al., Spine, 40(12):E675-93 (2015); The Lancet Neurology, editorial 18(7):P615(2019)).

[0118] By way of example and not limitation, these conditions also include plexus / radiculopathy, such as plexopathy, cervical radiculopathy, sciatica (lumbar radiculopathy). The incidence of plexopathy is 2 to 3 cases per 100,000 people. Current options include the use of anti-epileptic drugs and antidepressants to treat neuropathic pain, which indicates that there is an unmet need. The prevalence of cervical radiculopathy is 100 cases per 100,000 people in men and 60 cases per 100,000 people in women. (McCartney S et al., Br. J. Gen. Pract., 68(666):44-46 (2018)). The annual prevalence of sciatica is 1% to 5%, and although many cases are spontaneously relieved, sciatica responds less and less to treatment as the onset time increases. Treatment options include surgery, standard analgesics and steroids, which indicates that new treatments are needed. (Lewis R et al., Health Technology Assessment – ​​The Clinical Effectiveness and Cost-Effectiveness of Management Strategies for Sciatica: Systematic Review andEconomic Model, No. 15.39 NIHR Journals Library (2011)).

[0119] Other indications include diseases of the peripheral nervous system. By way of example and not limitation, they include peripheral neuropathy; peripheral neuropathy associated with recovery from surgery; carpal tunnel syndrome; chemotherapy-induced peripheral neuropathy; compression and trauma; diabetic neuropathy; peripheral neuropathy associated with herpes zoster (postherpetic neuralgia); complex regional pain syndrome; and trigeminal neuralgia. Peripheral neuropathy is a disorder of the peripheral nerves that affects at least 20 million people in the United States. Almost 60 percent of people with diabetes suffer from diabetic neuropathy, a type of peripheral neuropathy. ( http: / / www.healthcommunities.com / neuropathy / overview-of-neuropathy.shtml ). Carpal tunnel syndrome affects 3% to 6% of adults, and treatments include splints, steroids, and surgery. (LeBlanc KE et al., Am FamPhysician, 83(8):952-58 (2011)). Chemotherapy-induced peripheral neuropathy occurs in 40% to 60% of patients during and 3 months after chemotherapy, which is reported in 650,000 patients each year. Peripheral neuropathy leads to chemotherapy dose reduction or even cessation, affects quality of life, and no medication or supplement has been shown to prevent the disease. (JAMAOncology, 5(5):750, (2019)). Peripheral neuropathy related to compression and trauma occurs in 2% to 3% of trauma patients, which affects 3 million trauma patients in the United States. Although surgery is often effective, new medications are still needed. (AmericanAssociation for the Surgery of Trauma – Trauma Facts , available at http: / / www.aast.org / trauma-facts; and Novak CB, Medscape – Peripheral Nerve Injuries, (Oct 5, 2018), available at https: / / emedicine.medscape.com / article / 1270360- overview).

[0120] It has also been found that the methods of the present invention and blood products and plasma fractions containing plasma can be used to treat other peripheral nervous system indications including diabetic neuropathy. In the United States, there are approximately 30 million diabetic patients, of which 8% to 26% suffer from neuropathy. (Risson V et al., Incidence and prevalence of painful diabeticneuropathy and postherpetic neuralgia in major 5 European countries, the United States and Japan, Value in Health (20): A339-A811 PSY18 (2017), available at https: / / www.valueinhealthjournal.com / article / S1098-3015(17)31179-8 / pdfFDA-approved treatments for diabetic neuropathy include pregabalin, duloxetine, fluoxetine, and tapentadol, but many patients do not respond to these drugs, and none of them directly treat nerve damage.

[0121] It has also been found that peripheral neuropathy associated with herpes zoster (postherpetic neuralgia) can be treated by the methods and products of the present invention. Twenty percent of people with herpes zoster will suffer from postherpetic neuralgia, and there are 1 million cases in the United States each year. (See https: / / emedicine.medscape.com / article / 1143066-overview#a6 https: / / www.cdc.gov

[0122] / shingles / hcp / clinical-overview.html.) Gabapentin and pregabalin are approved for the treatment of this condition, but the pain is often difficult to treat. (Sacks GM, Am J Manag Care 19(1 Suppl):S207-13 (2013))

[0123] Other peripheral neuropathy indications, such as complex regional pain syndrome and trigeminal neuralgia, can be treated using the methods and compositions of the invention. They occur in 5.5 to 26 cases per 100,000 population. They are associated with severe pain and disability, and their response to treatment is variable, indicating a high unmet need. (Complex Region Pain Syndrome Fact Sheet, National Institutes of Health - National Institute of Neurological Disorders and Stroke, available at https: / / www.ninds.nih.gov / Disorders / Patient-Caregiver-Education / Fact-Sheets / Complex-Regional-Pain-Syndrome-Fact-Sheet ). Trigeminal neuralgia affects between 4.2 and 28.9 people per 100,000. It has a significant impact on quality of life and can become resistant to treatment over time, requiring patients to try multiple different treatments. (Wu N et al., J Pain, 18(Suppl 4):S69, (2017)). The only approved treatment is carbamazepine. Therefore, there is an unmet need for treatments for the pain these patients experience.

[0124] Other indications that can be treated using the methods and compositions of the invention include the following examples: central post-stroke pain; central pain in multiple sclerosis; post-traumatic headache; De Roche-Barre syndrome; optic neuritis; mitochondrial optic neuropathy; ischemic optic neuropathy; neuromyelitis optica; hereditary optic neuropathy; alcoholic neuropathy; Guillain-Barré syndrome; chronic inflammatory demyelinating polyradiculoneuropathy (CIDP); multifocal motor neuropathy (MNN); paraneoplastic autonomic neuropathy; sarcoidosis-associated peripheral neuropathy; rheumatoid arthritis-associated peripheral neuropathy; systemic lupus erythematosus-associated peripheral neuropathy; Sjögren's syndrome-associated peripheral neuropathy; celiac disease-associated peripheral neuropathy associated with leprosy; peripheral neuropathy associated with hepatitis B; peripheral neuropathy associated with hepatitis C; peripheral neuropathy associated with HIV / AIDS; peripheral neuropathy associated with amyloidosis; peripheral neuropathy associated with anti-MAG; peripheral neuropathy associated with cryoglobulinemia; peripheral neuropathy associated with POEMS; toxic peripheral neuropathy; peripheral neuropathy associated with nephropathy; peripheral neuropathy associated with vasculitis; peripheral neuropathy associated with vitamin and nutritional deficiency; Charcot-Marie-Tooth (CMT); idiopathic peripheral neuropathy; fibromyalgia; and paraneoplastic peripheral neuropathy.

[0125] It has also been found that the methods of the present invention and blood products and plasma fractions containing plasma can be used to treat indications related to wound healing. By way of example and not limitation, the wound can be an abrasion, avulsion, cut, laceration, and puncture. The indications can include chronic wounds and acute wounds. By way of example and not limitation, wound indications include: chronic wounds, such as diabetic ulcers, pressure ulcers, venous ulcers, arterial ulcers; and acute wounds, such as surgical wounds, traumatic wounds, and burns. However, any type of chronic wound or acute wound can be treated with the methods and compositions of the present invention.

[0126] Diabetic ulcers affect more than 2.2 million people in the United States, and the global prevalence is 6.4%. (Chun D et al., J Clin Med, 8:748 (2019)). Despite several treatment options such as debridement and medical dressings, many patients still suffer from infection and ultimately require amputation, highlighting the need for new treatments, especially drug therapies.

[0127] The overall incidence of pressure ulcers in hospitalized patients is 1.8%, with hundreds of thousands of cases each year. (Bauer K et al., Ostomy Wound Manage, 62(11):30-38 (2016)). Like diabetic ulcers, there are treatment options such as debridement and medical dressings, but many patients will become infected and the ulcers can lead to death.

[0128] Venous ulcers occur primarily in the legs and constitute a significant burden in the elderly, occurring in approximately 1% of the world's population. (Nelzen O, Phlebolymphology, 15(4) (2008)). Venous ulcers are difficult to heal compared to other chronic ulcers and have a significant tendency to recur. As with diabetic and pressure ulcers, treatment options such as debridement and medical dressings exist, but their recurrence highlights the need for new treatments, particularly drug-based ones. The incidence of arterial ulcers is approximately one-quarter that of venous ulcers. (Gabriel A, Vascular Ulcers, (2018), available at https: / / emedicine.medscape.com / article / 1298345-overview#a6 ). Treatment options also include debridement and medical dressings, but there is a lack of approved pharmaceutical agents.

[0129] Approximately 1.3 million patients sustain surgical wounds each year. (See MediWound – Innovating Solutionsfor Wound & Burn Care (2019) available at 19 http: / / ir.mediwound.com / static- files / cd547017-d1ed-460e-8cb2-0550b1e18a29 ). Surgical wounds are usually cuts or incisions made by a scalpel in the skin during surgery, but they may also be caused by the placement of drains during surgery. The healing of surgical wounds is a key outcome of surgery. Postoperative wound disruption or separation of wound layers with fascial disruption can be a serious complication. (See Hospital Harm Improvement Resource – Wound Disruption (2016), available at https: / / www.patientsafetyinstitute.ca / en / toolsResources / Hospital-Harm- Measure / Documents / Resource-Library / HHIR%20Wound%20Disruption.pdf ). In addition, surgical wounds in older patients take longer to heal than in younger individuals. (Gerstein AD, Dermatol Clin, 11(4):749-57 (1993)).

[0130] Traumatic wounds are primarily cuts, lacerations, puncture wounds, or abrasions that have caused damage to the skin and underlying tissue. Traumatic wounds are generally divided into three categories: acute wounds; lacerations, and penetrating wounds. Acute wounds are tears or lacerations of the skin that are jagged and often contain foreign matter such as glass, metal, gravel, sand, or dirt. Lacerations are caused by a sharp object penetrating the skin and underlying subcutaneous tissue. Penetrating wounds are the deepest and most serious of the three types. Typical examples are stab wounds and gunshot wounds. (See Traumatic Wounds, available at https: / / www.woundcarecenters.org / article / wound- types / traumatic-wounds; and Leaper DJ, BMJ, 332(7540):532-35 (2006)). Despite several physical treatment options (e.g., suturing), pharmacological intervention is still required.

[0131] The World Health Organization estimates that 180,000 people die each year from burns. The main cause of morbidity is non-fatal burns, including prolonged hospitalization. https: / / www.who.int / news-room / fact-sheets / detail / burns ). Typical treatment methods include surgical treatment and dressings. Drug treatment focuses on analgesia, infection control, sedation, replacement of circulating blood volume, anticoagulation and nutrition. (Green A et al., Clinical Pharmacist, 2:249-54 (2010)). The methods and compositions of the present invention can fill the unmet need for drug intervention to promote the healing of skin and underlying tissue injuries.

[0132] The methods and blood products and plasma fractions comprising plasma of the present invention can be used to treat conditions and indications associated with postoperative recovery at different time points. By way of example and not limitation, administration to a subject can be performed preoperatively, perioperatively (during a surgical procedure), or postoperatively.

[0133] One embodiment of the present invention is that the methods of the present invention and blood products and plasma fractions containing plasma can be used to treat pain. By way of example and not limitation, the pain can include acute pain and chronic pain. Another embodiment of the present invention is that the methods of the present invention and blood products and plasma fractions containing plasma can also be used to treat central pain or central neuropathy. Central pain includes neurological diseases caused by damage or dysfunction of the central nervous system (CNS), wherein the central nervous system (CNS) includes the brain, brainstem and spinal cord. It may affect a large part of the body or it may be limited to a specific area. The pain can be continuous or intermittent. The degree of pain can be moderate to severe. This pain may also be affected by touch, movement, emotions and temperature changes. The pain may also occur immediately after the causative event occurs, or it may be delayed for months or years. (See Central Pain Information Page - National Institute of Neurological Disorders and Stroke, Central Pain Syndrome Information Page, available at https: / / www.ninds.nih.gov / disorders / all-disorders / central-pain-syndrome-information-page; and Colloca L et al., Nat Rev Dis Primers, 3:17002 (2017)). Another embodiment of the invention is the use of the methods of the invention and blood products and plasma fractions comprising plasma to treat: spinal cord injury (SCI); myelopathy; plexopathy; cervical radiculopathy; sciatica (lumbar radiculopathy); central post-stroke pain; central pain in multiple sclerosis; post-traumatic headache; De-Roche syndrome; optic neuritis; mitochondrial optic neuropathy; ischemic optic neuropathy; neuromyelitis optica; and hereditary optic neuropathy.

[0134] In another embodiment of the present invention, the methods and blood products and plasma fractions comprising plasma of the present invention can also be used to treat peripheral pain or peripheral neuropathy. Peripheral neuropathy can refer to several conditions involving damage to the peripheral nervous system. More than 100 types of peripheral neuropathy have been identified, and these lesions can include motor nerves, sensory nerves, and autonomic nerves, depending on the type of nerve that is damaged. (See Central Page Information Page - National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy Fact Sheet, available at https: / / www.ninds.nih.gov / Disorders / Patient-Caregiver-Education / Fact- Sheets / Peripheral-Neuropathy-Fact-Sheet; and Colloca L et al., Nat Rev Dis Primers, 3:17002 (2017)). Other embodiments of the invention include using the methods of the invention and blood products and plasma fractions comprising plasma to treat: carpal tunnel syndrome; chemotherapy-induced peripheral neuropathy; compression and trauma; diabetic neuropathy; peripheral neuropathy associated with herpes zoster (postherpetic neuralgia); complex regional pain syndrome; trigeminal neuralgia; alcoholic neuropathy; Guillain-Barré syndrome; chronic inflammatory demyelinating polyneuropathy (CIDP); multifocal motor neuropathy (MNN); paraneoplastic autonomic neuropathy; sarcoidosis-associated peripheral neuropathy; rheumatoid arthritis-associated peripheral neuropathy; systemic lupus erythematosus-associated peripheral neuropathy; Sjögren's syndrome-associated peripheral neuropathy; chylomicronized Diarrhea-associated peripheral neuropathy; Bell's palsy; Lyme disease-associated peripheral neuropathy; leprosy-associated peripheral neuropathy; hepatitis B-associated peripheral neuropathy; hepatitis C-associated peripheral neuropathy; HIV / AIDS-associated peripheral neuropathy; amyloidosis-associated peripheral neuropathy; anti-MAG-associated peripheral neuropathy; cryoglobulinemia-associated peripheral neuropathy; POEMS-associated peripheral neuropathy; toxic peripheral neuropathy; nephropathy-associated peripheral neuropathy; vasculitis-associated peripheral neuropathy; vitamin and nutrient deficiency-associated peripheral neuropathy; Charcot-Marie-Tooth disease (CMT); idiopathic peripheral neuropathy; fibromyalgia; and paraneoplastic peripheral neuropathy.

[0135] One embodiment of the invention is that the methods of the invention and blood products and plasma fractions comprising plasma can treat wounds by promoting wound healing. Other embodiments of the invention include treating chronic or acute wounds with the methods of the invention and blood products and plasma fractions comprising plasma. Other embodiments of the invention include treating: diabetic ulcers; pressure ulcers; venous ulcers; arterial ulcers; surgical wounds; traumatic wounds; and burns.

[0136] M. Reagents, Devices, and Kits

[0137] The present invention also provides reagents, devices and kits thereof for implementing one or more than one of the above-mentioned dispensing. The reagents, devices and kits thereof of the present invention may vary greatly.

[0138] Reagents and devices of interest include those described above with respect to methods for preparing blood products containing plasma for transfusion to subjects requiring, for example, anticoagulants, cryoprotectants, buffers, isotonic solutions, and the like.

[0139] The kit may also include a blood collection bag, a catheter, a needle, a centrifuge tube, etc. In other embodiments, the kit of the present invention includes two or more plasma product containers, such as plasma protein component containers, such as three or more three, four or more four, five or more five, including six or more six plasma product containers. In some cases, the number of different plasma product containers in the kit may be 9 or more, 12 or more, 15 or more, 16 or more, 21 or more, 24 or more, 30 or more, including 36 or more, such as 48 or more. Each container may have associated therewith identification information, the identification information including various data about the plasma product contained therein, the identification information may include one or more of the donor age of the plasma product, the processing details of the plasma product, blood type information, etc., wherein the processing details of the plasma product, such as whether the plasma product has been processed to remove proteins above the average molecular weight (e.g., as described above). In some cases, each container in the test kit contains identification information about the plasma contained therein, wherein the identification information includes information about the donor age of the plasma product, for example, the identification information provides confirmation data related to the age of the plasma product donor (wherein the identification information can be the age of the donor when collected). In some cases, each container in the test kit contains plasma products from donors of substantially the same age, that is, all containers contain products from donors of substantially the same age or if different. Substantially the same age refers to that the age difference between each donor as the source of the plasma product in the test kit is 5 years or less than 5 years in some cases, for example, 4 years or less than 4 years, such as 3 years or less than 3 years, including 2 years or less than 2 years, for example 1 year or less than 1 year, such as 9 months or less than 9 months, 6 months or less than 6 months, 3 months or less than 3 months, including 1 month or less than 1 month. The identification information can be displayed on any convenient component of the container, such as a label, an RFID chip, etc. The identification information can be human-readable, computer-readable, etc. as required. The container can have any convenient configuration. The volume of the container can be changed, and in some cases the volume of the container is 10 mL to 5000 mL, for example, 25 mL to 2500 mL, for example, 50 mL to 1000 mL, including 100 mL to 500 mL. The container can be rigid or flexible, and can be made of any convenient material, such as a polymeric material, including a medical grade plastic material. In some cases, the container has a bag or bag-like structure. In addition to the container, the test kit can also include an application device such as described above. The components of the test kit can be arranged in any suitable packaging configured to accommodate the container and other test kit components, such as a box or similar structure.

[0140] In addition to the above components, the kit of the present invention can also include instructions for practicing the inventive method. These instructions can be present in the kit of the present invention in various forms, one of which or more than one may be present in the kit. One form in which these instructions can exist is as printed information on a suitable medium or substrate in a kit package, a package insert, etc., such as a sheet or several sheets of paper with information printed on it. Another way is a computer-readable medium having information recorded thereon, such as a floppy disk, a CD, a portable flash drive, etc. Another way that can exist is a website address that can read information at a remote site using the Internet. Any convenient way can exist in the kit.

[0141] N. Experimental Examples

[0142] 1. Pain Model

[0143] a) Pre-injury pain management

[0144] (1) Changes in neurological nerve damage

[0145] The chronic pain model of chronic constriction injury (CCI) was used to measure the pain levels experienced by 22-month-old C57BL / 6J mice that received the following treatments: (1) CCI followed by treatment with PPF1; (2) CCI followed by treatment with vehicle; or (3) sham surgery followed by treatment with vehicle. Using this model, the nervous system is conditioned to a state of sustained hyperresponsiveness, thereby lowering the pain threshold long after the initial injury occurs. (See, e.g., Safakhah, HA et al., Journal of Pain, 10:1457-66 and Suter MR et al., Anesthesiology Res and Practice (2011), the entire contents of which are incorporated herein by reference).

[0146] PPF1 is a PPF that contains approximately 88% normal human albumin (relative to total protein), 12% alpha globulin and beta globulin, and no more than 1% gamma globulin as determined by electrophoresis. Unless otherwise specified, PPF1 is administered in vivo using a 5% solution (weight / volume, 50 g / L) in this example. PPF2 is a PPF from a different batch than PPF1. PPF2 has the same protein content and concentration index as PPF1.

[0147] Figure 1The timeline of the CCI experiment is described. Twenty-three month old wild-type mice were subjected to CCI or sham surgery via ligation 24 hours prior to 7 consecutive days of 150 uL / day pulse dosing (iv tail vein) in PPF1 or vehicle control groups. Behavioral assessments were performed in the fourth week, and tissues were collected for histological examination in the fifth week.

[0148] Figure 2 Is a diagram of the location of CCI administration to wild-type mice at twenty-three months of age. As shown, a ligature was made on the sciatic nerve. This diagram is adapted from Suter MR et al., Anesthesiology Res and Practice, (2011), the entire contents of which are incorporated herein by reference.

[0149] Figure 3 Shows Figure 1 Data shown are from a mechanical von Frey allodynia test in wild-type mice 4 weeks after CCI or sham surgery. To determine the animals' tolerance to mechanical stress, the debilitated sciatic nerve of the subjects' hind paws was stimulated with von Frey filaments of varying thickness. Figure 3 The pressure exerted by mice when they withdrew their hind paws was measured and plotted in the Figure. The figure illustrates that mice treated with PPF1 after CCI experienced significantly less pain (could tolerate more pressure) than control mice treated with vehicle after CCI. Animals treated with sham surgery also experienced significantly less pain than animals treated with vehicle after CCI. The main finding was that PPF1 had a positive effect on the mechanical nociception deficits induced by CCI. ***P<0.001 PPF1 treatment after CCI vs. vehicle treatment after CCI, *P<0.05 Sham surgery vehicle vs. CCI vehicle; one-way ANOVA with Tukey post hoc test.

[0150] Figure 4 Shows Figure 1 Hippocampal histology data of wild-type mice shown. Neurogenesis was measured using the doublecortin (DCX) marker. Mice that underwent CCI surgery and were treated with PPF1 showed a significant increase in neurogenesis in the dentate gyrus region of the hippocampus compared to mice that received vehicle treatment. Mice that underwent sham surgery tended to have stronger neurogenesis relative to mice that underwent CCI surgery, and both groups received vehicle treatment post-surgery. Therefore, PPF1 has the ability to restore neurogenesis after chronic nerve injury. *P<0.05 PPF1 treatment after CCI versus vehicle treatment after CCI; unpaired T test.

[0151] Figure 5 Shows Figure 1Hippocampal histology data of wild-type mice are shown. Inflammatory markers were measured as CD68 expression. Our study showed that mice that underwent CCI surgery and vehicle treatment expressed significantly higher numbers of CD68-positive cells in the hippocampus compared to mice treated with PPF1 after CCI surgery. PPF1-treated animals had comparable levels of inflammation to the sham-operated group. This suggests that PPF1 helps to reduce neuroinflammation caused by chronic nerve injury. *P<0.05 PPF1 treatment after CCI vs. vehicle treatment after CCI, sham vehicle vs. CCI vehicle; one-way ANOVA with Tukey post hoc test.

[0152] Figure 6 Data are shown for mechanical von Frey allodynia in C57BL / 6J mice that underwent CCI or sham surgery and were followed by Figure 1 The tests were performed on the timeline shown. Twenty-two month old mice in the PPF1 group and vehicle control group were administered a pulse dosing regimen of 150 uL / day (intravenous injection into the tail vein) for 7 consecutive days. Another group received gabapentin, 75 mg / kg daily (administered intraperitoneally) for 7 consecutive days. All treatments began 24 hours after CCI or sham surgery. To determine the animals' tolerance to mechanical pressure, the hind paws of the subjects' sciatic nerves were stimulated by von Frey filaments of varying thickness. In the weeks after CCI or sham surgery, the pressure when the mice withdrew their hind paws was assessed, as Figure 6 . The figure illustrates that mice administered PPF1 after CCI surgery were significantly more tolerant to mechanical nociception at all time points evaluated compared to mice treated with vehicle after CCI. In contrast, mice administered gabapentin had significant improvements in mechanical nociception only 2 weeks after CCI surgery and had mechanical nociception similar to that of mice treated with vehicle at all other time points. Sham-operated mice had significant increases in mechanical nociception responses 3 and 5 weeks after surgical treatment. In summary, these data suggest that PPF1 improves peripheral pain for a longer period of time compared to the baseline of care treatment (gabapentin). ***, **** P<0.001, P<0.0001 PPF1 versus vehicle control; ANOVA and Tukey post hoc test. * P<0.05 Gabapentin versus vehicle control; ANOVA and Tukey post hoc test. *, ** P<0.05, P<0.01 Sham versus vehicle control; ANOVA and Tukey post hoc test.

[0153] Figure 7 Data from hot plate tests are shown for Figure 1The test was performed on wild-type mice as described by Woolfe and Macdonald. (Woolfe G. and Macdonald AD, J. Pharmacol. Exp. Ther. 80: 300-07 (1944), the entire contents of which are incorporated herein by reference). The temperature of the hot plate was set to 55°C. The mice were placed in a transparent cylinder for 30 minutes to adapt. The cylinder was placed on the hot plate and a timer was started. When pain response behavior (e.g., licking the hind paw or jumping on the hind paw) was first observed, the time was recorded as the latency. If no pain response behavior was observed, the animal was removed at a predetermined cutoff time, such as 30 seconds, to prevent tissue damage. Mice were tested only at 2 weeks and 5 weeks after CCI surgery because repeated exposure to the test has been shown to change sensitivity. Figure 7 Shown are the latency to hot plate pain responses 5 weeks after CCI or sham surgery. PPF1-treated mice showed a significant decrease in sensitivity to hot plate stimulation compared to controls treated with CCI vehicle, indicating a rescue effect of PPF1. ** P < 0.01 sham versus CCI, **** P < 0.0001 PPF1 versus vehicle-treated CCI mice. ANOVA and Tukey post hoc test.

[0154] (2) Prevention of spinal cord inflammation

[0155] A separate study similar to the above study (described above) was conducted on 22-month-old C57BL / 6J mice. Groups of mice were treated as follows: (1) with PPF (PPF2) after CCI; (2) with vehicle after CCI; (3) with recombinant human albumin (rhAlb) after CCI; or (4) with vehicle after sham surgery. Mice in the PPF2, rhAlb, or vehicle control groups were administered a pulsatile dosing regimen of 150 uL / day (intravenously injected into the tail vein) for 7 consecutive days. All treatments were initiated 24 hours after CCI or sham surgery.

[0156] Figure 8 Data from a hot plate test (described above) is shown. Figure 1 The timeline shown was tested thirty-five (35) days after CCI. PPF2 treated mice had significantly reduced sensitivity to hot plate stimulation compared to controls treated with CCI vehicle control. Mice treated with recombinant human albumin also had significantly reduced sensitivity to hot plate stimulation compared to controls treated with CCI vehicle, but not to the extent of mice treated with PPF2. * P<0.05 rhAlb vs. vehicle treated CCI mice, *** P<0.001 PPF2 vs. vehicle treated CCI mice. ANOVA and Tukey post hoc test.

[0157] Fig. 9 Data from the mechanical von Frey allodynia test for the same mice before (starting point) and at different time intervals after CCI are shown. In the weeks following CCI or sham surgery, mice were assessed for pressure when withdrawing their hind paws, as Fig. 9 Shown. The figure illustrates that mice administered PPF2 after CCI surgery were significantly more tolerant to mechanical nociception at all time points evaluated compared to mice treated with vehicle or recombinant human albumin (rhAlb) after CCI. This suggests that PPF (PPF2) improves pain for a longer period of time compared to vehicle and albumin controls, and albumin is the major protein component of PPF. Therefore, these effects do not appear to be mediated through albumin, but rather through other proteins present in PPF. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 relative to vehicle control; ANOVA and Tukey post hoc test.

[0158] Fig.10 Shown are relative levels of myelin basic protein (MBP, detected by Abcam, ab40390 anti-rabbit antibody) in the distal sciatic nerve five weeks after the last dose of PPF (PPF1) in another experiment similar to that described above in 22 month old mice. * P < 0.05; *** P < 0.001 vs. vehicle control; ANOVA and Tukey post hoc test.

[0159] Fig.11 Relative levels of the S-100 Schwann cell marker in these mice are shown. In both cases, PPF in CCI mice increased the relative levels of these markers compared to control CCI mice. This suggests that PPF promotes sciatic nerve repair mechanisms by increasing the expression of myelin proteins and S-100 protein. It also suggests that PPF induces myelination repair mechanisms. ** P<0.01; *** P<0.001 vs. vehicle control; ANOVA and Tukey post hoc test.

[0160] Fig.12 yes Fig.10 and Fig.11 The data conveyed are qualitative representations of fluorescence microscopy.

[0161] Fig.13 and Fig.14Detection of BDNF and CD68 in the dorsal horn of the spinal cord of mice treated 24 hours after CCI injury is shown respectively. Brain-derived neurotrophic factor (BDNF, detected by Abcam, ab108319 anti-rabbit antibody) is secreted by activated microglia, and has been shown to enhance spinal cord nociception (detection of pain stimulation) through the involvement of synaptic facilitation and central sensitization mechanisms. Neuropathic pain induced by peripheral injury is often accompanied by increased expression of BDNF in the spinal cord (Garraway SM et al., Neural Plast. Article ID 9857201 (2016)). CD68 levels (detected by Biorad MCA1957 GA anti-rat antibody) were also measured. CD68 is a marker for activated microglia. Fig.13 and Fig.14 PPF treatment 24 hours after CCI injury resulted in a significant reduction in BDNF and CD68 markers in the dorsal horn of the spinal cord, suggesting that it could prevent microglial activation and block the deleterious downstream events associated with the development of neuropathic pain. ** P < 0.01; *** P < 0.001 vs. vehicle control; ANOVA and Tukey post hoc test.

[0162] Fig.15 and Fig.16 They are Fig.13 and Fig.14 Fluorescence microscopy images of the data conveyed in . The rectangle highlights the dorsal horn of the spinal cord analyzed in the L4-L6 lumbar segments. The images on the right side of the figures are higher magnification images of the rectangular area on the left side of each figure.

[0163] b) Pain management within 14 days after injury

[0164] Fig.17 The protocol for 22-month-old C57BL / 6J mice is shown. Initial von Frey paw withdrawal thresholds, a measure of mechanical allodynia, were measured 3 to 4 days before CCI or sham surgery. Groups of mice were treated as follows: (1) PPF (PPF1) 14 days after CCI; (2) vehicle 14 days after CCI; (3) recombinant human albumin (rhAlb) 14 days after CCI; or (4) vehicle 14 days after sham surgery. Mice in the PPF1, rhAlb, or vehicle control groups were administered a pulsatile dosing regimen of 150 uL / day (intravenous injection into the tail vein) for 7 consecutive days. All treatments began 14 days after CCI or sham surgery.

[0165] Fig.18Von Frey paw withdrawal thresholds at baseline, 14 days post-CCI, 21 days post-CCI, 28 days post-CCI, 35 days post-CCI, 42 days post-CCI, and 49 days post-CCI are shown. At day 14, all groups except the sham group showed significant impairment, indicating that central sensitization occurred in all CCI groups 2 weeks after injury. This was not reversed until 7 days after cessation of PPF treatment (day 28), indicating that PPF did not act as a simple analgesic in this model. Instead, PPF treatment produced a mechanistic effect that was not observed with vehicle or recombinant human albumin (rh-albumin). This suggests that PPF resulted in a significant reduction in pain that was fully established prior to PPF treatment (which necessarily involves a central component) compared with vehicle controls. ** P<0.01; *** P<0.001; **** P<0.0001 vs. vehicle control; ANOVA and Tukey post hoc test.

[0166] Fig.19 and Fig. 20 The results showed that 35 days after CCI ( Fig.19 ) and 49 days ( Fig. 20 ) of the hot plate latency. Both sets of results indicate that PPF-treated mice had a sustained reduction in sensitivity to hot plate pain. This also supports the view that PPF acts through a mechanism other than simply providing analgesia. ** P < 0.01; ANOVA and Tukey post hoc test.

[0167] 2. Wound healing model

[0168] Using a diabetic mouse model (B6.BKS(D)-Lepr db / J) To investigate the effect of PPF1 on wound healing. Six-week-old B6.BKS(D)-Leprdb / J mice were shaved on the back on day -1. Two locations on the back of the mice were wounded on day 0. From day 0 (immediately after skin wounding) to day 6, mice received daily treatment (IV) with vehicle (150 µL) or PPF1 (150 µL).

[0169] The skin was wounded as follows: The day before the skin wounding, the animals were depilated using a depilatory cream and the skin was then gently cleaned with warm water. The animals were anesthetized using isoflurane for inhalation, and the surgical site was shaved and pretreated with povidone-iodine ("betadine") or chlorhexidine antiseptic products (or similar scrubs) and 70% ethanol. A hot water heating pad (or similar surgical product) was placed under the mouse. Two wound sites were marked on the dorsal skin with 5 mm diameter circles using a permanent marker. The dorsal skin was lifted with clean forceps and incised along the marked circles with fine surgical scissors. A 15 mm diameter silicone splint with a 6 mm diameter central incision was secured around the wound using Vetbond and nylon sutures. After the skin wounding, the mouse was weighed (initial weight) and placed in a clean home cage with a heating pad placed underneath and softened food. The mouse was kept on the heating pad and monitored until the mouse became bright, alert, and responsive.

[0170] Wound healing was assessed daily postoperatively until sutures were removed. Buprenorphine was injected intraperitoneally immediately postoperatively and every 12 hours for a total of three injections. Meloxicam was injected intraperitoneally before surgery and 24 hours after surgery. Softened food and clean H2O recovery gel were placed on the bottom of the cage after surgery. The weight of mice was measured daily after surgery. If the weight of mice decreased by more than 1 gram after surgery, 500 ul / day of normal saline was administered.

[0171] The amount of wound closure was assessed daily by measuring wound size using a caliper. Mice were sacrificed on days 10 and 14. Mice were deeply anesthetized with avertin (250 mg / kg) and cardiac puncture was performed, and blood samples were collected using a syringe prefilled with EDTA. The blood / EDTA was then placed in a microcentrifuge tube. The microcentrifuge tube was kept on ice and centrifuged at 1000 g (+4°C) for 15 minutes as quickly as possible to separate the plasma. The plasma from each mouse was aliquoted into 100 µL per vial, and the remaining plasma was placed in the next vial and stored at -80°C.

[0172] The skin of each mouse was collected, fixed with 4% paraformaldehyde, then washed twice in PBS and embedded in paraffin. The tissues were sectioned or dissolved and analyzed for inflammatory markers by standard histological and biochemical methods, including qRT-PCR, immunoblotting, ELISA, and immunohistochemistry.

[0173] Figure 21 is untreated ( Fig.21A ) and PPF1 treated ( Fig.21B ) of diabetic wounds (B6.BKS(D)-Lepr dbHistological comparison of PPF1 in diabetic mouse model (Figure 2A). The black bar indicates the wound bed thickness (epidermis plus granular layer). The arrow indicates the wound boundary. As determined by the wound bed thickness, the wound bed thickness of PPF1-treated mice increased. Therefore, it is demonstrated that PPF1 improves wound healing.

[0174] Figure 22 is untreated ( Fig.22A ) and PPF1 treated ( Fig. 22B ) of diabetic wounds (B6.BKS(D)-Lepr db Histological comparison of PPF1-treated wounds in a diabetic mouse model (Figure 2A). The black bar indicates the granular layer. The blue bar indicates the epidermis. The epidermis of PPF1-treated wounds was thicker than that of untreated wounds, while the granular layers of PPF1-treated wounds and untreated wounds showed a more obvious trend of difference (i.e., the granular layer of PPF1-treated wounds was thicker than that of untreated wounds).

[0175] Figure 23 to Figure 26 Results from the B6 ob / ob (B6.Cg-Lepob / J mice) diabetic mouse model are shown, evaluating the efficacy of PPF1 versus vehicle in diabetic wound healing. Nine-week-old male B6 OB / OB mice were used. Mice were weighed the day before wounding and fasted for 5 hours to determine fasting blood glucose from tail blood. Mice were equally divided into 2 different treatment groups based on body weight and blood glucose levels. For wounding, mice were shaved, depilatory cream (Nair™) was applied, wounded at two sites on the back (5 mm diameter incision), and then fitted with silicone rings (12 mm outer circumference, 6 mm inner circumference) soaked in 70% alcohol. The silicone rings were attached to the open wounds using Vectabond™. Four sutures were placed on each ring to ensure that the silicone ring remained attached to the wound throughout the experiment. 30 μL of PPF1 and control were applied directly to the wounds, and both wounds were sealed with a piece of Tegaderm™. Daily treatments were performed by injecting PPF1 and control into the Tegaderm™ covering the wounds. To image the wound, the Tegaderm™ covering the wound was cut away and the wound was resealed with a new piece of Tegaderm™. At sacrifice, the silicone ring was removed and the wound was dissected dorsally for histological examination.

[0176] Wound healing was assessed daily by measuring wound closure. Wound size was measured using a camera and precision ruler. Terminal tissue collection was performed on days 10 and 14. Tissues were sectioned or lysed and analyzed for inflammatory markers by standard histological methods, including immunohistochemistry, qRT-PCR, and H&E and other special stains.

[0177] Fig.23The overall design of the experiment is described. Blood was collected by dropping blood as indicated to measure fasting blood glucose levels. Skin wounds were made on day 2, and drugs were administered intravenously (iv) from day 1 to day 7. Fig.24 Shown is the percentage of wounds that remained open at several time points after wounding in the first study (Study 1). Mice were treated with PPF1 (150 µL) or saline control. After 10 days, open wound size was significantly reduced in animals treated with PPF1 compared to saline controls (** p < 0.006, unpaired T test).

[0178] Fig.25 Shown is the percentage of wounds that remained open at several time points after wounding in a second similar study (Study 2). Mice were treated with PPF1 (150 µL) or saline control. After 8 days, open wound size was significantly reduced in animals treated with PPF1 compared to saline controls. (** p< 0.0018, unpaired samples T test).

[0179] Fig.26 Data from Study 1 and Study 2 are combined to show the percentage of wounds that remained open 11 days after injury. PPF1-treated mice had a statistically significant reduction in the percentage of wounds that remained open after 11 days. (** p<0.006, unpaired T-test). The difference between PPF1-treated and vehicle-treated animals was also significant at day 10 (** p<0.006, unpaired T-test).

[0180] Fig. 27 and Fig.28 Shown are the results of a study in which PPF1 or vehicle was topically applied to wounds of B6 ob / ob (B6.Cg-Lepob / J mice). Fig. 27 A study paradigm of applying 30 μL of topical PPF1 or vehicle control to the wound daily is shown. Fig.23 Treat injuries as indicated. Fig.28 Results of a local study are shown as the percentage of the initial wound remaining after 10 days of treatment. Fig.28 It was shown that PPF1 significantly reduced the percentage of open wounds remaining after 10 days compared to vehicle controls.

[0181] In at least some of the foregoing embodiments, one or more than one element used in an embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. Those skilled in the art will recognize that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All of these modifications and changes will fall within the scope of the subject matter defined by the appended claims.

[0182] Those skilled in the art will understand that, in general, the terms used herein, especially in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "containing" should be interpreted as "containing at least", the term "including" should be interpreted as "including but not limited to", etc.). Those skilled in the art will also understand that if a specific number of an introduced claim description is intended, the intent will be explicitly described in the claim, and there is no such intent in the absence of an explicit description. For example, to aid understanding, the claims appended herein may contain the use of the introductory phrases "at least one" and "one or more than one" to introduce a claim description. However, the use of such phrases should not be interpreted as implying that a claim description introduced by a claim description without a quantifier limits any particular claim containing such an introduced claim description to only one embodiment of the description, even when the same claim includes the introductory phrases "one or more than one" or "at least one" and no quantifier is used (e.g., the no quantifier should be interpreted as "at least one" or "one or more than one"); the same is true for no quantifier when used to introduce a claim description. In addition, even if a specific number of the claims introduced is explicitly described, a person skilled in the art will recognize that such description should be interpreted as at least the number described (e.g., simply describing "two descriptions" without other modifications means at least two descriptions, or two or more than two descriptions). In addition, when using a convention similar to "at least one of A, B, and C, etc.", generally speaking, such a structure is intended to allow a person skilled in the art to understand such a convention (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). When using a convention similar to "at least one of A, B, or C, etc.", generally speaking, such a structure is intended to allow a person skilled in the art to understand such a convention (e.g., "a system having at least one of A, B, or C" will include, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). Those skilled in the art will also understand that, in fact, any antonymous conjunction and / or phrase presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one of the terms, one of the terms or all of the terms. For example, the phrase "A or B" should be understood to include the possibility of "A" or "B" or "A and B".

[0183] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0184] Those skilled in the art will understand that for any and all purposes, such as providing a written description, all ranges disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to fully describe and decompose the same range into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. In a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all languages ​​such as "at most", "at least", "greater than", "less than", etc. include the narrated numbers and refer to the ranges that can be subsequently decomposed into sub-ranges as described above. Finally, those skilled in the art will understand that the range includes each individual member. Therefore, for example, a group containing 1 to 3 items refers to a group containing 1, 2 or 3 items. Similarly, a group containing 1 to 5 items refers to a group containing 1, 2, 3, 4 or 5 items, and so on.

[0185] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0186] Therefore, the foregoing only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown herein, which embody the principle of the present invention and are included in the spirit and scope of the present invention.In addition, all examples and conditional language described herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote the development of the art, and should be interpreted as not being limited to the specifically described examples and conditions.In addition, all descriptions of the principles, aspects and embodiments of the present invention and its specific embodiments described herein are intended to include equivalents of their structure and function.In addition, such equivalents are expected to include currently known equivalents and equivalents of future development, that is, any element with the same function developed, regardless of its structure.In addition, any content disclosed herein is not intended to be provided to the public, regardless of whether the disclosure is explicitly mentioned in the claims.

[0187] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are presented by the appended claims. In the claims, only when the exact phrase "means" or the exact phrase "step" is described at the beginning of the limitation in the claim, it is an explicit reference to 35 USC §112(f) or 35 USC §112(6) in the claims; if the exact phrase is not used in the limitation in the claim, 35 USC §112(f) or 35 USC §112(6) is not cited.

Claims

1. Use of a plasma fraction in the preparation of a medicament for treating a subject diagnosed with pain, wherein an effective amount of the plasma fraction is administered to the subject.

2. The use according to claim 1, wherein the pain is chronic pain.

3. The use according to claim 1, wherein the pain is peripheral pain.

4. The use according to claim 1, wherein the pain is central pain.

5. The use according to any one of claims 1 to 4, wherein the plasma component is a plasma protein component.

6. The use according to claim 5, wherein the plasma protein fraction comprises 83% to 95% albumin.

7. The use according to claim 6, wherein the plasma protein fraction is a commercially available plasma protein fraction.

8. The use according to claim 1, wherein the plasma fraction is for administration by using a pulse dosing regimen.

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