Plasma fractions for liver regeneration

Abstract

Methods and compositions for treating aging-related diseases as well as liver regeneration, prevention of liver degeneration, and maintenance of liver are described. The composition used in the method comprises plasma having the efficacy of treating and / or preventing a disease and a plasma fraction derived from plasma.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of November 18, 2020, application number 202080080810.6, and invention name “Plasma fraction for liver regeneration”. I. Technical field

[0002] The present invention relates to methods and compositions for treating aging-related diseases and liver regeneration, preventing liver degeneration and maintaining the liver, and in particular to the use of plasma and plasma fractions derived from plasma having the efficacy of treating and / or preventing diseases. II. Background Technology

[0003] The present invention relates to prevention and treatment of diseases, including diseases associated with aging. The present invention relates to the use of blood products, such as plasma and plasma fractions to treat and / or prevent aging-related conditions associated with liver growth, maintenance and regeneration. According to 35 U.S.C. § 119 (e), this application claims priority to U.S. Provisional Patent Application Serial No. 62 / 937965 filed on November 20, 2019; and U.S. Provisional Patent Application Serial No. 62 / 975637 filed on February 12, 2020; the disclosures of these applications are incorporated herein by reference. III. Summary of the Invention

[0004] Liver failure is a large market with only one therapeutic intervention currently available – liver transplantation. Approximately 35 million people, or one in ten Americans, have some form of liver disease. This number is increasing due to the prevalence of cirrhotic liver disease in an increasingly obese population. NAFLD (non-alcoholic fatty liver disease) is a major risk factor for cirrhosis, which can ultimately lead to liver failure. Due to declining health in the general population, the number of organs available for transplant is low. As a result, only about one-third of patients on the liver transplant list receive a transplant. The list typically excludes patients over the age of 65 due to the increased risks associated with organ transplantation that are associated with aging. For patients with cirrhosis awaiting a liver transplant, the statistics are particularly dire. There are 300,000 cirrhosis-based hospitalizations and 36,000 cirrhosis-related deaths in the United States each year, with more than half of these patients ineligible for a transplant. The availability of alternative treatments could significantly impact the large proportion of patients with liver disease who would otherwise not have access to treatment.

[0005] Liver failure can be acute or chronic. Acute liver failure is often caused by viral infections (such as hepatitis B and C), overuse of drugs or toxins (such as acetaminophen), and metabolic or vascular diseases such as autoimmune hepatitis and Wilson's disease. Chronic liver failure is usually classified as cirrhosis and can be caused by viral infections, alcohol abuse, NAFLD (caused by obesity, high cholesterol and triglycerides, and high blood pressure), autoimmune diseases, blockage or damage to ducts such as the bile duct from the liver to the intestines, exposure to toxins or certain drugs, parasites, and heart failure causing blood to accumulate in the liver.

[0006] As mentioned above, liver resection and transplantation can be performed for liver failure, while transplantation is more commonly performed for chronic liver failure. In addition, these processes can be used for primary and secondary malignancies (i.e., cancer). The improved results of liver resection and transplantation are related to the focus on preoperative, perioperative and postoperative care. (Wrighton LJ et al., J Gastrointest Oncol., 3(1):41-47, (2012)). These include advances in surgical and anesthetic techniques, a better understanding of liver physiology, nutritional support, control of blood sugar and reduction of postoperative infection. (Same as above.) In the case of liver transplantation, immunosuppressants are most commonly used. The role of the attending physician in dealing with liver rejection and treating long-term complications such as hypertension and obesity has been an important part of improving prognosis. (See, Issa DH, Cleveland Clinic J of Med., 82(6):361-72 (2015)).

[0007] There is still a serious lack of new therapies to improve outcomes for patients with liver failure and / or undergoing resection or transplantation. In addition, even in the case of liver transplantation, there are not enough donor livers for all patients. (See, for example, Helwick C, The ASCO Post, (Sep 25, 2017) stating "The problem with transplantation is organ allocation.")

[0008] One compound being studied for the treatment of cirrhosis is albumin. This is to compensate for the loss of albumin production due to the reduction of cirrhosis's own albumin production. The basic principle is that albumin helps replace osmotic pressure, bind toxic substances, regulate homeostasis and has an anti-inflammatory effect. (Carvalho JR et al., Annals of Hepatology, 17(4):547-60(2018)). Therefore, the conventional idea in this field is that the purer the albumin and the higher the concentration, the better the efficacy. Therapeutic albumin is usually produced by plasma fractionation. Fractionation can produce an albumin solution, although it contains unwanted protein "contaminants" or "impurities".

[0009] The present invention provides novel therapeutics using plasma fractions to improve and accelerate the recovery of diseased, resected or transplanted livers in patients with liver disease. In addition, since the liver is the only internal organ capable of regeneration, the present invention provides methods for stimulating proliferation and regeneration of existing livers, which can, for example, facilitate recovery from surgery in some liver-related indications. Furthermore, the present invention utilizes substances that are considered "contaminants" or "impurities" derived from plasma fractionation to more effectively treat liver disease.

[0010] IV. 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. V. Description of the Figures

[0012] Figure 1 A graph showing the weight progression of 20 month old C57BL / 6 mice on a high fat diet ("HFD"). After 8.5 weeks of HFD, the mice gained significant weight.

[0013] Figure 2 Yes Figure 1 Oil Red O staining of the mouse liver. Compared with mice on a normal diet, mice on a HFD developed fatty liver.

[0014] Figure 3 The experimental design for evaluating the effect of PPF1 on 20-month-old C57BL / 6 mice with fatty liver is shown. Mice were placed on a 60% high-fat diet for 7 weeks before being treated with PPF1 or vehicle for 7 consecutive days. Surgery (70% hepatectomy) was performed the next day after the last dose of PPF1 or vehicle, and the median lobe and left lobe (resected) before surgery were removed during the hepatectomy. The right lobe and caudate lobe (residual) were harvested 48 hours after the hepatectomy.

[0015] Figure 4 Determined Figure 3 The treated mice had the resected lobe and residual lobe resulting from a 70% hepatectomy.

[0016] Figure 5 It shows that Figure 3 Results of determination of the ratio of liver resection weight to body weight in vehicle and PPF1 treated hepatectomized mice.

[0017] Figure 6 It shows that Figure 3 Weights of liver resections in vehicle and PPF1 treated mice.

[0018] Figure 7is a graph showing that serum ALT levels were normal and unaffected in PPF1 -treated HFD mice before hepatectomy.

[0019] Figure 8 It shows that Figure 3 The liver weight to body weight ratio was measured in vehicle and PPF1 treated mice 48 hours after hepatectomy.

[0020] Fig. 9 It shows that Figure 3 Liver weights in vehicle- and PPF1-treated mice 48 hours after hepatectomy.

[0021] Fig.10 It shows that Figure 3 Results of serum ALT levels in vehicle- and PPF1-treated mice 48 hours after hepatectomy.

[0022] Fig.11 Cell proliferation rates after hepatectomy are reported. EdU was delivered 24 hours after hepatectomy, and proliferation rates were tracked by Click-it labeling of EdU-positive cells. PPF1 significantly increased the number of EdU-positive cells per field compared to vehicle-treated animals.

[0023] Fig.12 Cell proliferation 48 hours after hepatectomy as measured by the number of Ki67-positive cells per field is reported. PPF1 significantly increased the number of Ki67-positive cells per field compared to vehicle-treated animals.

[0024] Fig.13 Cell proliferation rates in remnant livers by qPCR gene expression are reported. Relative expression of the cell cycle marker cyclin B1 is shown. In remnant liver sections, expression of cyclin B1 was significantly upregulated in PPF1-treated mice compared to vehicle-treated mice.

[0025] FIG. 14A to FIG. 14D qPCR expression of several markers in resected livers is reported. The cell cycle marker cyclin B1 ( Fig.14A ), cyclin A2 ( Fig. 14B ) and Ki67( Fig. 14C ). In resected liver sections taken during hepatectomy as preoperative controls, PPF1 surprisingly and significantly increased cell proliferation compared to vehicle controls with all three cell cycle markers. Fig.14D Relative expression levels of TNFα in resected liver sections are reported. TNFα is known to promote restoration of functional liver mass by driving hepatocyte proliferation during liver regeneration.

[0026] Fig.15 It shows that Fig. 14C Representative images of Ki67 immunostaining in resected livers reported in , which demonstrated that PPF1-treated resected livers had significantly higher numbers of Ki67-positive cells compared to vehicle controls.

[0027] Fig.16 Reported from Fig.15 Quantification of immunostaining shows an increase in the number of Ki67-positive cells in excised livers treated with PPF1.

[0028] Fig.17 is a graph showing the total number of animals undergoing hepatectomy and the survival rate for each treatment.

[0029] Fig.18 Two representative confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Histological sections were stained with GFAP antibody (astrocytic marker) and the colocalization level of EdU and GFAP was observed. Based on the lack of colocalization between EdU and GFAP, it was determined that cell proliferation associated with PPF1 administration did not occur in astrocytes.

[0030] Fig.19 Two representative confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with CD68 antibody (Kupffer cell marker) and the level of colocalization of EdU and CD68 was observed. The lack of colocalization between EdU and CD68 determined that no cell proliferation associated with PPF1 administration occurred in Kupffer cells.

[0031] Fig. 20 Representative confocal microscopy fields of remnant liver incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with HNF4a antibody (a hepatocyte marker), and the colocalization level of EdU and HNF4a was observed. Based on the lack of colocalization between EdU and HNF4a, it was determined that cell proliferation associated with PPF1 administration did not occur in hepatocytes.

[0032] Fig.21 Representative confocal microscopy fields of remnant liver incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with CD3 antibody (T cell marker) and the colocalization level of EdU and CD3 was observed. Based on the lack of colocalization between EdU and CD3, it was determined that no cell proliferation associated with PPF1 administration occurred in T cells.

[0033] Fig. 22 Two representative confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with CD31 antibody (a marker for sinusoidal endothelial cells), and the colocalization level of EdU and CD31 was observed. Based on the positive colocalization between EdU and CD31, it was determined that the cell proliferation caused by PPF1 administration was associated with liver sinusoidal endothelial cells (LSEC). Arrows indicate LSECs containing EdU-positive cells.

[0034] Fig.23 The experimental design for evaluating the effects of PPF1 (plasma fraction "PF") and recombinant human albumin (rhAlbumin) on 20-month-old C57BL / 6 mice is shown. Mice were placed on a 60% high-fat diet for 8 weeks before being treated with PPF1, rhAlbumin or vehicle for 7 consecutive days. Surgery (70% hepatectomy) was performed the next day after the last dose of PPF1, rhAlbumin or vehicle, and the median lobe and left lobe (resected) before surgery were removed during the hepatectomy. The right lobe and caudate lobe (residual) were harvested 48 hours after hepatectomy.

[0035] Fig.24 Cell proliferation in resected livers 48 hours after hepatectomy as measured by the number of Ki67-positive cells per field is reported. PPF1 significantly increased the number of Ki67-positive cells per field compared to vehicle-treated animals. In contrast, animals treated with rhAlbumin were not significantly different from those treated with vehicle.

[0036] Fig.25 reported the cell proliferation rate in the remnant liver after hepatectomy. EdU was delivered 24 hours after hepatectomy and the proliferation rate was tracked by click chemistry. PPF1 significantly increased the number of EdU-positive cells per field compared with vehicle-treated animals. In contrast, animals treated with rhAlbumin showed a less significant trend of proliferation compared with vehicle-treated animals.

[0037] Fig.26 The experimental design of the impact of PPF1 and HAS1 on C57BL / 6 mice of 20 months of age is shown. Before being treated with different plasma fractions (PF) PPF1 and HAS1 or vehicle for 7 consecutive days, mice were placed in a 60% high-fat diet for 8 weeks. Surgery (70% hepatectomy) was performed the second day after the last dose of PPF1, HAS1 or vehicle treatment, and the median lobe and left lobe (resected) before surgery were removed during the hepatectomy. The right lobe and caudate lobe (residual) were harvested 48 hours after hepatectomy.

[0038] Fig. 27Cell proliferation rates 24 hours after hepatectomy are reported. EdU was delivered 24 hours after hepatectomy. Both PPF1 and HAS1 significantly increased the number of EdU-positive cells compared to vehicle in the remnant liver.

[0039] Fig.28 Cell proliferation rate 48 hours after hepatectomy is shown. Ki67 immunostaining was performed on the remnant liver removed 48 hours after hepatectomy. Although both PPF1 and HAS significantly increased cell proliferation compared to control animals, PPF1 also induced significantly more proliferation compared to animals treated with HAS1.

[0040] Fig.29 Shown is the experimental design for evaluating the effects of PPF1 on 20 month old C57BL / 6 mice 2 hours after the last dose.

[0041] Fig.30 As shown above Fig.29 Results of TNFα gene expression analysis of the treated mice: PPF1 treated animals had significantly increased TNFα gene expression by QPCR compared to vehicle treated animals.

[0042] Fig.31 As shown above Fig.29 Results of Ki67 immunostaining analysis of the treated mice. PPF1-treated animals had significantly increased Ki67-positive cells 2 hours after the last dose compared to vehicle-treated animals, indicating that PPF1 increased hepatocyte proliferation. VI. Specific Implementation Methods

[0043] A. Introduction

[0044] The present invention relates to the treatment of liver disorders or diseases. Plasma fractions, including plasma fractionation products, have been shown to have significant activity in inducing liver recovery after hepatectomy. In addition, plasma fractions have been shown to activate cell proliferation in quiescent and intact livers (before hepatectomy). Compared with whole plasma serum, plasma fractions have several advantages because the plasma fractionation process can remove problematic coagulation factors and eliminate the need for cross-matching. In addition, in certain analyses, plasma fractions have shown unexpected improvements in efficacy compared to young plasma (see, e.g., U.S. Patent Application No. 15 / 499,694 and U.S. Patent Application No. 16 / 432,114; both are incorporated herein by reference in their entirety). Therefore, it is not reasonably predictable to predict the efficacy of plasma fractionation products from whole plasma serum.

[0045] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific method or composition described, and thus, of course, can vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be restrictive, because the scope of the present invention will be limited only by the appended claims.

[0046] The publications discussed herein are provided only for their disclosures prior to the date of filing of the present application. Any information herein should not be construed as admitting that the present invention cannot precede these publications because of prior inventions. In addition, the publication date provided may be different from the actual publication date, and the actual publication date may need to be independently confirmed.

[0047] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range is also explicitly disclosed, to one tenth of the lower limit unit, unless otherwise clearly specified herein. Each smaller range between any of the values ​​or intermediate values ​​in the range and any other values ​​or intermediate values ​​in the range is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the range or excluded from the range, and when one, none or both of the two limits of each range are included in the smaller range, the range is also included in the present invention, unless there is any clearly excluded limit in the range. When the range includes one or both of the limits, the range excluding one or both of these included limits is also included in the present invention.

[0048] It should also be noted that the claims may be drafted to exclude any optional elements. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0049] Those skilled in the art will appreciate upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0050] B. Definition

[0051] 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 method and material similar or equivalent to those described herein may be used in practice or testing the present invention, some possible 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 disclosures cited. It should be understood that if there is a conflict, the present disclosure replaces any disclosure incorporated into the publication.

[0052] It must be noted that as used herein and in the appended claims, quantifiers are not used to include plural referents unless the context clearly dictates otherwise. Thus, for example, one skilled in the art will appreciate that 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, such as polypeptides, and so forth.

[0053] 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 to be treated according to the disclosed methods. Mammals include, for example, humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice and rats. In certain embodiments, the object is a non-human mammal. In some embodiments, the object is a domestic animal. In other embodiments, the object is a pet. In some embodiments, the object is a mammal. In certain embodiments, the object is a human. Other objects 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, for example, as in animal models of diseases) and non-human primates (e.g., chimpanzees and monkeys). Thus, the objects of the present invention may include, but are not limited to, mammals, for example, humans and other primates, such as chimpanzees and other ape and monkey species, etc., wherein the object is a human in certain embodiments. The term object also means a person or organism including any age, weight or other physical characteristics, wherein the object may be an adult, a child, an infant or a newborn.

[0054] "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 individual serving as the source of the blood product containing young plasma is 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 individuals. In some cases, the object is a newborn, and the source of the plasma product is the umbilical cord, wherein the plasma product is harvested from the umbilical cord of the newborn. Thus, "young" and "young individuals" can refer to objects between 0 and 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" and "young individuals" can refer to biological (different from chronological) age, such as individuals who have not shown the inflammatory cytokine levels in the plasma shown in older individuals. In contrast, "young" and "young individuals" can refer to biological (as distinct from chronological) age, such as individuals that exhibit higher levels of anti-inflammatory cytokines compared to levels of anti-inflammatory cytokines in older individuals. As an 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 of other inflammatory cytokines between elderly individuals and young individuals can be used to refer to biological age. (See U.S. Patent Application No. 13 / 575,437, which is incorporated herein by reference). Typically, the individual is healthy, for example, the individual does not have a malignant blood disease or autoimmune disease at the time of collection.

[0055] As used herein, "treatment" refers to any of (i) preventing a disease or condition or (ii) alleviating or eliminating the symptoms of a disease or condition. Treatment can be performed prophylactically (before the onset of liver disease or liver failure) and includes: (a) preventing the subject from developing a condition; (b) inhibiting the condition, i.e., preventing the condition from occurring; or (c) alleviating the condition, i.e., causing the condition to subside. Treatment can cause a variety of different physical manifestations, such as regulation of gene expression, rejuvenation of tissues or organs, reduced inflammation, etc. The therapeutic agent can be administered before, during, or after the onset of the condition. The target therapy can be administered during the symptomatic phase of the condition, and in some cases, after the symptomatic phase of the condition. Treatment can also be performed by administering interventions such as plasma, plasma fractions, or blood products containing plasma components before, during, and / or after liver resection or transplantation.

[0056] "Improvement", "enhancement" or "increase" in relation to liver function, regeneration or recovery refers to any significant increase in liver function measured using standard methods known in the art. By way of example and not limitation, this may include measuring blood levels of certain proteins, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin and total protein, bilirubin, gamma-glutamyl transferase (GGT), L-lactate dehydrogenase (LD), prothrombin time (PT). Examples of normal levels of the proteins may be: ALT (7 U / L to 55 U / L); AST (8 U / L to 48 U / L); ALP (40 U / L to 129 U / L); albumin (3.5 g / dL to 5.0 g / dL); total protein (6.3 g / dL to 7.9 g / dL); bilirubin (0.1 mg / dL to 1.2 mg / dL); GGT (8 U / L to 61 U / L); LD (122 U / L to 222 U / L); and PT (9.4 seconds to 12.5 seconds).

[0057] Blood products containing plasma components.When implementing the target method, a blood product comprising a plasma component is administered to an individual in need, such as an individual suffering from one or more than one of the following conditions: liver disease or liver failure. Thus, the method according to an embodiment of the present invention includes administering a blood product comprising a plasma component from an individual ("donor individual" or "donor") to an individual suffering from one or more than one of the following conditions: liver disease or liver failure ("recipient individual" or "recipient"). "Blood products comprising plasma components" means any product (e.g., whole blood, plasma, or a fraction thereof) comprising plasma derived from blood. The term "plasma" is used in its conventional sense and refers to a pale yellow / light yellow liquid component of 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 plasma-containing blood products suitable for 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 ("leukoreduction"), blood products composed of plasmapheretically-derived or apheretically-derived plasma, fresh frozen plasma, blood products consisting essentially of purified plasma, and blood products consisting essentially of plasma fractions. In some cases, the plasma product used is a non-whole plasma product, meaning that the product is not whole blood, and therefore lacks one or more components found in whole blood, such as red blood cells, white blood cells, etc., at least relative to the extent of the components present in whole blood. In some cases, the plasma product is substantially acellular if not completely acellular, wherein in such cases, the cell content may be 5% or less by volume, such as 1% or less than 1%, including 0.5% or less than 0.5%, wherein in some cases the acellular plasma fraction is a composition that is completely devoid of cells, i.e., they do not include cells.

[0058] Collection of blood products containing plasma components. Embodiments of the methods described herein include administering a blood product comprising a plasma component that may be derived from a donor, including a human volunteer. The term "human-derived" may refer to such products. Methods for collecting blood products comprising plasma 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).

[0059] In one embodiment, the donation is obtained by venipuncture. In another embodiment, the venipuncture is only a single venipuncture. In another embodiment, saline volume replacement is not used. In a preferred embodiment, a plasmapheresis process is used to obtain a blood product containing plasma. Plasmapheresis can include removing a volume of plasma adjusted according to weight and returning cellular components to the donor. In a preferred embodiment, sodium citrate is used in the plasmapheresis process to prevent cell coagulation. After administration of citrate, the volume of plasma collected from the donor is preferably 690mL to 880mL, and is preferably coordinated with the weight of the donor.

[0060] C. Plasma fraction

[0061] During World War II, there was a need for a stable plasma expander that could be used on the battlefield when soldiers lost a lot of blood. Therefore, a method for preparing freeze-dried plasma was developed. However, since sterile water is required for reconstitution, it is difficult to use freeze-dried plasma in combat situations. 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 for treating shock. (See Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's method for purifying plasma fractions utilizes the denaturing effect of cold ethanol and utilizes changes in pH and temperature to achieve separation.

[0062] Embodiments of the methods described herein include administering plasma fractions to a subject. Fractionation is a method by which certain protein subgroups are separated from plasma. Fractionation techniques are known in the art and rely on steps 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). Several steps are involved in the process, each of which involves a specific ethanol concentration that results in selective protein precipitation and changes in pH, temperature, and osmotic molarity. The precipitate is also separated by centrifugation or precipitation. The original "Cohn fractionation method" involves separating proteins into five fractions by precipitation, referred to as fraction I, fraction II+III, fraction IV-1, fraction IV-4, and fraction V. Albumin is the end point (fraction V) product initially identified in the process. According to an embodiment of the present invention, each fraction (or the effluent from the previous separation step) contains or potentially contains a therapeutically useful protein fraction. (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. Pat. Nos. 3869431, 5110907, 5219995, 7531513 and 8772461, which are incorporated herein by reference). The above experimental parameters can be adjusted to obtain specific protein fractions.

[0063] Recently, fractionation has reached further complexity, and thus comprises additional embodiments of the present invention. This recent increase in complexity has occurred by the introduction of chromatography, resulting in the separation of new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and Cohn fractions; by integrating chromatography and ethanol fractionation processes to improve the recovery of IgG; and virus reduction / inactivation / removal (same). In order to capture proteins at physiological pH and ionic strength, anion exchange chromatography can be used. This maintains the functional activity of proteins and / or protein fractions. Heparin and monoclonal antibodies are also used for affinity chromatography. In addition, fractionation by gel filtration, fractionation by salt, and fractionation by polyethylene glycol are used. (Hosseini M Iran J Biotech, 14 (4): 213-20 (2016), which is incorporated herein by reference). One of ordinary skill in the art will recognize that the above parameters and techniques can be adjusted to obtain a specific desired protein-containing plasma fraction.

[0064] Plasma fractionation can also be based on ammonium sulfate. (See, e.g., Odunuga OO, Biochem Compounds, 1:3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), incorporated herein by reference). In addition to obtaining specific blood fractions, ammonium sulfate-based fractionation has also been used to reduce the bulk protein in plasma. (Saha S et al., J. Proteomics Bioinform, 5 (8) (2012), incorporated herein by reference).

[0065] In an embodiment of the present invention, plasma is graded in an industrial environment. Frozen plasma is thawed at 1°C to 4°C. Continuous refrigerated centrifugation is performed on the thawed plasma, and cryoprecipitate is separated. The recovered cryoprecipitate is frozen and stored at a temperature of -30°C or below -30°C. Poor cryoprecipitate ("poor cold") plasma is immediately processed to capture unstable coagulation factors (such as factor IX complex, and its components, and protease inhibitors, such as antithrombin and C1 esterase inhibitors) (by, for example, preliminary chromatography). Continuous centrifugation and precipitation separation can be used for subsequent steps. Such technology is known to those of ordinary skill in the art, and is described in, for example, U.S. Patent No. 4,624,780, No. 5,219,995, No. 5,288,853 and U.S. Patent Application No. 20140343255 and No. 20150343025, the disclosure of which is incorporated herein by reference in its entirety).

[0066] In an embodiment of the invention, the plasma fraction may include a plasma fraction containing a high concentration of albumin. In another embodiment of the invention, the plasma fraction may include a plasma fraction containing a high concentration of IgG or intravenous immunoglobulin (IGIV) (e.g. In another embodiment of the present invention, the plasma fraction may include an IGIV plasma fraction, for example, which has been substantially depleted of immunoglobulins (IgG) by methods known to those of ordinary skill in the art, such as protein A-mediated depletion. (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 fraction can be a plasma fraction in which substantially all coagulation factors are removed in order to retain the efficacy of the fraction and reduce the risk of thrombosis. For example, the plasma fraction can be a plasma fraction described in U.S. Pat. No. 62 / 376529, filed on August 18, 2016; the disclosure of which is incorporated herein by reference in its entirety.

[0067] D. Albumin products

[0068] For those of ordinary skill in the art, albumin plasma products ("APP") are divided into two major categories: plasma protein fraction ("PPF") and human albumin solution ("HAS"). PPF is derived from a process with a higher yield than HAS but a lower minimum albumin purity than HAS (>83% for PPF and >95% for HAS). (Production of human albumin solution: a continuously developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85(6):887-95, at888(2000)). In some cases, the albumin purity of PPF is 83% to 95%, or 83% to 96%. Albumin purity can be determined by electrophoresis or other quantitative determination methods, such as by spectroscopy. In addition, some people point out that PPF has disadvantages due to the presence of protein "contaminants" such as PKA. Same as above. Therefore, PPF preparations are no longer popular as albumin plasma products and have even been removed from the pharmacopoeia of some countries. Same as above. In contrast to these problems, the present invention advantageously utilizes these "contaminants." In addition to alpha, beta and gamma globulins and the above-mentioned PKA, the method of the present invention also utilizes additional proteins or other factors in the "contaminants" that promote, for example, cell proliferation and tissue regeneration.

[0069] Those skilled in the art will recognize that there are or have been several commercial sources of PPF ("commercial PPF preparations"). These commercial sources include Plasma-Plex TM PPF (Armour Pharmaceutical Co., Tarrytown, NY), Plasmanate TM PPF (Grifols, Clayton, NC), Plasmatein TM (Alpha Therapeutics, Los Angeles, CA) and Protenate TM PPF (Baxter Labs, Inc. Deerfield, IL).

[0070] Those skilled in the art will also recognize that there are or have been several commercial sources of HSA ("commercial HSA preparations"). These commercial sources include Albuminar TM (CSL Behring), AlbuRx TM (CSL Behring), Albutein TM(Grifols,Clayton,NC),Buminate TM (Baxatla, Inc., Bannockburn, IL), Flexbumin TM (Baxatla, Inc., Bannockburn, IL) and Plasbumin TM (Grifols, Clayton, NC).

[0071] 1. Plasma protein fraction (human) (PPF)

[0072] According to the U.S. Food and Drug Administration ("FDA"), "Plasma Protein Fraction (Human)" or PPF is a proprietary name for a product defined as "a sterile solution of proteins consisting of albumin and globulins derived from human plasma." (Code of Federal Regulations "CFR" 21 CFR 640.90, which is incorporated herein by reference). The source material for PPF is plasma recovered from whole blood prepared in accordance with the provisions of 21 CFR 640.1-640.5, which are incorporated herein by reference, or source plasma prepared in accordance with the provisions of 21 CFR 640.60-640.76, which are incorporated herein by reference.

[0073] The PPF was tested to determine compliance with the following standards pursuant to 21 CFR 640.92, which is incorporated herein by reference:

[0074] (a) the final product should be a 5.0 + / - 0.30 percent protein solution; and

[0075] (b) The total protein in the final product shall consist of at least 83 percent albumin and not more than 17 percent globulin. Gamma-globulin shall not exceed 1 percent of the total protein. The protein composition shall be determined by a method approved for each manufacturer by the Director of the Center for Biologics Evaluation and Research of the Food and Drug Administration.

[0076] As used herein, "plasma protein fraction" or "PPF" refers to a sterile solution of proteins consisting of albumin and globulin derived from human plasma, wherein the albumin content is at least 83% and the globulins (including α1 globulin, α2 globulin, β globulin and γ globulin) and other plasma proteins do not exceed 17%, and the γ globulin does not exceed 1%, as determined by electrophoresis. (Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2 (174) (1957)). PPF may also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting 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, eds. (1990)).

[0077] 2. Albumin (human) (HAS)

[0078] 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" 21CFR 640.80, which is incorporated herein by reference). The source material of albumin (human) is plasma recovered from whole blood prepared in accordance with the provisions of 21CFR 640.1-640.5 (which is incorporated herein by reference) or source plasma prepared in accordance with the provisions of 21CFR 640.60-640.76 (which is incorporated herein by reference). Other requirements for albumin (human) are listed in 21CFR 640.80-640.84 (incorporated herein by reference).

[0079] Albumin (human) was tested to determine compliance with the following standards in accordance with 21 CFR 640.82:

[0080] (a) Protein concentration. The final product should conform to one of the following concentrations: 4.0 + / - 0.25 percent; 5.0 + / - 0.30 percent; 20.0 + / - 1.2 percent; and 25.0 + / - 1.5 percent protein solution.

[0081] (b) Protein composition. At least 96 percent of the total protein in the final product shall be albumin as determined by a method approved for each manufacturer by the Director, Center for Biologics Evaluation and Research, Food and Drug Administration.

[0082] As used herein, "albumin (human)" or "HAS" refers to a sterile solution of proteins consisting of albumin and globulin derived from human plasma, wherein the albumin content is at least 95% and globulins (including α1 globulin, α2 globulin, β globulin and γ globulin) and other plasma proteins do not exceed 5%. HAS may also refer to a solid form, which has a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting albumin from the total protein.

[0083] As can be appreciated by those of ordinary skill in the art, PPF and HAS fractions may also be freeze-dried or in other solid forms. Such preparations with appropriate additives may be used to prepare, for example, tablets, powders, granules or capsules. The solid form may be formulated into a preparation for injection by dissolving, suspending or emulsifying the solid form in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a synthetic fatty acid glyceride, a higher fatty acid ester or a propylene glycol ester; and, if necessary, including conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0084] E. Fractions with reduced coagulation factors

[0085] Another embodiment of the present invention uses a plasma fraction from which substantially all coagulation factors are removed to retain the efficacy of the fraction and reduce the risk of thrombosis. Conveniently, the blood product can be derived from a young donor or a young donor group, and it can be made IgM-free to provide a young blood product compatible with ABO. At present, the plasma of the transfusion is matched with the ABO blood type because the presence of naturally occurring antibodies to A and B antigens can cause transfusion reactions. IgM presents a transfusion reaction that causes when the patient receives ABO-mismatched plasma. Removing IgM from the blood product or fraction helps eliminate the transfusion reaction of the object to which the blood product of the present invention and the plasma fraction are administered.

[0086] Therefore, in one embodiment, the present invention relates to a method for treating an object suffering from an undesirable condition / indication associated with any of the following liver diseases or liver failures. The method comprises: administering to the object a blood product or blood fraction derived from whole blood of an individual or a group of individuals, wherein the blood product or blood fraction 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 fraction is derived is a young individual. In some embodiments, the blood product is substantially free of at least one coagulation factor and IgM. In certain embodiments, the blood product is substantially free of fibrinogen (factor 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. U.S. Patent Application No. 62 / 376529, filed on August 18, 2016, further supports such embodiments of the present invention, and the patent application is incorporated herein by reference as a whole.

[0087] F. Treatment of protein-rich plasma products

[0088] Another embodiment of the present invention uses a plasma fraction with a reduced albumin concentration compared to PPF, but with an increased amount of globulins and other plasma proteins (referred to as "contaminants"). Like PPF, HAS, effluent I and effluent II / III, the embodiments are all effectively free of coagulation factors. Such plasma fractions are referred to as "protein-rich plasma protein preparations" hereinafter. For example, an embodiment of the present invention can use a protein-rich plasma protein preparation, which is composed of 82% albumin and 18% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention can use a protein-rich plasma protein preparation, which is composed of 81% albumin and 19% alpha globulin, beta globulin and gamma globulin and / or other plasma proteins. Another embodiment of the present invention can use a protein-rich plasma protein preparation, which is composed of 80% albumin and 20% alpha globulin, beta globulin and gamma globulin and / or other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 70% to 79% albumin and corresponding 21% to 30% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 60% to 69% albumin and corresponding 31% to 40% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 50% to 59% albumin and corresponding 41% to 50% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 40% to 49% albumin and corresponding 51% to 60% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 30% to 39% albumin and corresponding 61% to 70% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 20% to 29% albumin and corresponding 71% to 80% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 10% to 19% albumin and corresponding 81% to 90% alpha globulin, beta globulin and gamma globulin and other plasma proteins. Another embodiment of the present invention may use a protein-rich plasma protein preparation consisting of 1% to 9% albumin and corresponding 91% to 99% alpha globulin, beta globulin and gamma globulin and other plasma proteins.Another embodiment of the present invention may use a protein-enriched plasma protein preparation consisting of 0% albumin and 100% alpha, beta, and gamma globulins and other plasma proteins.

[0089] The above described embodiments of the invention may also have a total gamma globulin concentration of 1% to 5%.

[0090] The specific concentration of protein in the plasma fraction can be determined using techniques known to those of ordinary skill in the relevant art. Such techniques include, by way of example and not limitation, electrophoresis, mass spectrometry, ELISA analysis, and Western blot analysis.

[0091] G. Preparation of Plasma Fractions

[0092] Methods for preparing PPF and other plasma fractions are well known to those of ordinary skill in the art. Embodiments of the present invention allow blood for preparing human plasma protein fractions to be collected in flasks with citrate or anticoagulant citrate dextrose solution (or other anticoagulants) to inhibit coagulation, and further separated into 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 SANGUINIS2 (174) (1957), which is incorporated herein by reference). According to this method, the mixture can be collected to 2°C to 8°C. The plasma can then be separated by centrifugation at 7°C, removed, and stored at -20°C. The plasma can then be thawed and fractionated at 37°C, preferably within eight hours after being removed from -20°C storage.

[0093] Plasma can be separated from fraction I using 8% ethanol at pH 7.2 and a temperature of -2°C to -2.5°C, with a protein concentration of 5.1% to 5.6%. Cold 53.3 percent ethanol (176 mL / L plasma) and acetate buffer (200 mL 4M sodium acetate, 230 mL glacial acetic acid made up to 1 L with H2O) can be added using a nozzle at a rate of, for example, 450 mL / min while the temperature of the plasma is lowered to -2°C. Fraction I can be separated by ultracentrifugation and removed from the effluent (effluent I). Fibrinogen can be obtained from fraction I according to methods known to those of ordinary skill in the art.

[0094] Fraction II+III is separated from effluent I by adjusting the effluent to pH 6.8, 21% ethanol at -6°C and a protein concentration of 4.3%. During the process of lowering the temperature of effluent I to -6°C, cold 95% ethanol (176 mL / L effluent I) and 10 M acetic acid for pH adjustment can be added using a nozzle at a rate of, for example, 500 mL / min. The resulting precipitate (fraction II+III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from fraction II+III using methods known to those of ordinary skill in the art.

[0095] Fraction IV-1 is separated from effluent II+III ("effluent II / III") by adjusting the effluent to pH 5.2, 19 percent ethanol at -6°C, and a protein concentration of 3 percent. A nozzle can be used to add H2O and 10M acetic acid for pH adjustment while maintaining effluent II / III at -6°C for 6 hours. The precipitated fraction IV-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 is recovered from effluent IV-1 by adjusting the ethanol concentration to 30 percent, pH 4.65, temperature -7°C, and a protein concentration of 2.5 percent. This can be achieved by adjusting the pH of effluent IV-1 with cold acid-alcohol (two parts 2M acetic acid and one part 95% ethanol). While maintaining a temperature of -7°C, 170 mL of cold ethanol (95%) is added to each liter of adjusted effluent IV-1. The precipitated protein can be allowed to stand for 36 hours and then removed by centrifugation at -7°C. Fraction IV-4 paste can also be obtained using the Cohn fractionation process. In fact, fraction IV-4 and its production method have been previously described (Schopfer LM et al., PLoS ONE, 14 (1): e0209795 (2018), incorporated herein by reference in its entirety) (Schopfer LM et al., PLoS ONE, 14 (1): e0209795 (2018) and Bertolini J, Goss N, Curlin J eds., PRODUCTION OF PLASMAPROTEINS FOR THERAPEUTIC USE, 16.4: 231: 232 (2013), incorporated herein by reference in its entirety).

[0096] The recovered protein (stable plasma protein fraction) can be dried (e.g., by freeze drying) to remove ethanol and H2O. The resulting dry powder can be dissolved in sterile distilled water, for example, using 15 liters of water / kg of powder, and the pH of the solution is adjusted to 7.0 with 1M NaOH. The final concentration of acetyltryptophan can be adjusted to 0.004M for acetyltryptophan, 0.004M for octanoic acid, and 0.112M for sodium by adding sterile distilled water containing sodium acetyltryptophan, sodium octanoate, and NaCl to a final concentration of 5 percent for the protein. Finally, the solution can be filtered at 10°C to obtain a clear solution, and then heat treated at 60°C for at least 10 hours to inactivate pathogens.

[0097] Those of ordinary skill in the art will recognize that each of the above-described different fractions, effluents, or pastes can be used with the methods of the present invention to treat conditions such as liver disease or liver failure. For example, but not limited to, effluent I or effluent II / III can be used to treat conditions such as liver disease or liver failure and is an embodiment of the present invention. Another example is that a paste of fraction I, a paste of fraction II+III, a paste of fraction IV-1, a paste of fraction IV-4, and / or a paste of fraction V, or a suspension thereof, can be used to treat conditions such as liver disease or liver failure and is an embodiment of the present invention.

[0098] The aforementioned methods for preparing plasma fractions and plasma protein fractions (PPF) are exemplary only and relate only to embodiments of the present invention. One of ordinary skill in the art will recognize that these methods can be varied. For example, in different embodiments and methods of the present invention, pH, temperature and ethanol concentration can be adjusted, among others, to produce different variations of plasma fractions and plasma protein fractions. In another example, other embodiments of the present invention contemplate the use of nanofiltration to remove / inactivate pathogens from plasma fractions and plasma protein fractions.

[0099] Additional embodiments of the invention contemplate methods and compositions using and / or comprising additional plasma fractions. For example, the invention contemplates, among other things, that a particular concentration of albumin is not critical for treating conditions associated with liver disease or liver failure. Thus, the invention contemplates fractions having reduced albumin concentrations, such as fractions having less than 83% albumin.

[0100] H. Treatment

[0101] Aspects of the methods of the present invention described herein include treating subjects with blood products such as plasma fractions, for example, as described above. Embodiments include treating human subjects with blood products comprising plasma. Those skilled in the art will recognize that methods for treating subjects with blood products comprising plasma are recognized in the art. As an example and not limitation, one embodiment of the methods of the present invention described herein includes administering plasma fractions or fresh frozen plasma to subjects to treat liver-related conditions, such as acute failure or chronic failure. Other embodiments of the present invention include wherein acute liver failure is associated with infectious diseases such as hepatitis B or C, overuse of drugs or toxins (such as acetaminophen, isoniazid), and metabolic or vascular diseases such as autoimmune hepatitis and Wilson's disease. Other embodiments of the present invention include wherein chronic liver failure is associated with viral infection, alcoholism, NAFLD (caused by obesity, high cholesterol and triglycerides, and hypertension), autoimmune diseases, obstruction or damage of ducts such as the bile duct from the liver to the intestine, exposure to toxins or certain drugs, parasites, and heart failure leading to blood accumulation in the liver.

[0102] Additional embodiments of the present invention include liver diseases or liver failure associated with genetic diseases, including, for example, but not limited to: alpha-1 antitrypsin deficiency; bile acid synthesis disorders (Wilson disease, progressive familial intrahepatic cholestasis type 3); carbohydrate metabolism disorders (hereditary fructose intolerance, type IV glycogen storage disease); amino acid metabolism disorders (type I tyrosinemia); urea cycle disorders (argininosuccinate lyase deficiency, citrate deficiency-CTLN2, NICCD); lipid metabolism disorders (cholesterol ester storage disease); cystic fibrosis; hemochromatosis; Alstrom syndrome; and congenital hepatic fibrosis. (Scorza M et al., Int'l J Hepatology, 2014, Article ID 713754 (2014)).

[0103] Additional embodiments of the invention include liver disease or liver failure associated with infectious agents, such as, but not limited to: viruses (Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other herpes viruses, yellow fever, dengue fever, hepatitis B and hepatitis C); bacteria (typhoid fever, Mycobacterium tuberculosis, brucellosis, Q fever, leptospirosis, spirochetes - syphilis, Borrelia); parasites (schistosomiasis, malaria); fungi (Candida). (Talwani R et al., Clin Liver Dis., 15(1): 111-30 (2011)).

[0104] Additional embodiments of the invention include liver disease or liver failure associated with drugs or toxic substances, such as, but not limited to, acetaminophen, isoniazid, and drugs metabolized by the liver.

[0105] Additional embodiments of the invention include liver diseases, wherein the liver is partially or completely removed. Additional embodiments include liver diseases, wherein a donor liver is transplanted into a subject suffering from the liver disease. Additional embodiments include administering a therapeutic agent, such as a blood product or plasma fraction comprising plasma, to a subject preoperatively, perioperatively, and postoperatively.

[0106] In one embodiment, immediately, for example, within about 12 hours to 48 hours of collection from a donor, a blood product comprising plasma is administered to an individual suffering from liver disease or liver failure. In such cases, the product can be stored under refrigeration conditions of, for example, 0°C to 10°C. In another embodiment, fresh frozen plasma is plasma that has been frozen and stored (frozen) under conditions of -18°C or below -18°C. Before administration, fresh frozen plasma is thawed, and once thawed, administered to the subject 60 minutes to 75 minutes after the thawing process begins. Each subject preferably receives a single unit of fresh frozen plasma (200mL to 250mL), which is preferably derived from a donor in a predetermined age range. In one embodiment of the present invention, fresh frozen plasma is donated from (derived from) a young individual. In another embodiment of the present invention, fresh frozen plasma is donated from (derived from) a donor of the same sex. In another embodiment of the present invention, fresh frozen plasma is donated from (derived from) a donor aged 18 to 22 years old.

[0107] In an embodiment of the present invention, after donation by blood type, blood products comprising plasma are screened. In another embodiment of the present invention, according to the requirements of 21CFR 640.33 and the suggestions included in the FDA guidance documents, blood products comprising plasma are screened for infectious pathogens such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc.

[0108] In another embodiment of the present invention, the subject is treated with a plasma fraction. In an embodiment of the present invention, the plasma fraction is PPF, HAS, fraction IV-1, fraction IV-1 paste, fraction IV-4 or fraction IV-4 paste. In another embodiment of the present invention, the plasma fraction is a commercial PPF preparation in a commercial HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF, HAS, fraction IV-4 or fraction IV-4 paste derived from an individual of a specific age range, such as a young individual group, or a modified PPF, HAS, fraction IV-4 or fraction IV-4 paste fraction (e.g., PPF, HAS, fraction IV-1, fraction IV-1 paste, fraction IV-4 or fraction IV-4 paste) that has been subjected to additional fractionation or processing. In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulin (IgG). A blood fraction that is "substantially depleted" or has a particular protein, such as IgG, "substantially removed" refers to a blood fraction that contains less than about 50% of the amount present in a reference preparation or whole plasma, such as 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, as measured using standard assays well known in the art.

[0109] I. Application

[0110] Aspects of the methods of the present invention described herein include treating subjects with blood products, such as plasma or plasma fractions, such as those described above. Embodiments include treating human subjects with blood products comprising plasma. Those skilled in the art will recognize that methods of treating subjects with blood products comprising plasma are recognized in the art. As an example and not limitation, one embodiment of the methods of the present invention described herein includes administering fresh frozen plasma to subjects to treat, for example, liver disease or liver failure conditions. In one embodiment, immediately, for example, within about 12 hours to 48 hours of collection from a donor, a blood product comprising plasma is administered to an individual suffering from an undesirable condition, such as liver disease or liver failure. In such cases, the product can be stored under refrigerated conditions, such as 0°C to 10°C. In another embodiment, fresh frozen plasma is plasma that has been frozen and stored (cryopreserved) at or below -18°C. Prior to administration, fresh frozen plasma is thawed, and once thawed, administered to the subject 60 minutes to 75 minutes after the thawing process begins. Each subject preferably receives a single unit of fresh frozen plasma (200mL to 250mL), preferably derived from a donor in a predetermined age range. In one embodiment of the present invention, fresh frozen plasma is donated from (derived from) a young individual. In another embodiment of the present invention, fresh frozen plasma is donated from (derived from) a donor of the same sex. In another embodiment of the present invention, fresh frozen plasma is donated from (derived from) a donor aged 18 to 22 years old.

[0111] In an embodiment of the present invention, after donation by blood type, blood products comprising plasma are screened. In another embodiment of the present invention, according to the requirements of 21CFR 640.33 and the suggestions included in the FDA guidance documents, blood products comprising plasma are screened for infectious pathogens such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc.

[0112] In another embodiment of the present invention, the subject is treated with a plasma fraction. In an embodiment of the present invention, the plasma fraction is PPF or HAS. In another embodiment of the present invention, the plasma fraction is one of a commercial PPF preparation or a commercial HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF or HAS derived from an individual of a specific age range, such as a young individual group, or is a modified PPF or HAS fraction (e.g., a PPF or HAS in which one or more than one specific protein has been partially or substantially removed) that has been subjected to additional fractionation or processing. In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulins (IgG). A blood fraction that is "substantially depleted" or has a particular protein, such as IgG, "substantially removed" refers to a blood fraction that contains less than about 50% of the amount present in a reference preparation or whole plasma, as measured using standard assays well known in the art, such as 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.

[0113] Embodiments of the present invention include treating an object suffering from liver disease or liver failure by administering an effective amount of plasma or plasma fractions to the object. Another embodiment of the present invention includes administering an effective amount of plasma or plasma fractions, and subsequently monitoring the improved liver function, liver regeneration, the presence of markers, the reduction of pain or the reduction of inflammation of the object. Another embodiment of the present invention includes treating an object suffering from a related condition such as liver disease or liver failure by administering an effective amount of plasma or plasma fractions to the object, wherein the plasma or plasma fractions are administered in such a manner that, relative to the most recently administered dose, after reaching the average or median half-life of plasma proteins or plasma fraction proteins, liver function is improved, liver regeneration, markers are present, pain is reduced or inflammation is reduced (referred to herein as "pulse administration" or "pulse administration") (see U.S. Patent Application Nos. 15 / 499,697 and 62 / 701,411, which are incorporated herein by reference in their entirety). Another embodiment of the present invention includes administering plasma or plasma fractions by a dosing regimen of at least two consecutive days, and monitoring the improvement of liver function or HSC marker levels of the object for at least 3 days after the date of the last administration. Other embodiments of the present invention include administering plasma or plasma fractions by a dosing regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, and monitoring the improvement of liver function, liver regeneration, the presence of markers, the reduction of pain or the reduction of inflammation of the subject for at least 3 days after the date of the last administration. Another embodiment of the present invention includes administering plasma or plasma fractions by a dosing regimen of at least 2 consecutive days, and after the date of the last administration, monitoring the improvement of liver function when the average half-life of protein in plasma or plasma fractions has been reached, the presence of markers, the reduction of pain or the reduction of inflammation. Another embodiment of the present invention includes administering plasma or plasma fractions by a dosing regimen of non-continuous 2 to 14 days, wherein each gap between administrations can be 0 to 3 days respectively.

[0114] In some cases, pulse administration according to the present invention, for example as described above, includes administering a first group of doses, followed by a non-administration period, such as a "non-administration period", which is followed by administering another dose or another group of doses. The duration of the "non-administration period" can vary, but in some embodiments is 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 in some cases, the non-administration period is 15 days to 365 days, such as 30 days to 90 days, including 30 days to 60 days. Thus, the embodiment of the method includes non-long-term (i.e., non-continuous) administration of plasma products, such as non-long-term administration. In some embodiments, the pulse administration and subsequent non-administration period mode are repeated multiple times as needed, wherein in some cases, the mode is continued for 1 year or more than 1 year, such as 2 years or more than 2 years, up to and including the life of the subject. Another embodiment of the present invention includes administering plasma or plasma fractions by administering for 5 consecutive days, 2 days to 3 days of non-administration period, followed by continuous administration of 2 days to 14 days of dosing regimen:.

[0115] Additional embodiments of the invention include liver diseases in which the liver is partially or completely removed. Additional embodiments include liver diseases in which a donor liver is transplanted into a subject suffering from the liver disease. Additional embodiments include administering a therapeutic agent, such as a blood product or plasma fraction comprising plasma, to a subject preoperatively, perioperatively, and postoperatively. Methods for removing and transplanting a liver are well known in the art.

[0116] In biochemistry, an "effective amount" or "effective dose" of an active agent means an amount of the active agent that will inhibit, antagonize, reduce, decrease or eliminate by 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%, i.e., to a negligible amount, and in some cases, reverse an undesirable condition such as liver disease or liver failure.

[0117] J. Plasma protein fraction

[0118] In practicing the methods of the invention, a plasma fraction is administered to a subject. In embodiments, the plasma fraction is a plasma protein fraction (PPF). In further embodiments, the PPF is selected from a commercial PPF preparation.

[0119] In another embodiment, the PPF is composed of 88% normal human albumin, 12% alpha globulin and beta globulin, and no more than 1% gamma globulin as determined by electrophoresis. Additional embodiments for practicing the embodiments of the methods of the present invention include, for example, embodiments of a 5% PPF solution buffered with sodium carbonate and stabilized with 0.004M sodium octanoate and 0.004M acetyltryptophan. When practicing the methods of the present invention, additional formulations may be used, including formulations that vary 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 25%, from about 25% to 30%) and the concentrations of solvents and stabilizers.

[0120] K. Plasma fractions specific to donor age

[0121] Other embodiments of the present invention include administering plasma protein fractions derived from plasma of individuals of certain age ranges. Embodiments include administering PPF or HAS derived from plasma of young individuals. In another embodiment of the present invention, young individuals have a single specific age or a specific age range. In another embodiment, the average age of the donor is less than the age of the object or less than the average age of the object being treated.

[0122] Certain embodiments of the present invention include 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, a plasma protein fraction or a specific plasma protein fraction is obtained from a specific individual that meets a specific age range.

[0123] L. Indications

[0124] One embodiment of the present invention is to use plasma fractions and plasma fractionation products to subjects diagnosed with liver disease or liver failure that can benefit from improved liver proliferation or regeneration. Another embodiment of the present invention includes when the disease is associated with: acute liver failure, chronic liver failure or chronic acute liver failure. Chronic acute liver failure occurs in patients with relatively stable chronic liver disease but suddenly turns into acute liver failure. This usually leads to a very high mortality rate. Another embodiment of the present invention is where acute liver failure is associated with infectious diseases such as hepatitis B or C, overuse of drugs or toxins (such as acetaminophen, isoniazid) and metabolic or vascular diseases such as autoimmune hepatitis and Wilson's disease. Additional embodiments of the present invention include where chronic liver failure is associated with viral infection, alcoholism, NAFLD (caused by obesity, high cholesterol and triglycerides and high blood pressure), autoimmune diseases, obstruction or damage of ducts such as bile ducts from the liver to the intestines, exposure to toxins or certain drugs, parasites, and heart failure leading to blood accumulation in the liver.

[0125] Additional embodiments of the present invention include liver diseases or liver failure associated with genetic diseases, such as, but not limited to, alpha-1 antitrypsin deficiency; bile acid synthesis disorders (Wilson's disease, progressive familial intrahepatic cholestasis type 3); carbohydrate metabolism disorders (hereditary fructose intolerance, type IV glycogen storage disease); amino acid metabolism disorders (type I tyrosinemia); urea cycle disorders (argininosuccinate lyase deficiency, citrate deficiency-CTLN2, NICCD); lipid metabolism disorders (cholesterol ester storage disease); cystic fibrosis; hemochromatosis; Astrom syndrome; and congenital hepatic fibrosis. (Scorza M et al., Int'l J Hepatology, 2014, Article ID 713754 (2014)).

[0126] Additional embodiments of the invention include liver disease or liver failure associated with infectious agents, such as, but not limited to: viruses (Epstein-Barr virus, cytomegalovirus, herpes simplex virus and other herpes viruses, yellow fever, dengue fever, hepatitis B and hepatitis C); bacteria (typhoid fever, mycobacterium tuberculosis, brucellosis, Q fever, leptospirosis, spirochetes - syphilis, Borrelia); parasites (schistosomiasis, malaria); fungi (Candida). (Talwani R et al., Clin Liver Dis., 15(1): 111-30 (2011)).

[0127] Additional embodiments of the invention include liver disease or liver failure associated with drugs or toxic substances, such as, but not limited to, acetaminophen, isoniazid, and drugs metabolized by the liver.

[0128] Additional embodiments of the invention include liver diseases in which a portion of the liver is removed. Additional embodiments include liver diseases in which a donor liver is transplanted into a subject with the liver disease. Additional embodiments include administering a therapeutic agent, such as a blood product or plasma fraction comprising plasma, to a subject preoperatively, perioperatively, and postoperatively.

[0129] M. Reagents, Devices, and Kits

[0130] Also provided are reagents, devices and kits thereof for carrying out one or more than one of the above methods. The target reagents, devices and kits thereof can vary widely.

[0131] Reagents and devices of interest include those mentioned above with respect to the method of preparing a plasma-containing blood product for infusion into a subject in need thereof, such as anticoagulants, cryopreservatives, buffers, isotonic solutions, and the like.

[0132] The kit may also include blood collection bags, tubes, needles, centrifuge tubes, etc. In other embodiments, the kits described herein include two or more containers of plasma products, such as plasma protein fractions, such as three or more, four or more, five or more, including six or more containers of plasma products. In some cases, the number of different containers of plasma products in the kit may be 9 or more, 12 or more, 15 or more, 18 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 a variety of data about the plasma product contained therein, which may include one or more of the age of the donor of the plasma product, processing details about the plasma product, such as whether the plasma product has been treated to remove proteins with a molecular weight higher than the average (such as described above), blood type details, etc. In some cases, each container in the test kit includes identification information about the plasma contained therein, and the identification information includes information about the donor age of the plasma product, such as identification information provides age-related data confirming the plasma product donor (wherein, the identification information can be the age of the donor during collection). In some cases, each container of the test kit includes plasma products from donors of substantially the same age, that is, all containers include products from donors of substantially the same age if not the same. Substantially the same age means that the different donors of the plasma products from which the test kit is obtained differ in some cases by 5 years or less than 5 years, such as 4 years or less than 4 years, such as 3 years or less than 3 years, including 2 years or less than 2 years, such as 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 present on any convenient part of the container, such as a label, an RFID chip, etc. As required, the identification information can be human-readable, computer-readable, etc. The container can have any convenient configuration. The volume of container can be changed, but in some cases, the volume is 10ml to 5000mL, for example 25mL to 2500mL, for example 50ml to 1000mL, including 100mL to 500mL.Container can be rigid or flexible, and can be made of any convenient material, for example polymeric material, including medical grade plastic material.In some cases, container has bag or pouch configuration.In addition to container, described test kit can also include application device as described above.The assembly of described test kit can be provided with any suitable packaging, for example, box or similar structure, it is configured to hold the assembly of container and other test kits.

[0133] In addition to the above-mentioned components, the target test kit may also include instructions for implementing the target method. Instructions may be present in the target test kit in various forms, one of which or more than one may be present in the test kit. A form in which instructions may exist is printed information on a suitable medium or substrate, for example, one or more than one sheet of paper on which information is printed, in the packaging of the test kit, on a package insert, etc. Another way may be a computer-readable medium on which information is recorded, such as a floppy disk, a CD, a portable flash drive, etc. Another possible way is a website address, which can be used to access information at a remote site through the Internet. Any suitable device may be present in the test kit.

[0134] N. Experimental Examples

[0135] 1. Example 1

[0136] a) PPF1-induced functional recovery of liver in 20-month-old C57BL / 6 mice after 70% partial hepatectomy

[0137] Male C57BL / 6J mice (The Jackson Laboratory, Sacramento, CA) at 20 months of age were used. All mice were housed individually under specific pathogen-free conditions under a 12-hour light and 12-hour dark cycle, and the handling and use of all animals were in accordance with standard guidelines approved by the Institutional Animal Care and Use Committee. Animals were fed a 60% high-fat diet (Bio-Serv F3282) for 7 to 8 weeks and were randomly divided into a vehicle group and a PPF1 treatment group according to the body weight and ALT level after the diet to ensure that the average body weight and serum ALT level between the groups were the same. Surgery was performed with minor modifications as described above (Nevzorova, Y. et al., Lab. Anim. 49, 81-88 (2015)). The resected liver (left and median lobes) were used as preoperative controls, while the remnant liver (caudate and right lobes) were harvested 48 hours after surgery. Twenty-four (24) hours after surgery, EdU (2.5 mg / mL stock solution in saline) was injected intraperitoneally at 30 mg / kg body weight. Forty-six (46) hours after surgery, BrdU (10 mg / mL stock in saline) was injected intraperitoneally at 30 mg / kg body weight. 48 hours after surgery, all animals were weighed, and viscera (caudate and right lobes) and serum were collected for ALT analysis.

[0138] Commercially available PPF ("PPF1"), such as the commercial PPF preparation described above in a 5% solution, is stored at 4°C. PPF1 is a PPF having about 88% normal human albumin (relative to total protein), 12% alpha and beta globulins, and no more than 1% gamma globulin as determined by electrophoresis. Unless otherwise stated, PPF1 was administered using a 5% solution (weight / volume, 50 g / L) in the examples herein.

[0139] For qPCR analysis, RNA was extracted from excised or residual livers pulverized with liquid nitrogen and mortar / pestle. Extraction was then performed using Trizol (Ambion / Fisher 15-596-018) and RNeasy Mini Kit (Qiagen 74106). cDNA was amplified using iScript Reverse Transcription Supermix (Bio-Rad 1030). QPCR was amplified using SsoAdvancedUniversal SYBR green master mix (Bio-Rad 1725272) and performed using Quant Studio 6Flex (Applied Biosystem). Gene expression was normalized to RPS18 (ribosomal protein s18).

[0140] For immunostaining, 12 micron thick frozen liver sections embedded in OCT compound (Sakura 4583) were used for immunofluorescence staining. Antigens were repaired at 95°C for 30 minutes using citrate buffer (Sigma C9999). For EdU labeling, Click-IT Plus EdU Alexa Fluor 555 (Fisher C10638) was used. Hoechst 33342 was used to label the nuclei at 1 to 100,000 in ddH2O for 3 minutes (Fisher H3570). Oil Red O staining was performed to visualize triglyceride accumulation in the liver. In brief, frozen liver sections were immersed in isopropanol containing 0.375% Oil Red O for 5 minutes. The sections were rinsed under running tap water for 30 minutes and fixed with Prolong gold antifade reagent (Invitrogen P36934).

[0141] Figure 1 A graph showing the weight progression of 20 month old C57BL / 6 mice on a high fat diet ("HFD"). After 8.5 weeks of HFD, mice gained significant weight. Mean ± SEM, ****p<0.0001, ordinary one-way ANOVA.

[0142] Figure 2 Yes Figure 1 Oil Red O staining of the liver of the mice. Compared with mice fed a normal diet ("Normal Diet"), mice fed a HFD developed fatty liver.

[0143] Figure 3 Shown is the experimental design to evaluate the effects of PPF1 on 20 month old C57BL / 6 mice fed a high fat diet as described above.

[0144] Figure 4 Determined Figure 3 The treated mice had the resected lobe and residual lobe resulting from a 70% hepatectomy.

[0145] Figure 5 Shown in the Figure 3 The ratio of liver weight to body weight was determined for excised livers of vehicle and PPF1 treated mice.

[0146] Figure 6 Shown in the Figure 3 Liver weights of excised livers from vehicle and PPF1 treated mice.

[0147] Figure 7 is a graph showing that serum ALT levels were normal and unaffected in mice on a HFD treated with PPF1 before hepatectomy. ALT levels are considered normal when they are less than 50 units per liter to 60 units per liter. (See, for example, Mazzaccara C et al., PLoS ONE, 3(11): e3772 (2008)).

[0148] Figure 8 It shows that Figure 3 Results of determination of the ratio of liver weight to body weight in vehicle- and PPF1-treated mice with remnant livers 48 hours after hepatectomy. Liver weight was significantly increased 48 hours after hepatectomy in PPF1-treated animals compared to vehicle-treated animals. Mean ± SEM, **p < 0.001 using unpaired T-test with Whitney correction.

[0149] Fig. 9 It shows that Figure 3 Liver weight in vehicle- and PPF1-treated mice with remnant liver 48 hours after hepatectomy. Liver weight was significantly increased in PPF1-treated animals 48 hours after hepatectomy compared to vehicle-treated animals. Mean ± SEM, *p < 0.05 using unpaired T-test with Whitney correction.

[0150] Fig.10 It shows that Figure 3Results of serum ALT levels in vehicle- and PPF1-treated mice with remnant livers 48 hours after hepatectomy. Serum ALT levels were significantly reduced in PPF1-treated animals 48 hours after hepatectomy compared to vehicle-treated animals, indicating that the extent of liver damage was also significantly reduced in animals treated with PPF1. Mean ± SEM, **p < 0.001 using unpaired T-test with Whitney correction.

[0151] In contrast, differences in resected liver weight, liver weight / body weight ratio, and ALT levels between vehicle-treated and PPF1-treated animals failed to reach significance.

[0152] Fig.11 Cell proliferation rates after hepatectomy are reported. EdU was delivered 24 hours after hepatectomy, and proliferation rates were tracked by Click-it labeling of EdU-positive cells. PPF1 significantly increased the number of EdU-positive cells per field compared to the remnant liver of vehicle-treated animals.

[0153] Fig.12 Cell proliferation 48 hours after hepatectomy as measured by the number of Ki67-positive cells per field is reported. PPF1 significantly increased the number of Ki67-positive cells per field compared to the remnant liver of vehicle-treated animals.

[0154] Fig.13 Cell proliferation rates in remnant livers by qPCR gene expression are reported. Relative expression of the cell cycle marker cyclin B1 is shown. In remnant liver sections, expression of cyclin B1 was significantly upregulated in PPF1-treated mice compared to vehicle-treated mice. Mean ± SEM, *p < 0.05 using Whitney's unpaired T test.

[0155] FIG. 14A to FIG. 14D qPCR expression of several markers in resected livers is reported. The cell cycle marker cyclin B1 ( Fig.14A ), cyclin A2 ( Fig. 14B ) and Ki67( Fig. 14C ) in excised liver sections removed during hepatectomy as preoperative controls, PPF1 surprisingly and significantly increased cell proliferation compared to vehicle controls with all three cell cycle markers. Fig.14DRelative expression levels of TNFα in resected liver sections are reported. TNFα is known to promote restoration of functional liver mass by driving hepatocyte proliferation and liver regeneration. In summary, PPF1 treatment resulted in activation of cell proliferation in otherwise quiescent and intact livers (before hepatectomy) compared to vehicle controls. Mean ± SEM, *p < 0.05 unpaired T-test using Whitney correction.

[0156] Fig.15 It shows that Fig. 14C Immunostaining of Ki67 in resected livers reported in , which confirmed that PPF1-treated resected livers had significantly higher numbers of Ki67-positive cells compared to vehicle controls.

[0157] Fig.16 Reported from Fig.15 Quantification of immunostaining showing an increase in the number of Ki67-positive cells in excised livers treated with PPF1. Mean ± SEM, *p < 0.05 using unpaired T-test with Whitney correction.

[0158] Fig.17 is a graph showing the total number of animals that underwent hepatectomy and the survival of animals that received each treatment. Animals treated with PPF1 showed a trend toward increased survival compared to vehicle-treated animals.

[0159] Fig.18 Two confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Histological sections were stained with GFAP antibody (astrocytic marker) and the level of colocalization between EdU and GFAP was observed. As determined by the lack of colocalization between EdU and GFAP, cell proliferation associated with PPF1 administration did not occur in astrocytes.

[0160] Fig.19 Two confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with CD68 antibody (Kupffer cell marker) and the level of colocalization of EdU and CD68 was observed. As determined by the lack of colocalization between EdU and CD68, cell proliferation associated with PPF1 administration did not occur in Kupffer cells.

[0161] Fig. 20 Confocal microscopy of a liver remnant 24 hours after hepatectomy with EdU (see Figure 3). Tissue sections were stained with HNF4a antibody (a hepatocyte marker) and the colocalization level of EdU and HNF4a was observed. As determined by the lack of colocalization between EdU and HNF4a, cell proliferation associated with PPF1 administration did not occur in hepatocytes.

[0162] Fig.21 Confocal microscopy of a liver remnant 24 hours after hepatectomy with EdU (see Figure 3 ). Tissue sections were stained with CD3 antibody (T cell marker) and the colocalization level of EdU and CD3 was observed. As determined by the lack of colocalization between EdU and CD3, cell proliferation associated with PPF1 administration did not occur in T cells.

[0163] Fig. 22 Two confocal microscopy fields of a liver remnant incorporating EdU 24 hours after hepatectomy are shown (see Figure 3 ). Tissue sections were stained with CD31 antibody (a marker for sinusoidal endothelial cells), and the level of colocalization of EdU and CD31 was observed. Cell proliferation caused by PPF1 administration was associated with liver sinusoidal endothelial cells (LSECs), as determined by the positive colocalization between EdU and CD31. These results suggest that PPF1 increases the proliferation of capillary cells in the liver, allowing more blood to be brought to the site of injury, suggesting a mechanism for how tissue repair and regeneration occurs after PPF1 treatment. (Arrows indicate LSECs containing EdU-positive cells).

[0164] 2. Example 2

[0165] a) Effect of PPF1, recombinant human albumin and HAS1 on 20-month-old children after 70% partial hepatectomy

[0166] Effects on the liver of C57BL / 6 mice

[0167] Recombinant human albumin

[0168] Male C57BL / 6J were treated as described above in Example 1, except that an additional group was treated with recombinant human albumin ("rhAlbumin"). Comparisons between the PPF1-treated group, the rhAlbumin-treated group, and the vehicle-treated group were performed.

[0169] Fig.23The experimental design for evaluating the effects of PPF1 (type of plasma fraction "PF") and recombinant human albumin (rhAlbumin) on 20-month-old C57BL / 6 mice on a high-fat diet is shown. Mice were placed on a 60% high-fat diet for 8 weeks before being treated with PPF1, rhAlbumin or vehicle for 7 consecutive days. Surgery (70% hepatectomy) was performed the next day after the last dose of PPF1, rhAlbumin or vehicle, and the median lobe and left lobe (resected) before surgery were removed during the hepatectomy. The right lobe and caudate lobe (residual) were harvested 48 hours after hepatectomy.

[0170] Fig.24 Cell proliferation in resected livers 48 hours after hepatectomy, measured by the number of Ki67-positive cells per field, is reported. PPF1 significantly increased the number of Ki67-positive cells per field compared to vehicle-treated animals. In contrast, animals treated with rhAlbumin were not significantly different from those treated with vehicle. Mean ± SEM, ***p < 0.0001 using unpaired T-test with Whitney correction, ns = not significant.

[0171] Fig.25 Cell proliferation rates in the remnant liver after hepatectomy are reported. EdU was delivered 24 hours after hepatectomy and proliferation rates were tracked by click chemistry. PPF1 significantly increased the number of EdU-positive cells per field compared to vehicle-treated animals. In contrast, animals treated with rhAlbumin did not reach significance in proliferation compared to vehicle-treated animals.

[0172] These results suggest that albumin, the most abundant component of PPF1, may not be the component of PPF1 responsible for its proliferation, tissue repair and regeneration activities. Instead, this suggests that other components of PPF1, which are usually considered impurities left over from plasma fractionation, surprisingly drive these activities of PPF1.

[0173] Human Albumin Solution (HAS)

[0174] Male C57BL / 6J were again treated as described above in Example 1, except that an additional group was treated with human albumin solution ("HAS1"). Comparisons between the PPF1-treated group, the HAS1-treated group, and the vehicle-treated group were performed.

[0175] Fig.26The experimental design of the impact of PPF1 and HAS1 on 20-month-old C57BL / 6 mice on a high-fat diet is shown. Before being treated with PPF1, rhAlbumin or vehicle for seven consecutive days, mice were placed in a 60% high-fat diet for 8 weeks. Surgery (70% hepatectomy) was performed the second day after the last dose of PPF1, HAS1 or vehicle, and the median lobe and left lobe (resected) before surgery were removed during the hepatectomy. The right lobe and caudate lobe (residual) were harvested 48 hours after hepatectomy.

[0176] Fig. 27 Cell proliferation rates 24 hours after hepatectomy are reported. EdU was delivered 24 hours after hepatectomy. Both PPF1 and HAS1 significantly increased the number of EdU positive cells compared to vehicle in the remnant liver. *p<0.05, Welch's t-test. Error bars = SEM HAS1 is a commercially available HAS, such as the commercial HAS preparation described above in a 5% solution, and stored at 4°C.

[0177] Fig.28 Cell proliferation rate 48 hours after hepatectomy is shown. Ki67 immunostaining was performed on remnant livers removed 48 hours after hepatectomy. While both PPF1 and HAS significantly increased cell proliferation compared to control animals, PPF1 also induced significantly more proliferation compared to HAS1-treated animals. *p<0.05, **p<0.005, ***p<0.0005 Welch's t-test. Error bars = SEM

[0178] These results show that plasma fraction-induced hepatocyte proliferation is observed in fatty livers treated with HAS1 and PPF1. However, although two different plasma fractions (HAS1 and PPF1) significantly induced proliferation after 24 hours compared with the vehicle, the effect of PPF1 after 48 hours is more significant than HAS1, and even more significant than the vehicle. This shows again that, compared with PPF1, the purer and more abundant albumin in HAS1 is surprisingly not the main component that causes the plasma fraction to the influence of proliferation, tissue repair and regeneration. On the contrary, this shows that the impurities left by other components of the plasma fraction or plasma fractionation unexpectedly drive these activities of PPF1.

[0179] 3. Example 3

[0180] a) Effects of PPF1 on the liver of 20-month-old C57BL / 6 mice two hours after the last treatment administration

[0181] Twenty-month-old male C57BL / 6J mice were randomly divided into vehicle group and PPF1 treatment group according to body weight to ensure the same average body weight between groups. Mice were raised on a normal diet instead of a high-fat diet. Animals were administered 150 μL of vehicle or PPF1 daily for 7 consecutive days by intravenous injection. On the seventh day, the liver was removed 2 hours after administration. For gene expression analysis, RNA was extracted and reverse transcribed, and SYBR qPCR was performed for TNFα. Gene expression was normalized to RPS18. For immunostaining, proliferating cells in the harvested liver were labeled with Ki67 antibody.

[0182] Fig.29 Shown is the experimental design for evaluating the effects of PPF1 on 20 month old C57BL / 6 mice 2 hours after the last dose.

[0183] Fig.30 As shown above Fig.29 Results of TNFα gene expression analysis of the treated mice. PPF1 treated animals had significantly increased TNFα gene expression by QPCR compared to vehicle treated animals. **p<0.005. Welch's t-test, error bars = SEM

[0184] Fig.31 As shown above Fig.29 Results of Ki67 immunostaining analysis of treated mice. PPF1 treated animals had significantly increased Ki67 positive cells 2 hours after the last dose compared to vehicle treated animals, indicating that PPF1 increased hepatocyte proliferation. *p<0.05. Welch's t test, error bars = SEM

[0185] These results suggest that plasma fractions such as PPF1 can effectively induce proliferation and regeneration even in healthy, non-steatotic aged livers.

[0186] 4. Example 4

[0187] a) Effects of Fraction IV-1 Paste Suspension on Liver of 20-month-old C57BL / 6 Mice after 70% Partial Hepatectomy

[0188] Male C57BL / 6J mice (The Jackson Laboratory, Sacramento, CA) aged 20 months were used. All mice were housed individually under specific pathogen-free conditions under a 12-hour light and 12-hour dark cycle, and the handling and use of all animals were in accordance with standard guidelines approved by the Institutional Animal Care and Use Committee. Animals were fed a 60% high-fat diet (Bio-Serv F3282) for 7 to 8 weeks and were randomly divided into a vehicle group and a fraction IV-1 paste suspension treatment group according to the body weight and ALT level after the diet to ensure that the average body weight and serum ALT level between the groups were the same. Surgery was performed with minor modifications as described above (Nevzorova, Y. et al., Lab. Anim. 49, 81-88 (2015)). The resected liver lobes (left and median lobes) were used as preoperative controls, and the residual liver lobes (caudate and right lobes) were harvested 48 hours after surgery. Twenty-four (24) hours after surgery, EdU was injected intraperitoneally at 30 mg / kg body weight (2.5 mg / mL stock in normal saline). Forty-six (46) hours after surgery, BrdU was injected intraperitoneally at 30 mg / kg body weight (10 mg / mL stock in normal saline). 48 hours after surgery, all animals were weighed and the viscera (caudate and right lobes) and serum were collected for ALT analysis. The effects of the Fraction IV-1 paste suspension on these experimental contents were compared to vehicle. In addition, the effects of the Fraction IV-1 paste suspension on these experimental contents were compared to other plasma fractions.

Claims

1. Use of a plasma fraction in the preparation of a medicament for treating a subject diagnosed with a liver disease.

2. The use according to claim 1, wherein the plasma fraction is a plasma protein fraction.

3. Use according to claim 2, wherein the plasma protein fraction comprises 83% to 95% albumin.

4. The use according to claim 1, wherein the plasma fraction comprises a fraction IV-1 paste.

5. The use according to any one of claims 1 to 4, wherein the plasma fraction is administered by a dosing regimen using pulse dosing.

6. The use according to any one of claims 1 to 4, wherein the liver disease is chronic liver failure.

7. The use according to claim 6, wherein the chronic liver failure is associated with viral infection, alcoholism, NAFLD caused by obesity, high cholesterol and triglycerides and high blood pressure, autoimmune diseases, obstruction or damage of ducts, exposure to toxins or certain drugs, or parasites.

8. The use according to claim 7, wherein the duct is the bile duct from the liver to the intestine.

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