CN117355313A

CN117355313A - Plasma fraction for liver regeneration

Info

Publication number: CN117355313A
Application number: CN202280036050.8A
Authority: CN
Inventors: 维多利亚·海费茨; 班森·鲁
Original assignee: Universal Solvent Co ltd
Current assignee: Universal Solvent Co ltd
Priority date: 2021-05-19
Filing date: 2022-05-05
Publication date: 2024-01-05
Also published as: EP4340849A1; CA3218096A1; AU2022275694A1; WO2022245552A1

Abstract

Methods and compositions for treating aging-related disorders, liver regeneration, preventing liver degeneration, and maintaining the liver are described. The composition used in the method comprises plasma and plasma derived plasma fractions having efficacy in the treatment and/or prevention of disease.

Description

Plasma fraction for liver regeneration

I. Cross-reference to related applications

The present application claims priority from U.S. patent application Ser. No. 17/324359, filed on day 2021, 5, and 19, which is a partial continuation of U.S. patent application Ser. No. 16/951891, filed on day 2020, 11, and 18, which is hereby incorporated by reference in its entirety, according to 35 U.S. C. ≡119 (e), claims priority from U.S. provisional patent application Ser. No. 62/937965, filed on day 2019, 11, and U.S. provisional patent application Ser. No. 62/975637, filed on day 2020, 2, and 12.

II background art

The present invention relates to the 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, for the treatment and/or prevention of conditions associated with aging and with liver growth, maintenance and regeneration.

III summary of the invention

Liver failure is a vast market, and currently there is only one therapeutic intervention, liver transplantation. In one tenth of americans, approximately 3500 tens of thousands suffer from some form of liver disease. This number is increasing due to the prevalence of liver cirrhosis in increasingly obese people. NAFLD (non-alcoholic fatty liver disease) is a major risk factor for cirrhosis, which can ultimately lead to liver failure. The number of organs available for transplantation is small due to the reduced health of the general population. As a result, only about 1/3 of the patients on the liver transplant list received the transplant. This list generally excludes patients over 65 years old, as the increased risk associated with organ transplantation is age-related. Statistics are especially terrible for cirrhosis patients awaiting liver transplantation. 300000 cases of liver cirrhosis-based hospitalization were performed annually in the united states, and 36000 cases of liver cirrhosis deaths were not met by more than half of the patients. The availability of alternative treatment methods can significantly affect the majority of liver disease patients who would not otherwise have available treatment.

Liver failure may be acute or chronic. Acute liver failure is typically caused by viral infections (e.g., hepatitis b and c), overuse of drugs or toxins (e.g., acetaminophen), and metabolic or vascular diseases such as autoimmune hepatitis and wilson's disease. Chronic liver failure is generally classified as cirrhosis and can be caused by viral infection, alcoholism, NAFLD (caused by obesity, high cholesterol and triglycerides, and hypertension), autoimmune diseases, obstruction or damage of the ducts, e.g. bile ducts from the liver to the intestinal tract, exposure to toxins or certain drugs, parasites, heart failure leading to blood accumulation in the liver.

As described above, liver failure can be resected and transplanted, and transplantation is more commonly done for chronic liver failure. In addition, these processes can be used for primary and secondary malignancies (i.e., cancers). Improved outcome of liver resections and transplants has been associated with concerns for pre-operative, perioperative and post-operative care. (Wright ton LJ et al, J Gastroitest Oncol.,3 (1): 41-47, (2012)). These include advances in surgical and anesthesia techniques, better understanding of liver physiology, nutritional support, glycemic control, and reduction of post-operative infections. In the case of liver transplants, immunosuppressants are most commonly used (supra). The role of the attending physician in the management of liver rejection and in the treatment of long-term complications such as hypertension and obesity has been an important part of the improvement of prognosis. (see, issa DH, cleveland Clinic J of Med.,82 (6): 361-72 (2015)).

There is still a serious lack of new therapies to improve the prognosis of liver failure and/or patients receiving resection or transplantation. Furthermore, even in the case of liver transplantation surgery, there is not enough donor liver for all patients. ( For example, see notes that "the problem of transplantation is organ allocation. "Helwick C, the ASCO Post, (Sep 25,2017) )

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 albumin production by cirrhosis itself. The basic principle is that albumin is helpful for replacing osmotic pressure, combining toxic substances, regulating internal balance and playing an anti-inflammatory role. (Carvalho JR et al Annals of Hepatology,17 (4): 547-60 (2018)). Thus, the general idea in this field is that the purer the albumin, the higher the concentration, the better the efficacy produced. Therapeutic albumin is typically produced by plasma fractionation. Fractionation can produce an albumin solution, albeit with unwanted protein "contaminants" or "impurities" contained therein.

The present invention provides novel therapies that use plasma fractions to improve and accelerate recovery of diseased, resected or transplanted liver in patients with liver disease. Furthermore, since liver is the only organ capable of regeneration, the present invention provides a means to stimulate existing liver proliferation and regeneration, for example, in some liver-related indications, which may facilitate surgical resumption. Furthermore, the present invention utilizes substances derived from plasma fractionation that are considered "contaminants" or "impurities" to more effectively treat liver diseases.

IV. incorporated by reference

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 drawings

Figure 1 shows a graph of body weight progression of high fat diet ("HFD") 20 month old C57BL/6 mice. After 8.5 weeks of HFD, mice gain significantly weight.

FIG. 2 is an oil red O staining of mouse livers as described in FIG. 1. Mice with HFD showed fatty liver compared to normal diet mice.

FIG. 3 shows the experimental design for assessing the effect of PPF1 on C57BL/6 mice at 20 months of age with fatty liver. Mice were placed on a 60% high fat diet for 7 weeks prior to treatment with PPF1 or vehicle for 7 consecutive days. Surgery was performed the next day after the last dose of PPF1 or vehicle (70% hepatectomy) and pre-operative median lobes and Zuo She (resected) were removed during hepatectomy. Right and tail leaves (residual) were harvested 48 hours after hepatectomy.

Figure 4 determines excised leaves and residual leaves resulting from a 70% hepatectomy of mice treated as described in figure 3.

Fig. 5 shows the results of the determination of the weight to weight ratio of liver resections in vehicle-treated and PPF 1-treated liver resected mice treated as described in fig. 3.

Figure 6 shows the weight of liver resections in vehicle-treated and PPF 1-treated mice treated as described in figure 3.

Fig. 7 is a graph showing that serum ALT levels were normal and unaffected in PPF1 treated HFD mice prior to hepatectomy.

Fig. 8 shows the results of liver weight to body weight ratio determinations in vehicle-and PPF 1-treated mice at 48 hours post hepatectomy as described in fig. 3.

Figure 9 shows liver weight in vehicle-and PPF 1-treated mice 48 hours after hepatectomy as described in figure 3.

Fig. 10 shows the results of serum ALT levels in vehicle-and PPF 1-treated mice 48 hours after hepatectomy as described in fig. 3.

Fig. 11 reports the cell proliferation rate after hepatectomy. 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.

Fig. 12 reports cell proliferation 48 hours after hepatectomy as measured by Ki67 positive cell number per field. PPF1 significantly increased the number of Ki67 positive cells per field compared to vehicle treated animals.

FIG. 13 reports the cell proliferation rate in residual liver expressed by qPCR genes. The relative expression of the cyclin B1 marker is shown. In the liver residual section, PPF 1-treated mice were significantly upregulated in cyclin B1 expression compared to vehicle-treated mice.

Fig. 14A-14D report qPCR expression of several markers in resected liver. The relative expression of the cyclin B1 (fig. 14A), cyclin A2 (fig. 14B) and Ki67 (fig. 14C) markers is shown. PPF1 surprisingly significantly increased cell proliferation in resected liver sections taken during hepatectomy as pre-operative control compared to vehicle controls with all three cell cycle markers. Fig. 14D reports the relative expression levels of tnfα in resected liver sections. Tnfα is known to promote the restoration of functional liver quality by driving hepatocyte proliferation during liver regeneration.

Fig. 15 shows representative images of Ki67 immunostaining in resected liver as reported in fig. 14C, confirming that PPF1 treated resected liver has significantly higher numbers of Ki67 positive cells compared to vehicle control.

Figure 16 reports quantification of immunostaining from figure 15 showing an increase in the number of Ki67 positive cells in PPF1 treated resected liver.

Fig. 17 is a graph showing the total number of animals subjected to hepatectomy and the survival rate of each treatment.

Fig. 18 shows two representative confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Histological sections were stained with GFAP antibodies (astrocyte markers) and observed for co-localization levels of EdU and GFAP. It was determined that no cell proliferation associated with PPF1 administration occurred in astrocytes based on lack of co-localization between EdU and GFAP.

Fig. 19 shows two representative confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD68 antibody (kupfu cell marker) and observed for co-localization levels of EdU with CD 68. Cell proliferation associated with PPF1 administration did not occur in kupfu cells as determined by lack of co-localization between EdU and CD 68.

Fig. 20 shows representative confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with HNF4a antibodies (hepatocyte markers) and observed for co-localization levels of EdU with HNF4 a. It was determined that no cell proliferation associated with PPF1 administration occurred in hepatocytes based on the lack of co-localization between EdU and HNF4 a.

Fig. 21 shows representative confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD3 antibodies (T cell markers) and observed for co-localization levels of EdU with CD 3. Cell proliferation associated with PPF1 administration did not occur in T cells as determined by lack of co-localization between EdU and CD 3.

Fig. 22 shows two representative confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD31 antibody (sinus endothelial marker) and observed for co-localization levels of EdU with CD 31. Cell proliferation induced by PPF1 administration was determined to be associated with Liver Sinus Endothelial Cells (LSEC) based on positive co-localization between EdU and CD 31. Arrows indicate LSECs containing EdU positive cells.

FIG. 23 shows the experimental design for assessing the effect of PPF1 (plasma fraction "PF") and recombinant human albumin (rhAlbumin) on C57BL/6 mice at 20 months of age. Mice were placed in a 60% high fat diet for 8 weeks prior to treatment 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 pre-operative median lobes and Zuo She (resected) were removed during hepatectomy. Right and tail leaves (residual) were harvested 48 hours after hepatectomy.

Figure 24 reports cell proliferation in resected liver 48 hours after hepatectomy as measured by Ki67 positive cell number per field. PPF1 significantly increased the number of Ki67 positive cells per field compared to vehicle treated animals. In contrast, the rhAlbumin treated animals were not significantly different from the vehicle treated animals.

Fig. 25 reports the cell proliferation rate in the residual liver after hepatectomy. EdU was delivered 24 hours after hepatectomy and proliferation rate was followed 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 showed less pronounced proliferation tendencies compared to animals treated with vehicle.

FIG. 26 shows the experimental design for assessing the effect of PPF1 and HAS1 on C57BL/6 mice at 20 months of age. Mice were placed in a 60% high fat diet for 8 weeks before being treated with different Plasma Fractions (PF) PPF1 and HAS1 or vehicle for 7 consecutive days. The next day after treatment with the last dose of PPF1, HAS1 or vehicle, surgery was performed (70% hepatectomy) and pre-operative median lobes and Zuo She (resected) were removed during hepatectomy. Right and tail leaves (residual) were harvested 48 hours after hepatectomy.

Fig. 27 reports the cell proliferation rate 24 hours after hepatectomy. EdU was delivered 24 hours after hepatectomy. Both PPF1 and HAS1 significantly increased the number of EdU positive cells compared to the vehicle in the residual liver.

Fig. 28 shows the cell proliferation rate 48 hours after hepatectomy. Residual liver removed 48 hours after hepatectomy was Ki67 immunostained. Although both PPF1 and HAS significantly increased cell proliferation compared to control animals, PPF1 also significantly induced more proliferation compared to HAS1 treated animals.

Fig. 29 shows the experimental design for assessing the effect of PPF1 on C57BL/6 mice at 20 months of age 2 hours after the last dose.

FIG. 30 shows the results of a TNFα gene expression analysis on mice treated as described above in FIG. 29. PPF1 treated animals significantly increased tnfα gene expression by QPCR compared to vehicle treated animals.

FIG. 31 shows the results of Ki67 immunostaining analysis on mice treated as described above in FIG. 29. Compared to vehicle treated animals, PPF1 treated animals significantly increased Ki67 positive cells 2 hours after the last dose, indicating that PPF1 increased hepatocyte proliferation.

Figure 32 reports basal levels of cell proliferation in resected liver at hepatectomy. Male mice of twenty months of age were continuously treated with vehicle control or fraction IV-4 paste suspension for seven days using a pulse dosing regimen.

Fig. 33 reports the level of cell proliferation of residual liver lobes 48 hours after hepatectomy. Male mice of twenty months of age were continuously treated with vehicle control or fraction IV-4 paste suspension for seven days using a pulse dosing regimen.

Fig. 34 shows the proliferation level of hepatocytes in residual liver lobes 48 hours after hepatectomy by determining the ratio of ki67+ cells to hnf4a+ cells in 10 fields. Male mice of twenty months of age were continuously treated with vehicle control or fraction IV-4 paste suspension for seven days using a pulse dosing regimen.

Figure 35 shows the effect of high fat diet loading agent control, high fat diet plus PPF1 and normal diet on p53 gene relative RNA expression.

FIG. 36 shows the relative RNA expression of the same mouse group p21 gene as described in FIG. 35.

FIG. 37 shows the same view as FIG. 35The same mouse group p16 ^ink4a Relative RNA expression of genes.

VI. Detailed description of the preferred embodiments

A. Introduction to the invention

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 following hepatectomy. In addition, the plasma fraction was demonstrated to activate cell proliferation in resting and intact livers (prior to hepatectomy). The plasma fraction has several advantages over whole plasma serum, as the plasma fractionation process can remove problematic coagulation factors and eliminate the need for cross-matching. In addition, in certain assays, plasma fractions have shown unexpected improvements in efficacy compared to young plasma (see, e.g., U.S. patent application No. 15/499694 and U.S. patent application No. 16/432114; both of which are incorporated herein by reference in their entirety). Thus, predicting the efficacy of plasma fractionation products from whole plasma serum is not reasonably predictable.

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. No information herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates may be provided differently from the actual publication dates which may need to be independently confirmed.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also explicitly disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the ranges or excluded in the range, and each range is also encompassed within the invention, subject to any specifically excluded limit in the stated range, unless both, either or both of the two limits in the range are included in the smaller ranges. Where the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention.

It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, such recitations are intended to serve as a basis for the use of such exclusive terminology as "only," "only," and the like, or the use of a "negative" limitation in conjunction with the recitation of claim elements.

Those of skill in the art will upon reading this disclosure, it will be apparent that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any stated method may be performed in the order of stated events or in any other order that is logically possible.

B. Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some of the 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 in connection with which the publications are cited. It should be understood that this disclosure supersedes any disclosure of the incorporated publications if there is a conflict.

It must be noted that as used herein and in the appended claims, the use of numerical terms including plural referents is not used unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a peptide" includes reference to one or more than one peptide and equivalents thereof, such as polypeptides, and the like, as known to those skilled in the art.

In describing the methods of the present invention, the terms "host," "subject," "individual," and "patient" are used interchangeably and refer to any mammal in need of treatment 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 subject is a non-human mammal. In some embodiments, the subject is a livestock animal. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In certain embodiments, the subject is a human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys). Thus, subjects of the invention may include, but are not limited to, mammals, e.g., humans and other primates, e.g., chimpanzees, and other apes and monkey species, and the like, wherein in certain embodiments the subject is a human. The term subject is also intended to include a person or organism of any age, weight or other physical characteristic, wherein the subject may be an adult, child, infant or neonate.

"young person" or "young subject" refers to a subject that 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 a chronological order. In some cases, the individual serving as a source of the blood product comprising young blood plasma is 10 years old or less than 10 years old, e.g., 5 years old or less than 5 years old, including individuals 1 year old or less than 1 year old. In some cases, the subject is a neonate and the source of the plasma product is an umbilical cord, wherein the plasma product is harvested from the umbilical cord of the neonate. Thus, "young" and "young subject" may refer to subjects from 0 years to 40 years old, such as subjects from 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 subject" may refer to biological (as opposed to chronological) ages, such as subjects that have not exhibited inflammatory cytokine levels in plasma that are exhibited in older subjects. Conversely, "young" and "young subject" may refer to biological (as opposed to chronological) age, e.g., subjects exhibiting higher levels of anti-inflammatory cytokines than in older subjects. By way of example and not limitation, the inflammatory cytokine is an eosinophil-activating chemokine and the fold difference between a young subject or young subject and an elderly subject is at least 1.5 fold. Similarly, fold-differences in other inflammatory cytokines between aged and young individuals can be used to refer to biological age. (see U.S. patent application Ser. No. 13/575437, incorporated herein by reference). Typically, an individual is healthy, e.g., the individual is not suffering from a hematological malignancy or autoimmune disease at the time of collection.

As used herein, "treating" refers to any of (i) preventing a disease or disorder or (ii) alleviating or eliminating symptoms of a disease or disorder. Treatment may be performed prophylactically (prior to onset of liver disease or failure) and includes: (a) preventing a condition in a subject; (b) inhibiting the disorder, i.e., preventing the disorder from occurring; or (c) alleviating the condition, even if the condition subsides. Treatment may result in a variety of different physical manifestations, e.g., modulation of gene expression, tissue or organ rejuvenation, reduced inflammation, etc. The therapeutic agent may be administered before, during or after the onset of the condition. The targeted therapy may be administered during and, in some cases, after the symptomatic phase of the disorder. Treatment may also be performed by administering interventions such as plasma, plasma fractions or blood products containing plasma components prior to, during and/or after hepatectomy or transplantation.

"improvement", "increase" or "increase" in connection with liver function, regeneration or restoration 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 protein may be: ALT (7U/L to 55U/L); AST (8U/L to 48U/L); ALP (40U/L to 129U/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 (8U/L to 61U/L); LD (122U/L to 222U/L); and PT (9.4 seconds to 12.5 seconds).

A blood product comprising a plasma component.In practicing the subject methods, a blood product comprising a plasma component is administered to an individual in need thereof, e.g., an individual suffering from one or more of the following conditions: liver disease or liver failure. Thus, a method according to an embodiment of the invention comprises administering a blood product comprising a plasma component from an individual ("donor individual" or "donor") to an individual suffering from one or more of the following conditions: liver disease or liver failure ("subject" or "receptor"). By "blood product comprising a plasma component" is meant any product derived from blood comprising plasma (e.g. whole blood, plasma or fractions thereof). The term "plasma" is used in its conventional sense to refer to the pale yellow/pale yellow liquid component of blood, which consists of about 92% water, 7% proteins, such as albumin, gamma globulin, antihemophilic factor, and other clotting factors, and 1% mineral salts, sugars, fats, hormones, and vitamins. Non-limiting examples of blood products containing plasma suitable for use in the subject methods include whole blood treated with anticoagulants (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products produced by filtering whole blood to remove leukocytes ("leukocyte removal"), blood products consisting of plasmapheresis-derived (plasmapheresis-derived) or apheresis-derived (apheresis-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 preparation used is a non-whole plasma preparation, meaning that the preparation is not whole blood, and therefore lacks one or more components found in whole blood, such as red blood cells, at least to the extent that such components are present in whole blood White blood cells, and the like. In some cases, the plasma preparation is substantially cell-free if not completely cell-free, wherein in such cases the cell content may be 5% or less than 5% by volume, such as 1% or less than 1%, including 0.5% or less than 0.5%, wherein in some cases the cell-free plasma fraction is a composition that is completely cell-free, i.e., they do not include cells.

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

In one embodiment, the donation is obtained by venipuncture. In another embodiment, the venipuncture is a single venipuncture only. In another embodiment, no brine volume displacement is used. In a preferred embodiment, a plasmapheresis procedure is used to obtain a blood product comprising plasma. Plasmapheresis may involve removing a weight-adjusted volume of plasma and returning the cellular components to the donor. In a preferred embodiment, sodium citrate is used during the plasmapheresis process to prevent cell coagulation. The volume of plasma collected from the donor after administration of citrate is preferably 690mL to 880mL and is preferably coordinated with the weight of the donor.

C. Plasma fraction

The need for a stable plasma expander for use in the battlefield when soldiers lose significant amounts of blood has arisen during world war ii. Thus, methods for preparing lyophilized plasma have been developed. However, since reconstitution requires sterile water, it is difficult to use lyophilized plasma in combat situations. Alternatively, e.j.cohn doctor suggested that albumin could be used and prepared a ready-to-use stable solution that could be immediately introduced for the treatment of shock. (see Johan, current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed.,1st ed. 1991)). The method of purifying plasma fractions of Cohn doctor uses denaturation of cold ethanol and changes in pH and temperature to effect separation.

Embodiments of the methods described herein include administering a plasma fraction to a subject. Fractionation is a method whereby certain protein subgroups are separated from plasma. Fractionation techniques are known in the art and rely on the steps developed by Cohn et al in the 1940 s. (E.Cohn, preparation and properties of serum and plasma proteins.IV.Asystem for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids.68J Amchem Soc 459 (1946), incorporated herein by reference). The process involves several steps, each involving specific changes in ethanol concentration and pH, temperature and osmolality that result in the precipitation of the selective protein. The precipitate is also separated by centrifugation or sedimentation. The initial "Cohn fractionation method" involved separating the protein by precipitation into five fractions, designated fraction I, fraction II+III, fraction IV-1, fraction IV-4 and fraction V, respectively. Albumin is the end point (fraction V) product initially identified in the process. According to an embodiment of the invention, each fraction (or effluent from a previous separation step) comprises or potentially comprises a therapeutically useful protein fraction. (see Thierry Burnouf, modern Plasma Fractionation,21 (2) Transfusion Medicine Reviews (2007); fail Denizli, plasma fractionation: conventional and chromatographic methods for albumin purification,4J.biol. & chem.315, (2011); and T.Brodniewicz-Proba, human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-modified Products,5Blood Reviews 245 (1991), and U.S. Pat. Nos. 3869431, 5110907, 5219995, 7531513, and 8772461, which are incorporated herein by reference). The above experimental parameters may be adjusted to obtain a specific protein fraction.

Recently, fractionation has reached further complexity and thus encompasses additional embodiments of the present invention. Recently this increase in complexity has occurred by: introducing chromatography, resulting in separation of new proteins from existing fractions such as cryoprecipitate, cryoprecipitate-poor plasma (cryo-pool plasma) and Cohn fraction; the recovery rate of IgG is improved by integrating the chromatographic and ethanol fractionation processes; and virus reduction/inactivation/removal (supra). To capture proteins at physiological pH and ionic strength, anion exchange chromatography can be used. This maintains the functional activity of the protein and/or protein fraction. 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 may be adjusted to achieve a particular desired plasma fraction containing protein.

Plasma fractionation may also be based on ammonium sulphate. (see, e.g., odunga OO, biochem Compounds,1:3 (2013); wingafield PT, curr Protoc Protein Sci, appx.3 (2001), incorporated herein by reference). In addition to obtaining specific blood fractions, ammonium sulfate-based fractionation is also used to reduce the amount of protein in plasma. (Saha S et al, j.proteomics bioenform, 5 (8) (2012), incorporated herein by reference).

In an embodiment of the invention, the plasma is fractionated in an industrial setting. Frozen plasma was thawed at 1 to 4 ℃. The thawed plasma is subjected to continuous freeze centrifugation and cryoprecipitate is isolated. The recovered cryoprecipitate was frozen at-30℃or below-30℃and stored. Cryoprecipitate-depleted ("cryoprecipitate-depleted") plasma is immediately processed to capture (by, for example, primary chromatography) unstable clotting factors, such as factor IX complexes, and components thereof, as well as protease inhibitors, such as antithrombin and C1 esterase inhibitors. Continuous centrifugation and precipitation separation can be used in subsequent steps. Such techniques are known to those of ordinary skill in the art and are described, for example, in U.S. patent nos. 4624780, 5219995, 5288853, and U.S. patent application nos. 20140343255 and 20150343025, the disclosures of which are incorporated herein by reference in their entirety).

In embodiments of the invention, the plasma fraction may include a plasma fraction containing high concentrations of albumin. In another embodiment of the invention, the plasma fraction may include a fraction containing high concentrations of IgG or intravenous immunoglobulin (IGIV) (e.g) Is a plasma fraction of (a) of (b). In another embodiment of the invention, the plasma fraction may comprise an IGIV plasma fraction, e.g. substantially depleted of immunoglobulins (IgG) by methods well known to the person skilled in the art, e.g. 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,14at 2375-93 (2015)). In further embodiments, the plasma fraction may be one in which substantially all of the clotting factors are removed in order to preserve the efficacy of the fraction and reduce the risk of thrombosis. For example, the plasma fraction may be that described in U.S. patent 62/376529 filed 8/18/2016; the disclosure of which is incorporated herein by reference in its entirety.

D. Albumin preparation

For one of ordinary skill in the art, there are two broad categories of albumin plasma preparations ("APP"): plasma protein fraction ("PPF") and human albumin solution ("HAS"). PPF originates from a process with higher yield than HAS but with a minimum albumin purity below HAS (for PPF >83%, for HAS > 95%). (Production of human albumin solution: acontinually developing colloid, P.Matejtschuk et al, british J.of Anaethesia 85 (6): 887-95, at888 (2000)). In some cases, the albumin purity of PPF is 83% to 95%, or 83% to 96%. Albumin purity may be determined by electrophoresis or other quantitative measurement, for example by spectroscopy. In addition, some have indicated that PPF has disadvantages due to the presence of protein "contaminants" such as PKA. As before. Thus, PPF formulations have no longer become popular as albumin plasma preparations and have even been disnamed from pharmacopoeias in certain countries. As before. In contrast to these problems, the present invention advantageously exploits these "contaminants". In addition to the α, β and γ globulins and PKA described above, the methods of the present invention also utilize additional proteins or other factors in the "contaminants" that promote, for example, cell proliferation and tissue regeneration.

Those skilled in the art will recognize that there are or have been several commercial sources of PPF ("commercial PPF formulations"). 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)。

Those skilled in the art will also recognize that there are or have been several commercial sources of HAS ("commercial HAS formulations"). These commercial sources include albuminur ^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 Plasburn ^TM (Grifols,Clayton,NC)。

1. Plasma protein fraction (human) (PPF)

According to the U.S. food and drug administration ("FDA"), "plasma protein fraction (human)" or PPF is the proper name for the preparation defined as "sterile solution of proteins consisting of albumin and globulin from human plasma". (Federal regulations code "CFR"21CFR 640.90, which is incorporated herein by reference). The source material for PPF is plasma recovered from whole blood prepared as specified by 21cfr 640.1-640.5 (which is incorporated herein by reference) or source plasma prepared as specified by 21cfr 640.60-640.76 (which is incorporated herein by reference).

PPF was tested to determine that it meets the following criteria according to 21cfr 640.92 (which is incorporated herein by reference):

(a) The final product should be a 5.0+/-0.30 percent protein solution; and

(b) The total protein in the final product should consist of at least 83 percent albumin and no more than 17 percent globulin. The proportion of gamma-globulin in the total protein should not exceed 1 percent. Protein composition was determined by methods that have been approved by the U.S. food and drug administration biological product evaluation and research center owner for each manufacturer.

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 globulins, α2 globulins, β globulins, and γ globulins) and other plasma proteins are no more than 17% and γ globulins are no more than 1%, as determined by electrophoresis. (Hink, J.H., jr. Et al Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS2 (174) (1957)). PPF may also refer to a solid form, having 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)).

2. Albumin (human) (HAS)

According to the FDA, "albumin (human)" (also referred to herein as "HAS") is the proprietary name of the preparation defined as "sterile solution of albumin derived from human plasma". (U.S. Federal regulations code "CFR"21CFR 640.80, which is incorporated herein by reference). The source material for albumin (human) is plasma recovered from whole blood prepared as specified by 21cfr 640.1-640.5 (which is incorporated herein by reference) or source plasma prepared as specified by 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).

Albumin (human) was tested to determine that it meets the following criteria according to 21cfr 640.82:

(a) Protein concentration. The final product should meet 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.

(b) Protein composition. At least 96 percent of the total protein in the final product should be albumin as determined by methods that have been approved by the U.S. food and drug administration biological product evaluation and research center for each manufacturer.

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 the globulins (including α1, α2, βand γglobulins) 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.

The PPF and HAS fractions may also be freeze-dried or in other solid forms, as will be appreciated by those of ordinary skill in the art. Such formulations with suitable additives may be used for the preparation of, for example, tablets, powders, granules or capsules. The solid forms may be formulated into formulations for injection by dissolving, suspending or emulsifying the solid forms in an aqueous or non-aqueous solvent, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol esters; and, if necessary, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

E. Reduced coagulation factor fraction

Another embodiment of the invention uses a plasma fraction from which substantially all of the clotting factors are removed to preserve the efficacy of the fraction and reduce the risk of thrombosis. Conveniently, the blood product may be derived from a young donor or a young donor population and may be made IgM free to provide a young blood product compatible with ABO. Currently, infused plasma matches the ABO blood group because the presence of naturally occurring antibodies to the a and B antigens can lead to transfusion reactions. IgM presents transfusion reactions that lead to when patients receive ABO mismatched plasma. Removal of IgM from the blood product or fraction helps to eliminate transfusion reactions in subjects to whom the blood product and plasma fraction of the invention are administered.

Thus, in one embodiment, the invention relates to a method of treating a subject suffering from an undesired condition/indication associated with any of the following liver diseases or liver failure. The method comprises the following steps: administering to the subject a blood product or blood fraction derived from whole blood of the individual or 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 further embodiments, the blood product is substantially free of erythrocytes and/or leukocytes. In further embodiments, the blood product is substantially cell-free. In other embodiments, the blood product is derived from plasma. Such embodiments of the present invention are further supported by U.S. patent application Ser. No. 62/376529, filed 8/18/2016, which is incorporated herein by reference in its entirety.

F. Treatment of protein-rich plasma protein preparations

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

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

The specific concentration of protein in the plasma fraction can be determined using techniques well known to those of ordinary skill in the relevant art. By way of example and not limitation, such techniques include electrophoresis, mass spectrometry, ELISA analysis, and western blot analysis.

G. Preparation of plasma fractions

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 used to prepare human plasma protein fractions to be collected in flasks with citrate or anti-coagulated citrate dextrose solution (or other anticoagulants) to inhibit coagulation and further isolate fractions I, II +III, IV and PPF according to the method disclosed by Hink et al. (see Hink, J.H., jr. Et al, preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIIS 2 (174) (1957), incorporated herein by reference). According to this method, the mixture can be collected to a temperature of 2℃to 8 ℃. The plasma can then be separated by centrifugation at 7 ℃, removed and stored at-20 ℃. The plasma may then be thawed and fractionated at 37 ℃, preferably within eight hours after removal from storage at-20 ℃.

The plasma can be separated from fraction I using 8% ethanol at pH 7.2 and a temperature of-2 ℃ to-2.5 ℃ with a protein concentration of 5.1% to 5.6%. Cold 53.3 percent ethanol (176 mL/L plasma) and acetate buffer (with H) can be added at a rate of, for example, 450 mL/min using a nozzle during the plasma temperature is reduced to-2 °c ₂ O200 mL of 4M sodium acetate, 230mL of glacial acetic acid were fixed to 1L). Fraction I can be separated by ultracentrifugation and removed from the effluent (effluent I). Fibrinogen may be obtained from fraction I according to methods well known to those of ordinary skill in the art.

Fractions ii+iii were separated from effluent I by adjusting the effluent to pH 6.8, 21 percent ethanol at a temperature of-6 ℃ and a protein concentration of 4.3 percent. In dropping the temperature of effluent I to-6 ℃, a nozzle may be used to add cold 95% ethanol (176 mL/L effluent I) and 10M acetic acid for pH adjustment at a rate of, for example, 500 mL/min. The resulting precipitate (fraction II+III) can be removed by centrifugation at-6 ℃. Gamma globulin can be obtained from fractions ii+iii using methods well known to those of ordinary skill in the art.

Fraction IV-1 was isolated from effluent ii+iii ("effluent II/III") by adjusting the effluent to pH 5.2, 19 percent ethanol at a temperature of-6 ℃ and a protein concentration of 3 percent. H can be added using a nozzle ₂ O and 10M acetic acid for pH adjustment while maintaining the effluent II/III at-6℃for 6 hours. The precipitated fraction IV-1 may be left to stand at-6 ℃ for 6 hours and then separated from the effluent by centrifugation at the same temperature. The stable plasma protein fraction was recovered from the effluent IV-1 by adjusting the ethanol concentration to 30 percent, pH 4.65, temperature-7 ℃ and protein concentration to 2.5 percent. This can be achieved by adjusting the pH of the effluent IV-1 with cold acid-alcohol (two parts 2M acetic acid and one part 95% ethanol). 170mL of cold ethanol (95%) was added to each liter of conditioned effluent IV-1 while maintaining a temperature of-7 ℃. The precipitated protein may be left to stand for 36 hours and then removed by centrifugation at-7 ℃.

Fraction IV-4 supernatants and pastes can also be obtained using Cohn fractionation procedures and other blood fractionation procedures known to those of ordinary skill in the art. In fact, fractions IV-4 supernatant and IV-4 solid paste and methods of their manufacture 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 PLASMA PROTEINS FOR THERAPEUTIC USE,16.4:231:232 (2013), incorporated herein by reference in its entirety). Using a conventional Cohn fractionation procedure, fraction IV-4 fractions can be obtained as follows: plasma was collected from the donor, adjusted to 8% ethanol, -3 ℃, pH 7.2, 5.1% protein and 0.14 ionic strength, and then separated into soluble and insoluble components using centrifugation or filtration to give fraction I supernatant and solid form; fraction I supernatant was adjusted to 25% ethanol, -5 ℃, pH 6.9, 3% protein and ionic strength of 0.09, which was then separated into soluble and insoluble components using centrifugation or filtration to give fraction ii+iii supernatant and solid form; fraction ii+iii supernatant was adjusted to 18% ethanol, -5 ℃, pH 5.2, 1.6% protein and 0.09 ionic strength, which was then separated into soluble and insoluble components using centrifugation or filtration to give fraction IV-1 supernatant and solid form; fraction IV-1 supernatant was adjusted to 40% ethanol, -5 ℃, pH 5.8, 1.0% protein and 0.09 ionic strength, which was then separated into soluble supernatant and insoluble (paste) components using centrifugation or filtration. (Vandersond J (1991), 'Current Approaches to the Preparation of Plasma Fractions,', the Biotechnology of blood. Butterworth-Heinemann, stonham, mass., pages 165-176, edited by Goldstein J, incorporated herein by reference in its entirety).

The recovered protein (stabilized plasma protein fraction) may be dried (e.g., by freeze drying) to remove ethanol and H ₂ O. The resulting dry powder may be dissolved in sterile distilled water, for example using 15 liters of water per kg of powder, and the pH of the solution adjusted to 7.0 with 1M NaOH. The final concentration of protein was adjusted to 5 percent by adding sterile distilled water containing sodium acetyl tryptophan, sodium octanoate and NaCl to a final concentration of 0.004M acetyl tryptophan, 0.004M octanoate and 0.112M sodium. Finally, the solution may be filtered at 10 ℃ to obtain a clear solution, and then heat treated at 60 ℃ for at least 10 hours to inactivate pathogens.

One of ordinary skill in the art will recognize that each of the different fractions, effluents or pastes described above can be used with the methods of the 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 are embodiments of the invention. Another example is that fraction I paste, fraction II+III paste, fraction IV-1 paste, fraction IV-4 paste and/or fraction V paste or suspensions thereof may be used for the treatment of conditions such as liver disease or liver failure and are embodiments of the invention. Interestingly, during fractionation, these pastes, particularly fraction IV-1 and fraction IV-4 pastes, were typically discarded, indicating that their utility has not been considered therapeutically valuable. (Bertolini J, goss N, curlin J eds., PRODUCTION OF PLASMA PROTEINS FOR THERAPEUTIC USE,16.4:231 (2013), incorporated herein by reference in its entirety).

Other embodiments of the invention contemplate suspending proteins in various plasma fractions, including pastes and/or supernatants suspended in buffers, for storage, transport, and preparation for administration to patients suffering from liver-related disorders. These buffers, by way of example and not limitation, have an osmotic pressure comparable to human plasma and comprise: sodium carbonate stabilized with 0.004M sodium octoate plus 0.004M acetyl tryptophan; 130mmol/L to 160mmol/L sodium, less than or equal to 2mmol/L potassium, 0.064mmol/g to 0.096mmol/g protein N-acetyl-DL-tryptophan and 0.064mmol/g to 0.096mmol/g protein octanoic acid; physiological saline solution, such as 0.9% sodium chloride or 0.9% HEPES, or 5% dextrose solution.

Embodiments of the invention also contemplate different concentrations of proteins from various plasma fractions suspended in a buffer, including pastes and/or supernatants. By way of example and not limitation, these concentrations include: 10g/L (1% protein solution); 50g/L (5% protein solution); 100g/L (10% protein solution); 150g/L (15% solution); 20g/L (20% solution); 25g/L (25% solution); 30g/L (30% solution), etc. For example, a 5% solution of fraction IV-4 paste suspension would be expected to have 50 grams of protein paste in 1L of buffer.

The foregoing methods of preparing the plasma fraction and the Plasma Protein Fraction (PPF) are merely exemplary and are directed to only embodiments of the present invention. One of ordinary skill in the art will recognize that these methods may vary. For example, in various embodiments and methods of the invention, the pH, temperature, and ethanol concentration may be adjusted, among other things, to produce different changes in the plasma fraction and the plasma protein fraction. In another example, additional embodiments of the present invention contemplate the use of nanofiltration to remove/inactivate pathogens from plasma fractions and plasma protein fractions.

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

H. Treatment of

Aspects of the methods of the invention described herein include treating a subject with a blood product, such as a plasma fraction, for example, as described above, comprising plasma. Embodiments include treating a human subject with a blood product comprising plasma. Those skilled in the art will recognize that methods of treating a subject with a blood product comprising plasma are well known in the art. By way of example and not limitation, one embodiment of the methods of the invention described herein includes administering a plasma fraction or fresh frozen plasma to a subject to treat a liver-related disorder, such as acute or chronic failure. Additional embodiments of the invention include those wherein acute liver failure is associated with excessive use of infectious diseases such as hepatitis b or c, drugs or toxins (e.g., acetaminophen, isoniazid), and metabolic or vascular diseases such as autoimmune hepatitis and wilson's disease. Additional embodiments of the invention include where 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 the ducts, e.g., bile ducts from liver to intestinal tract, exposure to toxins or certain drugs, parasites, heart failure, resulting in blood accumulation in the liver.

Additional embodiments of the invention include liver disease or failure associated with genetic disease, including for example but not limited to: alpha-1 antitrypsin deficiency; disorders of bile acid synthesis (wilson's disease, progressive familial intrahepatic cholestasis type 3); disorders of glucose metabolism (inherited fructose intolerance, glycogen storage disease type IV); disorders of amino acid metabolism (type I tyrosinemia); urea cycle disorders (argininosuccinate lyase deficiency, citrate deficiency-CTLN 2, NICCD); lipid metabolism disorders (cholesterol ester storage disease); cystic fibrosis; hemochromatosis; alstrom syndrome; and congenital liver fibrosis. (Scorza M et al, int' l J Hepatology,2014,Article ID 713754 (2014)).

Additional embodiments of the invention include liver disease or failure associated with infectious agents, such as, but not limited to: viruses (epstein barr virus, cytomegalovirus, herpes simplex virus and other herpesviruses, yellow fever, dengue, hepatitis b and hepatitis c); bacteria (typhoid, mycobacterium tuberculosis, brucellosis, Q heat, leptospirosis, spirochete-syphilis, borrelia); parasites (schistosomiasis, malaria); fungi (candida). (Talwani R et al, clin Liver Dis.,15 (1): 111-30 (2011)).

Additional embodiments of the invention include liver disease or failure associated with a drug or toxic substance such as, but not limited to: acetaminophen, isoniazid and drugs metabolized by the liver.

Additional embodiments of the invention include liver diseases in which the liver is partially or completely resected. Additional embodiments include liver disease, wherein the donor liver is transplanted into a subject having 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.

In one embodiment, the blood product comprising plasma is administered immediately, e.g., within about 12 hours to 48 hours of collection from the donor, to an individual suffering from a liver disease or liver failure condition. In such cases, the article may be stored under refrigerated conditions, for example, 0 ℃ to 10 ℃. In another embodiment, the fresh frozen plasma is plasma that has been stored frozen (cryopreserved) at-18 ℃ or below-18 ℃. Fresh frozen plasma was thawed prior to administration, and once thawed, administered to the subject 60 minutes to 75 minutes after the thawing process began. Each subject preferably receives a single unit of fresh frozen plasma (200 mL to 250 mL), which is preferably derived from a donor in a predetermined age range. In one embodiment of the invention, fresh frozen plasma is donated (derived) from a young subject. In another embodiment of the invention, fresh frozen plasma is donated (derived) from a donor of the same sex. In another embodiment of the invention, fresh frozen plasma is donated (derived) from a donor aged 18 to 22 years.

In an embodiment of the invention, blood products comprising plasma are screened after donation by blood group. In another embodiment of the invention, blood products comprising plasma are screened for infectious disease pathogens such as HIV I & II, HBV, HCV, HTLV I & II, anti-HBc according to the requirements of 21cfr 640.33 and recommendations contained in the FDA guidelines documents.

In another embodiment of the invention, the subject is treated with a plasma fraction. In an embodiment of the invention, the plasma fraction is PPF, HAS, fraction IV-1 paste, fraction IV-4 or fraction IV-4 paste. In a further embodiment of the invention, the plasma fraction is a commercial PPF formulation of a commercial HAS formulation. In another embodiment of the invention, the plasma fraction is PPF, HAS, fraction IV-4 or fraction IV-4 paste derived from individuals of a particular age range, e.g., a young population of individuals, or is a modified PPF, HAS, fraction IV-4 or fraction IV-4 paste fraction that HAS been subjected to additional fractionation or processing (e.g., PPF, HAS, fraction IV-1 paste, fraction IV-4 or fraction IV-4 paste in which one or more specific proteins have been partially or substantially removed). In another embodiment of the invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulins (IgG). By "substantially depleted" or "substantially removed" a blood fraction of a particular protein, e.g., igG, is meant a blood fraction containing less than about 50% of the amount present in a reference preparation or whole blood plasma, e.g., 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 known in the art.

I. Application of

Aspects of the methods of the invention described herein include treating a subject with a blood product, such as plasma or a plasma fraction, such as described above, comprising plasma. Embodiments include treating a human subject with a blood product comprising plasma. Those skilled in the art will recognize that methods of treating a subject with a blood product comprising plasma are well known in the art. By way of example and not limitation, one embodiment of the methods of the invention described herein includes administering fresh frozen plasma to a subject to treat, for example, liver disease or liver failure conditions. In one embodiment, the blood product comprising plasma is administered immediately, e.g., within about 12 hours to 48 hours of collection from the donor, to an individual suffering from an undesired condition, such as liver disease or liver failure. In such cases, the article may be stored under refrigerated conditions, for example, 0 ℃ to 10 ℃. In another embodiment, the fresh frozen plasma is plasma that has been stored frozen (cryopreserved) at-18 ℃ or below-18 ℃. Fresh frozen plasma was thawed prior to administration, and once thawed, administered to the subject 60 minutes to 75 minutes after the thawing process began. Each subject preferably receives a single unit of fresh frozen plasma (200 mL to 250 mL), which is preferably derived from a donor in a predetermined age range. In one embodiment of the invention, fresh frozen plasma is donated (derived) from a young subject. In another embodiment of the invention, fresh frozen plasma is donated (derived) from a donor of the same sex. In another embodiment of the invention, fresh frozen plasma is donated (derived) from a donor aged 18 to 22 years.

In another embodiment of the invention, the subject is treated with a plasma fraction. In an embodiment of the invention, the plasma fraction is PPF or HAS. In further embodiments of the invention, the plasma fraction is one of a commercial PPF formulation or a commercial HAS formulation. In another embodiment of the invention, the plasma fraction is PPF or HAS derived from an individual of a particular age range, e.g., a population of young individuals, or is a modified PPF or HAS fraction that HAS been subjected to additional fractionation or processing (e.g., PPF or HAS in which one or more specific proteins have been partially or substantially removed). In another embodiment of the invention, the plasma fraction is an IGIV plasma fraction that has been substantially depleted of immunoglobulins (IgG). By "substantially depleted" or "substantially removed" a blood fraction of a particular protein, e.g., igG, is meant a blood fraction containing less than about 50% of the amount present in a reference preparation or whole blood plasma, e.g., 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.

Embodiments of the invention include treating a subject suffering from a liver disease or liver failure condition by administering to the subject an effective amount of plasma or a plasma fraction. Another embodiment of the invention comprises administering an effective amount of plasma or plasma fraction and subsequently monitoring the subject for improved liver function, liver regeneration, the presence of a marker, a reduction in pain or a reduction in inflammation. Another embodiment of the invention includes treating a subject suffering from a related disorder, such as liver disease or liver failure, by administering to the subject an effective amount of plasma or a plasma fraction in a manner such that liver function is improved, liver regeneration, marker presence, pain reduction, or inflammation reduction (referred to herein as "pulsed administration" or "pulsed administration") after the mean or median half-life of the plasma protein or plasma fraction protein has been reached relative to the dose administered recently (see U.S. patent application nos. 15/499697 and 62/701411, which are incorporated herein by reference in their entirety). Another embodiment of the invention comprises administering plasma or plasma fractions by a dosing regimen of at least two consecutive days and monitoring the subject for improvement in liver function or HSC marker levels at least 3 days after the date of the last administration. Additional embodiments of the 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 in succession, and monitoring the subject for improvement in liver function, liver regeneration, presence of markers, reduction in pain or reduction in inflammation for at least 3 days after the date of the last administration. Another embodiment of the invention comprises administering plasma or plasma fractions by a dosing regimen of at least 2 consecutive days and monitoring, after the date of the last administration, for improvement of liver function, presence of markers, reduction of pain or reduction of inflammation when the average half-life of proteins in the plasma or plasma fractions has been reached. Another embodiment of the invention includes administering plasma or plasma fractions via a discontinuous dosing regimen of 2 to 14 days, wherein each gap between administrations can be 0 to 3 days each.

In some cases, pulsed administration according to the present invention, e.g., as described above, includes administration of a first set of doses followed by a non-administration period, e.g., "no-administration period," which is followed by administration of another dose or set of doses. The duration of the "no dosing period" may 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 no dosing period is 15 days to 365 days, such as 30 days to 90 days, including 30 days to 60 days. Thus, embodiments of the method include non-chronic (i.e., discontinuous) administration, e.g., non-chronic administration, of the plasma preparation. In some embodiments, the pattern of pulsatile dosing followed by no dosing periods is repeated as many times as desired, wherein in some cases the pattern is continued for 1 year or more than 1 year, e.g., 2 years or more than 2 years, up to and including the lifetime of the subject. Another embodiment of the invention comprises administering plasma or plasma fractions by a continuous 5 day administration, 2 to 3 day no dosing period, followed by a continuous 2 to 14 day dosing regimen: .

Additional embodiments of the invention include liver diseases in which the liver is partially or completely resected. Additional embodiments include liver disease, wherein the donor liver is transplanted into a subject having 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 of resecting and transplanting liver are well known in the art.

Biochemically, an "effective amount" or "effective dose" of an active agent means an amount of the active agent that will inhibit, antagonize, reduce or eliminate by about 20% or more than 20%, such as 30% or more than 30%, 40% or more than 40% or 50% or more than 50%, in some cases 60% or more than 60%, 70% or more than 70%, 80% or more than 80% or 90% or more than 90%, in some cases about 100%, i.e., to a negligible amount, and in some cases reverse an undesired condition such as liver disease or liver failure.

J. Plasma protein fraction

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 commercial PPF formulations.

In another embodiment, the PPF is comprised of 88% normal human albumin, 12% alpha and beta globulins, and no more than 1% gamma globulins as determined by electrophoresis. Additional embodiments for practicing embodiments of the methods of the invention include, for example, embodiments of a 5% PPF solution buffered with sodium carbonate and stabilized with 0.004M sodium octoate and 0.004M acetyl tryptophan. Additional formulations may be used in practicing the methods of the invention, including formulations that vary the percentage of PPF in solution (e.g., about 1% to about 10%, about 10% to about 20%, about 20% to 25%, about 25% to 30%) as well as the concentration of solvent and stabilizer.

K. Plasma fraction of specific donor age

Additional embodiments of the invention include administering plasma protein fractions derived from the plasma of individuals of certain age ranges. Embodiments include administering PPF or HAS derived from plasma of a young subject. In another embodiment of the invention, the young subject has a single specific age or specific age range. In another embodiment, the average age of the donor is less than the age of the subject or less than the average age of the subject receiving the treatment.

Certain embodiments of the invention include pooling blood or plasma from individuals of a particular 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 invention, the plasma protein fraction or the specific plasma protein fraction is obtained from a specific individual meeting a specific age range.

L. indications

One embodiment of the invention is the use of plasma fractions and plasma fractionation preparations for administration to a subject diagnosed with liver disease or liver failure that may benefit from improved liver proliferation or regeneration. Another embodiment of the invention includes when the disease is associated with: acute liver failure, chronic liver failure, or chronic plus acute liver failure. Chronic acute liver failure occurs in patients with relatively stable chronic liver disease, but suddenly changes to acute liver failure. This typically results in very high mortality rates. Another embodiment of the present invention is where acute liver failure is associated with excessive use of infectious diseases such as hepatitis B or C, drugs or toxins (e.g., acetaminophen, isoniazid), and metabolic or vascular diseases such as autoimmune hepatitis and Wilson's disease. Additional embodiments of the invention include where 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 the ducts, e.g., bile ducts from liver to intestinal tract, exposure to toxins or certain drugs, parasites, heart failure, resulting in blood accumulation in the liver.

Additional embodiments of the invention include liver disease or failure associated with genetic diseases such as, but not limited to: alpha-1 antitrypsin deficiency; disorders of bile acid synthesis (wilson's disease, progressive familial intrahepatic cholestasis type 3); disorders of glucose metabolism (inherited fructose intolerance, glycogen storage disease type IV); disorders of amino acid metabolism (type I tyrosinemia); urea cycle disorders (argininosuccinate lyase deficiency, citrate deficiency-CTLN 2, NICCD); lipid metabolism disorders (cholesterol ester storage disease); cystic fibrosis; hemochromatosis; astrom syndrome; and congenital liver fibrosis. (Scorza M et al, int' l J Hepatology,2014,Article ID 713754 (2014)).

Additional embodiments of the invention include liver disease or failure associated with a drug or toxic substance, such as, but not limited to: acetaminophen, isoniazid and drugs metabolized by the liver.

Additional embodiments of the invention include liver diseases in which the liver is partially resected. Additional embodiments include liver disease wherein the donor liver is transplanted into a subject having 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.

M, reagent, device and kit

Reagents, devices and kits thereof for carrying out one or more of the above methods are also provided. The target reagents, devices and kits thereof may vary widely.

Target reagents and devices include the reagents and devices mentioned above in connection with the method of preparing a blood product comprising plasma for infusion into a subject in need thereof, e.g., anticoagulants, cryo-storage agents, buffers, isotonic solutions, and the like.

The kit may also include blood collection bags, tubes, needles, centrifuge tubes, and the like. In other embodiments, the kits described herein comprise two or more containers of plasma preparations, e.g., plasma protein fractions, e.g., three or more containers of three, four or more four, five or more five, including six or more plasma preparations. In some cases, the number of different containers of plasma preparations in a kit may be 9 or more than 9, 12 or more than 12, 15 or more than 15, 18 or more than 18, 21 or more than 21, 24 or more than 24, 30 or more than 30, including 36 or more than 36, e.g., 48 or more than 48. Each container may have associated therewith identification information including a variety of data regarding the plasma product contained therein, which may include one or more of a donor age of the plasma product, processing details regarding the plasma product, such as whether the plasma product was treated to remove proteins above average molecular weight (e.g., as described above), blood group details, and the like. In some cases, each container in the kit includes identification information about the plasma contained therein, and the identification information includes information about the age of the donor of the plasma product, e.g., the identification information provides age-related data identifying the donor of the plasma product (where the identification information may be the age of the donor at the time of collection). In some cases, each container of the kit contains a plasma preparation from a donor of substantially the same age, i.e., all containers contain preparations from donors of substantially the same age, if not the same age. By substantially the same age is meant that the different donors from which the plasma preparations of the kit are obtained in some cases each differ by 5 years old or less than 5 years old, such as 4 years old or less than 4 years old, such as 3 years old or less than 3 years old, including 2 years old or less than 2 years old, such as 1 year old or less than 1 year old, 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 may be present on any convenient component of the container, such as a tag, RFID chip, etc. The identification information may be human readable, computer readable, etc., as desired. The container may have any convenient configuration. Although the volume of the container may vary, in some cases the volume is from 10mL to 5000mL, such as from 25mL to 2500mL, such as from 50mL to 1000mL, including from 100mL to 500mL. The container may be rigid or flexible and may be made of any convenient material, such as a polymeric material, including medical grade plastics materials. In some cases, the container has a pouch or pouch configuration. In addition to the container, the kit may also comprise an applicator, for example as described above. The components of the kit may be provided in any suitable package, for example, a cartridge or similar structure configured to house the container and other components of the kit.

In addition to the components described above, the subject kits may also include instructions for performing the subject methods. The instructions may be present in the subject kit in a variety of forms, one or more of which may be present in the kit. One form in which the instructions may exist is printing information on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, in the packaging of a kit, on a package insert, or the like. Another way may be a computer readable medium, such as a floppy disk, CD, portable flash drive, etc., on which information is recorded. Another possible way is a website address, which can be used over the internet to access information at a remote site. Any suitable device may be present in the kit.

Experimental examples n

1. Example 1

a) PPF 1-induced functional recovery of liver of C57BL/6 mice at 20 months of age after 70% partial hepatectomy

Male C57BL/6J mice (The Jackson Laboratory, sarcopto, calif.) were used at 20 months of age. All mice were individually housed under specific pathogen-free conditions under a 12-hour light, 12-hour dark cycle, and all animals were treated and used in compliance with institutional animal care and use committee approved guidelines. Animals were fed a 60% high fat diet (Bio-Serv F3282) for 7 to 8 weeks and were randomly divided into vehicle and PPF1 treated groups based on body weight and ALT levels after diet to ensure the average body weight and serum ALT levels were the same between groups. Surgery was performed with minor modifications as described previously (Nevzorova, Y. Et al, lab. Anim.49,81-88 (2015)). Resected liver (left She Hezheng leaf) served as preoperative control, while residual liver (tail and right leaf) was harvested 48 hours post-surgery. Twenty-four (24) hours after surgery, edU (2.5 mg/mL stock in saline) was injected intraperitoneally at 30mg/kg body weight. Forty-six (46) hours after surgery, brdU (10 mg/mL stock in saline) was injected intraperitoneally at 30mg/kg body weight. 48 hours post-surgery, all animals were weighed and residual dirt (tail and right lobes) and serum were collected for ALT analysis.

Commercially available PPF ("PPF 1"), such as the commercial PPF formulation described above in 5% solution, is stored at 4 ℃. PPF1 is PPF with about 88% normal human albumin (relative to total protein), 12% alpha and beta globulins, and no more than 1% gamma globulins, as determined by electrophoresis. PPF1 was administered using a 5% solution (weight/volume, 50 g/L) in the examples herein, unless otherwise indicated.

For qPCR analysis, RNA was extracted from resected or residual liver powdered with liquid nitrogen and mortar/pestle. Followed by extraction with Trizol (Ambion/Fisher 15-596-018) and RNeasy Mini Kit (Qiagen 74106). The cDNA was amplified using iScript Reverse Transcription Supermix (Bio-Rad 1030). QPCR was amplified using SsoAdvanced Universal SYBR green master mix (Bio-Rad 1725272) and performed using Quant Studio 6Flex (Applied Biosystem). Gene expression was normalized to RPS18 (ribosomal protein s 18).

For immunostaining, frozen liver sections 12 microns thick embedded in OCT compound (Sakura 4583) were used for immunofluorescent staining. Antigens were repaired using citrate buffer (Sigma C9999) for 30 min at 95 ℃. For the EdU tags, click-IT Plus EdU Alexa Fluor 555 (Fisher C10638) was used. Nuclei were labeled with Hoechst 33342 in ddH2O at 1 to 100000 for 3 min (Fisher H3570). Oil red O staining was performed to visualize triglyceride accumulation in the liver. Briefly, frozen liver sections were immersed in isopropanol containing 0.375% oil red O for 5 minutes. Sections were rinsed under running tap water for 30 min and fixed with Prolong gold antifade reagent (Invitrogen P36934).

Figure 1 shows a graph of body weight progression of high fat diet ("HFD") 20 month old C57BL/6 mice. After 8.5 weeks of HFD, mice gain significantly weight. Mean ± SEM, p <0.0001, common single factor anova.

FIG. 2 is an oil red O staining of mouse livers as described in FIG. 1. Mice with HFD present fatty liver compared to normal diet mice ("normal diet").

Figure 3 shows the experimental design for assessing the effect of PPF1 on a 20 month old C57BL/6 mouse on a high fat diet as described above.

Fig. 5 shows the results of the determination of the ratio of liver weight to body weight in vehicle-treated and PPF 1-treated resected livers from mice treated as described in fig. 3.

Figure 6 shows liver weight in vehicle-treated and PPF 1-treated resected livers from mice treated as described in figure 3.

Fig. 7 is a graph showing that serum ALT levels were normal and unaffected in PPF1 treated HFD mice prior to hepatectomy. ALT levels are considered normal when they are below 50 units per liter to 60 units per liter. (see, e.g., mazzaccara C et al, PLoS ONE,3 (11): e3772 (2008)).

Fig. 8 shows the results of the determination of the ratio of liver weight to body weight in vehicle-and PPF 1-treated mice with residual liver at 48 hours post hepatectomy as described in fig. 3. Compared to vehicle treated animals, PPF1 treated animals had a significant increase in liver weight at 48 hours post hepatectomy. Mean ± SEM, p <0.001 unpaired T test using Whitney correction.

Fig. 9 shows liver weights in vehicle-and PPF 1-treated mice with residual liver at 48 hours post hepatectomy as described in fig. 3. Compared to vehicle treated animals, PPF1 treated animals had a significant increase in liver weight at 48 hours post hepatectomy. Mean ± SEM, p <0.05 unpaired T test using Whitney correction.

Fig. 10 shows the results of serum ALT levels in vehicle-and PPF 1-treated mice with residual liver at 48 hours post hepatectomy as described in fig. 3. Serum ALT levels were significantly reduced in PPF1 treated animals at 48 hours post hepatectomy compared to vehicle treated animals, indicating that the extent of liver damage was also significantly reduced in PPF1 treated animals. Mean ± SEM, p <0.001 unpaired T test using Whitney correction.

In contrast, the differences in resected liver weight, liver weight/weight ratio and ALT levels between vehicle-treated and PPF 1-treated animals failed to reach significant levels.

Fig. 11 reports the cell proliferation rate after hepatectomy. 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 residual liver of vehicle treated animals.

Fig. 12 reports cell proliferation 48 hours after hepatectomy as measured by Ki67 positive cell number per field. PPF1 significantly increased the number of Ki67 positive cells per field compared to the residual liver of vehicle treated animals.

FIG. 13 reports the cell proliferation rate in residual liver expressed by qPCR genes. The relative expression of the cyclin B1 marker is shown. In the liver residual section, PPF 1-treated mice were significantly upregulated in cyclin B1 expression compared to vehicle-treated mice. Mean ± SEM, p <0.05 unpaired T test using Whitney.

Fig. 14A-14D report qPCR expression of several markers in resected liver. The relative expression of the cyclin B1 (fig. 14A), cyclin A2 (fig. 14B) and Ki67 (fig. 14C) markers is shown. PPF1 surprisingly significantly increased cell proliferation in resected liver sections removed during hepatectomy as a pre-operative control compared to vehicle controls with all three cell cycle markers. Fig. 14D reports the relative expression levels of tnfα in resected liver sections. Tnfα is known to promote restoration of functional liver quality by driving hepatocyte proliferation and liver regeneration. In summary, PPF1 treatment resulted in activation of cell proliferation in other quiescent and intact livers (prior to hepatectomy) compared to vehicle controls. Mean ± SEM, p <0.05 unpaired T test using Whitney correction.

Figure 15 shows immunostaining of Ki67 in resected liver as reported in figure 14C, confirming that PPF1 treated resected liver has significantly higher number of Ki67 positive cells compared to vehicle control.

Figure 16 reports quantification of immunostaining from figure 15 showing an increase in the number of Ki67 positive cells in PPF1 treated resected liver. Mean ± SEM, p <0.05 unpaired T test using Whitney correction.

Fig. 17 is a graph showing the total number of animals receiving hepatectomy and the survival rate of animals receiving each treatment. Animals treated with PPF1 showed a trend of increased survival compared to animals treated with vehicle.

Fig. 18 shows two confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Histological sections were stained with GFAP antibodies (astrocyte markers) and observed for co-localization levels between EdU and GFAP. Cell proliferation associated with PPF1 administration did not occur in astrocytes as determined by lack of co-localization between EdU and GFAP.

Fig. 19 shows two confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD68 antibody (kupfu cell marker) and observed for co-localization levels of EdU with CD 68. Cell proliferation associated with PPF1 administration did not occur in kupfu cells as determined by the lack of co-localization between EdU and CD 68.

Fig. 20 shows confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with HNF4a antibodies (hepatocyte markers) and observed for co-localization levels of EdU with HNF4 a. Cell proliferation associated with PPF1 administration did not occur in hepatocytes as determined by the lack of co-localization between EdU and HNF4 a.

Fig. 21 shows confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD3 antibodies (T cell markers) and observed for co-localization levels of EdU with CD 3. Cell proliferation associated with PPF1 administration did not occur in T cells as determined by the lack of co-localization between EdU and CD 3.

Fig. 22 shows two confocal microscopy fields of residual liver bound to EdU 24 hours after hepatectomy (see fig. 3). Tissue sections were stained with CD31 antibody (sinus endothelial marker) and observed for co-localization levels of EdU with CD 31. Cell proliferation induced by PPF1 administration was correlated with Liver Sinus Endothelial Cells (LSEC) as determined by positive co-localization between EdU and CD 31. These results indicate that PPF1 increases proliferation of liver capillary cells, allowing more blood to be brought to the site of injury, which suggests a mechanism how tissue repair and regeneration occurs after PPF1 treatment. (arrows indicate LSEC containing EdU positive cells).

2. Example 2

a) Effect of PPF1, recombinant human albumin and HAS1 on liver of 20 month old C57BL/6 mice after 70% segmental hepatectomy

Recombinant human albumin

Male C57BL/6J was treated as described in example 1 above, except that the other group was treated with recombinant human albumin ("rhAlbumin"). A comparison was made between PPF1 treated group, rhAlbumin treated group and vehicle treated group.

Figure 23 shows the experimental design of assessing the effect of PPF1 (type of plasma fraction "PF") and recombinant human albumin (rhAlbumin) on high fat diet of 20 month old C57BL/6 mice. Mice were placed in a 60% high fat diet for 8 weeks prior to treatment with PPF1, rhAlbumin or vehicle for 7 consecutive days. The next day after the last dose of PPF1, rhAlbumin or vehicle was operated on (70% hepatectomy) and pre-operative median lobes and Zuo She (resected) were removed during hepatectomy. Right and tail leaves (residual) were harvested 48 hours after hepatectomy.

Figure 24 reports cell proliferation in resected liver 48 hours after hepatectomy as measured by Ki67 positive cell number per field. PPF1 significantly increased the number of Ki67 positive cells per field compared to vehicle treated animals. In contrast, the animals treated with rhAlbumin were not significantly different from the animals treated with vehicle. Mean ± SEM, p <0.0001 unpaired T-test with Whitney correction, ns = insignificant.

Fig. 25 reports the cell proliferation rate in the residual liver after hepatectomy. EdU was delivered 24 hours after hepatectomy and proliferation rate was followed 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 terms of proliferation compared to vehicle treated animals.

These results indicate that albumin as the most abundant component of PPF1 may not be the component of PPF1 responsible for its proliferative, tissue repair and regenerative activities. Instead, this suggests that other components of PPF1, which are generally considered as impurities left over from plasma fractionation, surprisingly drive these activities of PPF 1.

Human Albumin Solution (HAS)

Male C57BL/6J was also treated as described in example 1 above, except that another group was treated with human albumin solution ("HAS 1"). A comparison was made between PPF1 treated group, HAS1 treated group and vehicle treated group.

FIG. 26 shows the experimental design for assessing the effect of PPF1 and HAS1 on high fat diet of 20 month old C57BL/6 mice. Mice were placed in a 60% high fat diet for 8 weeks prior to treatment with PPF1, rhAlbumin or vehicle for seven consecutive days. The next day after the last dose of PPF1, HAS1 or vehicle, surgery (70% hepatectomy) was performed, and pre-operative median lobes and Zuo She (resected) were removed during hepatectomy. Right and tail leaves (residual) were harvested 48 hours after hepatectomy.

Fig. 27 reports the cell proliferation rate 24 hours after hepatectomy. EdU was delivered 24 hours after hepatectomy. Both PPF1 and HAS1 significantly increased the number of EdU positive cells compared to the vehicle in the residual liver. * p <0.05, welch t test. Error bars = s.e.m. HAS1 is a commercially available HAS, such as the commercial HAS formulation in 5% solution described above, and stored at 4 ℃.

Fig. 28 shows the cell proliferation rate 48 hours after hepatectomy. Residual liver removed 48 hours after hepatectomy was Ki67 immunostained. Although both PPF1 and HAS significantly increased cell proliferation compared to control animals, PPF1 also significantly induced more proliferation compared to HAS1 treated animals. * p <0.05, < p <0.005, < p <0.0005 virgini t-test. Error line = s.e.m.

These results indicate that plasma fraction induced hepatocyte proliferation was observed in both HAS1 and PPF1 treated fatty liver. However, although both different plasma fractions (HAS 1 and PPF 1) induced proliferation significantly after 24 hours compared to the vehicle, the effect of PPF1 was more pronounced compared to HAS1 after 48 hours, and even more pronounced compared to the vehicle. This again shows that the purer and more abundant albumin in HAS1 is surprisingly not the main component leading to the effect of the plasma fraction on proliferation, tissue repair and regeneration compared to PPF 1. On the contrary, this suggests that other components of the plasma fraction or impurities left from the plasma fractionation unexpectedly drive these activities of PPF 1.

3. Example 3

a) Effect of PPF1 on liver of 20 month old C57BL/6 mice two hours after last treatment administration

Twenty month old male C57BL/6J mice were randomly split into vehicle and PPF1 treated groups according to body weight to ensure the average body weight was the same between groups. Mice were fed on a normal diet rather than a high fat diet. Animals were dosed with 150 μl of vehicle or PPF1 daily by intravenous injection for 7 consecutive days. On day seven, the liver was removed 2 hours after dosing. For gene expression analysis, RNA was extracted and reverse transcribed, and SYBR qPCR was performed on tnfα. Gene expression was normalized to RPS18. For immunostaining, proliferating cells in harvested livers were labeled with Ki67 antibody.

FIG. 30 shows the results of a TNFα gene expression analysis on mice treated as described above in FIG. 29. PPF1 treated animals significantly increased tnfα gene expression by QPCR compared to vehicle treated animals. * P <0.005. Wilgi's t-test, error line = s.e.m.

FIG. 31 shows the results of Ki67 immunostaining analysis on mice treated as described above in FIG. 29. Compared to vehicle treated animals, PPF1 treated animals significantly increased Ki67 positive cells 2 hours after the last dose, indicating that PPF1 increased hepatocyte proliferation. * p <0.05. Wilgi's t-test, error line = s.e.m.

These results indicate that plasma fractions such as PPF1 are effective in inducing proliferation and regeneration even in healthy, non-steatophilic aged livers.

4. Example 4

a) Effect of fraction IV-4 paste suspension on liver of 20 month old C57BL/6 mice after 70% partial hepatectomy

Male C57BL/6J mice (The Jackson Laboratory, sarcopto, calif.) were used at 20 months of age. All mice were individually housed under specific pathogen-free conditions under a 12-hour light, 12-hour dark cycle, and all animals were treated and used in compliance with institutional animal care and use committee approved guidelines. Animals were fed a 60% high fat diet (Bio-Serv F3282) for 7 to 8 weeks and randomly divided into vehicle groups and fraction IV-4 paste suspension treatment groups based on body weight and ALT levels after diet to ensure the average body weight and serum ALT levels were the same between groups. Surgery was performed with minor modifications as described previously (Nevzorova, Y. Et al, lab. Anim.49,81-88 (2015)). Resected liver leaves (left She Hezheng leaf) served as preoperative controls, while residual liver leaves (tail and right leaf) were harvested 48 hours post-operative. Twenty-four (24) hours after surgery, edU (2.5 mg/mL stock in saline) was injected intraperitoneally at 30mg/kg body weight. Forty-six (46) hours after surgery, brdU (10 mg/mL stock in saline) was injected intraperitoneally at 30mg/kg body weight. 48 hours post-surgery, all animals were weighed and residual dirt (tail and right lobes) and serum were collected for ALT analysis. The effect of fraction IV-4 paste suspension on the content of these experiments was compared with vehicle.

Figure 32 reports basal levels of cell proliferation in resected liver at hepatectomy. Fraction IV-4 paste suspension (resuspended in HEPES buffered 0.9% saline) significantly induced hepatocyte proliferation compared to control vehicle measured as the average number of ki67+ cells in 10 Fields (FOV) (x=p <0.05Welch t-test, all data averages ± SEM). By measuring the ratio of ki67+ cells to hnv4a+ cells (data not shown), the condition of specific proliferation of hepatocytes in resected liver at hepatectomy was determined. However, resected livers treated with the fraction IV-4 paste suspension were not significantly different from vehicle controls.

Fig. 33 reports the level of cell proliferation of residual liver lobes 48 hours after hepatectomy. In this experiment, fraction IV-4 paste suspension significantly enhanced liver regeneration at 48 hours compared to vehicle control. (=p <0.05Welch t-test, all data mean ± SEM). In this case, most of the proliferating cells are known to be hepatocytes. Fig. 34 shows the proliferation level of hepatocytes in residual liver lobes 48 hours after hepatectomy by determining the ratio of ki67+ cells to hnf4a+ cells in 10 fields. Herein, fraction IV-4 paste suspension significantly enhanced hepatocyte proliferation compared to vehicle control. (=p <0.05Welch t-test, all data mean ± SEM). These data indicate that the fraction IV-4 paste suspension can affect proliferation of various different types of hepatocytes depending on the time after hepatectomy, thereby promoting liver regeneration.

5. Example 5

a) Effect of PPF on liver senescence markers in 12 month old C57BL/6 mice on normal and high fat diets

Male C57BL/6J mice of 12 months of age were fed with 60% High Fat Diet (HFD) (Bio-Serv #F3282) for 4 weeks, while control mice were fed with a standard fat diet (normal diet or NC). Mice with HFD were then dosed with 150 μl of PPF1 (n=15 mice) or vehicle (n=15 mice) by 7 consecutive intravenous injections and a high fat diet was continued for the remainder of the experiment. As a control, vehicle was administered to normal diet mice (n=15 mice) and normal diet continued for the remainder of the experiment. Four weeks after the last intravenous administration, all animals were sacrificed and liver tissues were harvested to obtain total RNA.

QPCR

RNA was extracted from resected or residual liver powdered with liquid nitrogen and mortar/pestle. Followed by extraction with Trizol (Ambion/Fisher 15-596-018) and RNeasy Mini Kit (Qiagen 74106). The cDNA was amplified using iScript Reverse Transcription Supermix (Bio-Rad 1030). QPCR was amplified with SsoAdvanced Universal SYBR green master mix (Bio-Rad 1725272) and amplified with Quant Studio 6Flex (Applied)Biosystem) for QPCR. The gene expression was normalized to RPS18 (ribosomal protein s 18) and expressed as relative gene expression (relative to RPS 18). Three markers of cellular senescence, p53, p21 and p16, were detected ^ink4a 。

Figure 35 shows the effect of high fat diet and normal diet on p53 gene RNA expression, and the effect of PPF1 on p53 expression in mice with significantly reduced high fat diet. The differences between normal diet mice and HFD diet mice treated with the control vehicle were insignificant, while p53 expression was significantly reduced in HFD diet mice administered PPF1 compared to vehicle control HFD diet mice. Similar reductions were also observed between mice on the HFD diet of PPF1 and normal diet. P <0.01, welch T-test, all data mean ± SEM).

FIG. 36 shows RNA expression of the p21 gene from the same group of mice. P21 expression was significantly reduced in both normal and PPF 1-administered mice compared to vehicle-controlled HFD-fed mice. P <0.01, welch T test, all data mean ± SEM).

FIG. 37 shows the same group of mice p16 ^Ink4a RNA expression of the gene. Normal diet mice and PPF 1-administered HFD diet mice were p16 compared to vehicle-controlled HFD diet mice ^Ink41 The gene expression is significantly reduced. (. P)<0.05，**p<0.01, welch T-test, all data mean.+ -. SEM).

The decrease associated with the plasma fraction PPF1 suggests that PPF1 is at least partially responsible for targeting the aging pathway (e.g., p53, p21 and p16 ^Ink4a Pathways) improve liver regeneration in the injured liver. This is a surprising finding because plasma fractions such as PPF1 from plasma fractionation exhibit similar characteristics to small molecule anti-aging compounds such as ABT-737, dasatinib, quercetin, feissuers, 17-DMAG, navitoclax and catechins. Unexpectedly, factors in human plasma fractions reverse aging as do these compounds. Anti-senescence compounds have been shown to induce cell death by apoptosis and down regulate expression of senescence genes, such as p21 and p 53. Treatment with the anti-aging compound ABT-737 has been demonstrated to improve liver function and promoteRegeneration (Birch J et al, genes Dev.,34:463-64 (2020)). PPF1 also appears to down-regulate senescence-associated genes, thereby promoting regeneration of damaged livers (see, e.g., fig. 11).

Claims

1. A method of treating a subject diagnosed with liver disease, the method comprising administering an effective amount of a fraction IV-4 paste suspension.

2. The method of claim 1, wherein administration is using a dosing regimen of pulsed dosing.

3. The method according to any one of the preceding claims, wherein the liver disease is acute liver failure.

4. The method according to claim 1 or 2, wherein the liver disease is chronic liver failure.

5. The method according to claim 1 or 2, wherein the liver disease is chronic acute liver failure.

6. A method of improving recovery from hepatectomy in a subject, the method comprising administering to the subject an effective amount of a fraction IV-4 paste suspension.

7. The method of claim 6, further comprising the step of administering after surgery.

8. A method of improving recovery of a subject from liver transplant surgery, the method comprising administering to the subject an effective amount of a fraction IV-4 paste suspension.

9. The method of claim 8, further comprising the step of administering after surgery.

10. The method of any one of the preceding claims, wherein the suspension is 10 wt/vol%.