WO2020026227A1 - Methods and uses of alpha 1-antitrypsin for preservation of explanted organs - Google Patents

Abstract

The present invention provides methods for prevention or reduction of ischemic reperfusion injury (IRI) in organs involved in organ transplantation, by administering alpha 1-antitrypsin (AAT) during machine perfusion.

Description

METHODS AND USES OF ALPHA 1-ANTITRYPSIN FOR PRESERVATION

OF EXPLANTED ORGANS FIELD OF THE INVENTION

The present invention relates to methods of protecting organs susceptible to ischemic reperfusion injury (IRI) by administering alpha 1 -antitrypsin (AAT) during machine perfusion.

BACKGROUND OF THE INVENTION

AAT is a heavily glycosylated plasma protein of 52 kDa in size. AAT is produced by the liver and secreted into the circulation, and is also produced locally by lung epithelial cells. Circulating levels of AAT increase during an acute phase response. This increase is due to the presence of IL-l and IL-6 responsive elements inside the promoter region of the AAT encoding gene. AAT functions as a serine protease inhibitor that primarily targets elastase, trypsin, and proteinase-3, three inflammatory and immune cell-derived enzymes that are involved in protease-activated receptor (PAR) activation and the onset and progression of inflammation (Vergnolle N. 2009. Pharmacol Ther l23(3):292-309). Important pro-inflammatory mediators such as IL-l b, IL-6, IL-8, and TNFa are enhanced by these serine proteases and hence blocked by serine protease inhibitors, in particular by AAT (Pott G B et al. 2009. J Leukoc Biol. 85(5):886-95). Moreover, AAT induces the production and release of anti-inflammatory mediators such as IL-10 and IL-l-receptor antagonist (IL-IRa) (Lewis E C et al. 2008. Proc Natl Acad Sci USA. 105(42): 16236-41).

International application W02005/027821 to the applicant of the present invention teaches a novel composition of purified, stable, active alpha- 1 antitrypsin (AAT) for intravenous administration and inhalation, a process for its preparation, and its use for treating pulmonary disease. AAT is currently administered intravenously by using intravenous formulations indicated for augmentation therapy in patients having congenital deficiency of AAT with clinically evident emphysema.

AAT has been evaluated in the treatment of graft-versus-host disease in murine models of allogeneic bone marrow transplantation, and has demonstrated immune- modulating effects. Interim results from a phase Eli clinical study demonstrated that AAT therapy had important immune-modulating activity with resolution of GVHD in some subjects. Beneficial effects of AAT therapy are observed across several animal models in the settings of autoimmunity, alloimmunity, and xenoimmunity. As an example, AAT has been shown to prolong islet allograft survival in mice, an effect that was associated with reduced cellular infiltration and diminished pro-inflammatory responses, accompanied by the emergence of tolerogenic semi-mature dendritic cells (DCs) and regulatory T cells (Tregs).

Significant effort has been directed towards the development of strategies to reduce the shortage of organs for transplantation. The most consequential progress has been made as a result of the increased use of extended criteria, "marginal”, donor (ECD) livers by transplant centers. Approximately 10% of livers harvested are deemed unsuitable for transplant at the time of recovery, thus representing a significant potential to expand the donor pool and thus save thousands of lives.

Marginal livers include those that previously would have been rejected based on certain characteristics that put them at increased risk for failure or dysfunction. The specific criteria include increased donor age, prolonged warm ischemia time (WIT), cold ischemia time (CIT), or extensive steatosis. Compared to standard organs, extended criteria organs harbor an increased risk of ischemic injury. This is particularly true for livers after donor cardiac death (DCD), although donors after brain death (DBD) are occasionally rejected, usually because of donor age or degree of steatosis. The Organ Procurement and Transplantation Network (OPTN) reports that the use of "high risk" livers has increased dramatically over the past several years, comprising 25% of the livers transplanted nationally from deceased donors in 2015, compared to approximately 10% just 5 years ago.

This marked increase in the use of DCD and marginal livers has been instrumental in the expansion of the donor pool. However, this expansion has come at the cost of a notably increased risk of ischemia/reperfusion injury (IRI). The sequelae that follow IRI, including ischemic biliary injury, primary graft non-function, and early graft dysfunction are a significant barrier to successful transplantation. Ischemic injury disproportionately affects the cells of the biliary tract rather than the hepatocytes, manifesting as the development of non- anastomotic biliary strictures (NAS). Clinically significant NAS occur in up to 30% of DCD donors and double the need for re transplantation compared to standard DBD donors, where the incidence of NAS is 5- 15%. Unfortunately, prediction of which DCD donors will prove to be non-viable has been elusive. Additionally, no intervention has proven to be reliably effective in the prevention or treatment of hepatic IRI. These factors have led to hesitation in transplanting DCD organs, thus limiting full utilization of this population of potential grafts. Therefore, development of interventions that prevent and treat IRI are critical in order to realize the full potential of extended criteria donors and effectively reduce the organ shortage.

There is an unmet need for prevention of IRI and for improving the preservation of the explanted organ.

SUMMARY OF THE INVENTION

The present invention provides methods for protecting and preserving organs susceptible to ischemic reperfusion injury. Particularly, the present invention provides methods for prevention and reduction of injury and IRI by administering AAT during machine perfusion.

The present invention discloses for the first time that AAT-treated livers have improved hemodynamics, reduced cellular injury, and superior energy restoration compared to un-treated controls.

The present invention is based in part on the findings that AAT is an effective and safe therapy for reducing injury and IRI during ex-vivo machine perfusion.

According to one aspect, the present invention provides a method of protecting an organ susceptible to ischemic reperfusion injury (IRI), the method comprises administering to said organ, an effective amount of alpha 1 -antitrypsin (AAT) during ex-vivo machine perfusion.

According to certain embodiments, the administration to said organ occurs before said organ has been transplanted into a subject in need of organ transplantation.

According to certain embodiments, the administration to the organ occurs after said organ has been harvested from the donor.

According to certain embodiments, the administration to the organ occurs before said organ has been harvested from the donor.

According to another aspect, the present invention provides a method of preserving a harvested organ; the method comprises administering to said organ a preservation fluid for perfusion prior to the implantation of said organ in a patient requiring such implantation, wherein said preservation fluid contains alpha 1 -antitrypsin (AAT) at a concentration sufficient to reduce organ injury and IRI.

According to a further aspect, the present invention provides a method of prophylaxis of ischemic reperfusion injury (IRI) in an organ at risk of IRI, comprising administering to said organ, an effective amount of alpha 1 -antitrypsin (AAT) during machine perfusion. According to certain embodiments, the prophylaxis of IRI results in prophylaxis of IRI-associated organ dysfunction.

According to certain embodiments, the AAT is naturally occurring AAT purified from an unpurified mixture of proteins by a process comprising of chromatography on a plurality of ion exchange resins, comprising a first anion exchange resin followed by a cation and a second anion exchange resins.

According to certain embodiments, the organ is a solid organ.

According to certain embodiments, the solid organ is selected from the group consisting of liver, lung, kidney, pancreas, intestine, and heart.

According to certain embodiments, the organ is a liver.

According to certain embodiments, the IRI is ischemic biliary injury.

According to certain embodiments, the organ is an allotransplant or a xenotransplant.

According to certain embodiments, the effective amount of AAT is sufficient to maintain a target concentration between about 0.2 mg/mL to about 20 mg/ niL for the duration of perfusion.

According to certain embodiments, the duration of perfusion is from about 1 hour to about 24 hours.

According to certain embodiments, the AAT is recombinant or transgenic AAT.

According to certain embodiments, the method results in reduced frequency of transplant rejection.

According to certain embodiments, the method results in reduced apoptosis or reduced injury to the organ.

According to certain embodiments, the method results in a decreased organ discard rate.

According to certain embodiments, the method results in enablement of increase in organ preservation time.

According to some embodiments, said administration involves perfusing or flushing the organ with a preservation fluid containing AAT.

According to some embodiments, said preservation fluid contains at least one component selected from the group consisting of electrolytes, hormones, steroids, blood substitutes, oxygen carriers, and cell-protecting agents.

According to some embodiments, the transplant organ originates from a deceased donor.

According to some embodiments, the transplant organ originates from a donor after brain death (DBD).

According to some embodiments, the transplant organ originates from a donor after circulatory death (DCD).

According to some embodiments, AAT is added to a ready-made preservation solution just before use. Alternatively, a suitable preservation solution containing AAT may be prepared beforehand.

Other objects, features, and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates stable resistance, and improved hemodynamics in blood vessels of the AAT treated lobes as compared to the control treated lobes.

FIG. 2 demonstrates higher total flow rate in the AAT treated lobes as compared to the control treated lobes.

FIG. 3 demonstrates lower levels of Aspartate transaminase (AST) in the AAT treated lobes as compared to the control treated lobes. FIG. 4 demonstrates lower levels of Alanine transaminase (ALT) in the AAT treated lobes as compared to the control treated lobes.

FIG. 5 demonstrates elevated energy restoration (ATP: AMP ratio) in the AAT treated lobes as compared to the control treated lobes.

FIG. 6 demonstrates reduced cytokine levels (IL-8) in the AAT treated lobes as compared to the control treated lobes.

FIG. 7 shows the results of apoptosis assay (TUNEL) of liver biopsy specimens obtained from the AAT treated lobes as compared to the control treated lobes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses methods for prevention or reduction of ischemic reperfusion injury (IRI) by administering alpha 1 -antitrypsin (AAT) during machine perfusion.

Improved approaches to ex-vivo organ care are provided. More particularly, various embodiments are directed to improved methods and solutions relating to preserving a solid organ in an ex-vivo environment.

Disclosed herein are solutions for machine perfusion of an organ. The solutions can be used for perfusion of an organ, for example, for organ preservation during manipulation, treatment, storage, and/or transport of an organ for transplantation in a recipient.

Definitions

As used herein the term "about" refers to the designated value ± 10%.

As used herein, the term“Alpha- 1 Antitrypsin” (AAT) refers to a glycoprotein that in nature is produced by the liver and secreted into the circulatory system, as well as by lung and intestinal epithelial cells, and certain immune cells to regulate the local concentration of AAT. AAT belongs to the Serine Proteinase Inhibitor (Serpin) family of proteolytic inhibitors. This glycoprotein consists of a single polypeptide chain containing one cysteine residue and 12-13% of the total molecular weight of carbohydrates. AAT has three N-glycosylation sites at asparagine residues 46, 83, and 247, which are occupied by mixtures of complex bi- and triantennary glycans. This gives rise to multiple AAT isoforms, having isoelectric points in the range of 4.0 to 5.0. The glycan monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, and sialic acid. AAT serves as a pseudo-substrate for elastase; elastase attacks the reactive center loop of the AAT molecule by cleaving the bond between methionine358 - serine359 residues to form an AAT-elastase complex. This complex is rapidly removed from the blood circulation and the lung airways. AAT is also referred to as “alpha- 1 Proteinase Inhibitor” (API). It is to be explicitly understood that any AAT as is or will be known in the art, including plasma-derived AAT, recombinant AAT and transgenic AAT can be used according to the teachings of the present invention.

The term "subject," as used herein, refers to any animal, individual, or patient to which the methods described herein are applied. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and non-human primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

The term "glycoprotein" as used herein refers to a protein or peptide covalently linked to a carbohydrate. The carbohydrate may be monomeric or composed of oligosaccharides.

The terms "prevent" or "preventing" includes alleviating, ameliorating, halting, restraining, slowing, delaying, or reversing the progression, or reducing the severity of pathological conditions described above, or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.

"Amelioration" or "ameliorate" or "ameliorating" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

The term "preserving" as used herein refers to maintaining the state of an organ that is already functioning at a level that is suitable for use as a transplant, with no or minimal (e.g. about 10% or less, or about 5% or less) loss of any measurable parameter.

The term "reanimation" as used herein refers to an organ that is not fully functional, or is not functioning at a level that is suitable for use as a transplant, for a period of time and under conditions that permit organ repair (rejuvenation, regeneration, etc.)·

The term "dosage" as used herein refers to the amount, frequency and duration of AAT, which is given during a therapeutic period.

The term "dose" as used herein, refers to an amount of AAT, which is given in a single administration.

The term "Ischemia" as used herein refers to an absolute or relative shortage of the blood supply to an organ (i.e. a shortage of oxygen, glucose and other blood-borne fuels). The relative shortage results in tissue damage because of a lack of oxygen and nutrients. Ultimately, this can cause severe damage because of the potential for a build up of metabolic wastes.

The term“Ischemic Reperfusion Injury (IRI)” as used herein, refers to cellular damage caused to a tissue or organ when blood supply returns to the tissue after a period of ischemia. The absence of blood oxygen and nutrients during the ischemic period creates a condition in which the restoration of circulation results in oxidative damage, including cellular dysfunction, apoptosis, and necrosis. IRI can occur in any body tissue as a result of inter alia surgery, wounds, trauma, obstructions, implantations, and transplantations.

As used herein, the term "standard criteria donor (SCD)" is a donor who is under 50 years of age and has suffered brain death from any number of causes. This would include donors under the age of 50 who suffer from traumatic injuries or other medical problems such as a stroke. Pediatric donors are considered standard criteria donors. Other possible standard criteria donors could be between the ages of 50 and 59 (inclusive) without two or more of the following: a history of high blood pressure, terminal serum creatinine level greater than 1.5 mg/dl, or cerebrovascular cause of brain death.

As used herein, the term "Transplant" or various grammatical forms thereof, refer to the physical act of providing a patient with organs from a living source distinct from the patient. The transplant can be either a primary graft or a regraft. As used herein, the term "perfusate" refers to the fluid that has been caused to flow over and/or through an organ.

The term "pulsatile flow" means the rhythmic, intermittent propagation of a fluid, in contrast to smooth propagation, which produces laminar flow.

As used herein, the term“perfusion” refers to circulation of a fluid (also referred to as a perfusion solution or perfusate) through an organ to supply the needs of the organ to retain its viability (for example, in an ex vivo system). In some examples, the perfusion solution includes an oxygen carrier (for example, a hemoglobin-based oxygen carrier).“Machine perfusion” refers to the introduction and removal of a perfusion solution through an organ by a mechanical device. Such devices may include one or more chambers for holding an organ and a perfusion solution, one or more pumps for delivery of the perfusion solution to the organ, one or more means to regulate temperature of the perfusion solution, and one or more means to oxygenate the perfusion solution. In some examples, machine perfusion includes introduction of an oxygen carrying fluid into an organ and removal of oxygen depleted fluid from the organ by circulation of the oxygen carrying fluid through the organ.

In some embodiments, the perfusion can be pulsatile, with periodic increases and decreases of flow, in order to mimic the arterial blood flow from a beating heart. In other embodiments, the perfusion can be continuous, with a substantial absence of flow rate variations, to mimic venous blood flow under most physiological conditions.

As used herein, the term "Donor Organ" refers to a harvested organ that has been removed from the host. A donor organ may include any transplantable organ of the body.

“Organ Perfusion Solutions” as used herein are solutions for perfusion (for example, machine perfusion) of an organ. The solutions can be used for ex vivo perfusion of an organ, for example, for organ preservation during manipulation, treatment, and storage and/or transport of an organ for transplantation in a recipient. In some embodiments, the solutions have characteristics such as oxygen-carrying capacity, pH, osmolality, and/or COP that make them particularly suitable for machine perfusion of an organ at sub-normothermic temperatures (such as about l2-25.degrees C). In some embodiments, the solutions include an oxygen carrier (such as acellular cross-linked hemoglobin) in a physiologically acceptable medium. In particular examples, the characteristics of the solution are provided as those when the solution is under storage conditions (for example, in a container at room temperature). The solution may have a pH of about 7.0-8.0 (such as about 7.2-7.9, for example, about 7.4-7.85) at room temperature, an osmolality of about 290-360 mOsm/kg, and a COP of about 18-75 mm Hg. Some exemplary, non-limiting, perfusion solutions are provided herein, for example in Table 1.

Devices or systems that can be used in the disclosed methods are also known to one of ordinary skill in the art. Such devices include one or more chambers for holding an organ and a perfusion solution (such as the solutions disclosed herein) and one or more pumps (for example, one or more rotary pumps or peristaltic pumps) for delivery of the perfusion solution to the organ. Such devices also include one or more means to regulate the temperature of the perfusion solution, such as one or more heat exchangers and one or more means to oxygenate the perfusion solution (such as an oxygenator in the perfusion circuit). In one example, the device includes a disposable unit including one or more pump heads (such as one or more centrifugal pump heads with magnetic coupling), one or more oxygenators with an integrated heat exchanger, one or more pressure sensors, connecting tubing, and a reservoir which holds the organ and perfusion solution. The organ is connected to the connecting tubing by one or more cannulas. The one or more pumps can perfuse the organ via pulsatile or continuous (such as non-pulsatile) pressure. The device also includes one or more thermo-electric elements (for example, Peltier devices) in combination with a water pump. Water of the required temperature is pumped through the heat exchanger in the oxygenator in the disposable unit. In some examples, the machine perfusion device includes a perfusion liquid reservoir containing a perfusion solution disclosed herein.

Exemplary devices are available from Organ Assist, Groningen, Netherlands (such as Kidney Assist or Liver Assist), Organ Recovery Systems, Itasca, Ill. (such as LifePort kidney transporter or liver transporter), Transmedics, Andover, Mass (such as the heart or lung Organ Care System), OrganOx, Oxford, UK (such as OrganOx Metra), and XVIVO Perfusion Engelwood, Colo. Exemplary devices and systems are also described in U.S. Pat. Nos. 6,994,954; 6,953,655; 6,977,1420; 7,678,563; 7,811,808; 7,897,357; 8,268,547; 8,268,612; and 8,287,580; U.S. Pat. Publ. No. 2010/0028850; and International Pat. Publ. No. WO 2009/041806; all of which are incorporated herein by reference in their entirety. One of ordinary skill in the art can identify additional organ perfusion devices or systems that can be used with the methods and solutions disclosed herein.

A "method of prophylaxis of ischemic reperfusion injury (IRI)" refers to preventing, attenuating or reducing the damage caused by IRI, for example, preventing, attenuating or reducing cellular death and/or apoptosis and/or necrosis and/or oxidative stress.

As used herein, "an organ at risk of IRI" refers to an organ experiencing temporary cessation of blood flow or temporary global hypoxia. In a non-limiting example, a temporary cessation of blood flow may be due to thrombosis, vasoconstriction, and pressure on blood vessels for any reason or removal of the organ from the body with subsequent transplantation.

As used herein, "delayed graft function" or "DGF" refers to organ dysfunction following organ transplantation.

As used herein, the term“organ” refers to a part of the body, tissue, or portion thereof that can be transplanted or preserved ex vivo. Organs include, but are not limited to liver, kidney, heart, lung, pancreas, small intestine, and limbs (such as arm or leg, or portion thereof), or extremities (such as hand, foot, finger, toe, or a portion thereof). As used herein, "organ" also includes other tissues, such as tissue grafts (also referred to as composite tissue allografts herein).

"Physiological temperature" refers to temperatures between about 25 degrees C and about 37 degrees C.

As used herein, the term "cold storage" refers to storage at a temperature of about 0 degrees C or less, for example, storage on ice. Such storage reduces the rate of energy consumption, for example, by the organ.

As used herein, the term“Elevated liver enzymes” may indicate inflammation or damage to cells in the liver. Inflamed or injured liver cells leak higher than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated liver enzymes on blood tests. To "increase" means to increase, improve, or augment an activity, function, or amount as compared to a reference.

In some embodiments, by "increase" is meant the ability to cause an overall increase of 5% or greater, of 10% or greater, of 20% or greater, of 30% or greater, of 40% or greater, of 50% or greater, of 60% or greater, of 70% or greater, of 80% or greater, of 90% or greater, of 100% or greater relative to a reference value. In some embodiments, by "increase" is meant the ability to cause an overall increase of 5% to 50%, of 10% to 20% of 50% to 100%, of 25% to 70% relative to a reference value. In some embodiments, by "increase" is meant the ability to cause an overall increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%, or greater.

To "decrease" means to decrease, reduce, or arrest an activity, function, or amount as compared to a reference.

In some embodiments, by "decrease" is meant the ability to cause an overall decrease of 5% or greater, of 10% or greater, of 20% or greater, of 30% or greater, of 40% or greater, of 50% or greater, of 60% or greater, of 70% or greater, of 80% or greater, of 90% or greater, of 100% or greater relative to a reference value. In some embodiments, by "decrease" is meant the ability to cause an overall decrease of 5% to 50%, of 10% to 20% of 50% to 100%, of 25% to 70% relative to a reference value. In some embodiments, by "decrease" is meant the ability to cause an overall decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%, or greater.

To "maintain" means an activity, function, or amount as compared to a reference is maintained at a similar value or level.

In some embodiments, by "maintain" is meant within 0.5% above, 0.5% below, 1% above, 1% below, 2% above, 2% below, 3% above, 3% below, 4% above, 4% below, 5% above, 5% below, 10% above, 10% below, 15% above, 15% below, 20% above, or 20% below a reference value. In some embodiments, by "maintain" is meant within 20% or less above, 20% or less below, 15% or less above, 15% or less below, 10% or less above, 10% or less below, 5% or less above, 5% or less below, 1% or less above, or 1% or less below a reference value. A "reference" as used herein, refers to any sample, standard, or level that is used for comparison purposes.

In some embodiments, an amount is increased, decreased, or maintained over a period of time, relative to a control organ over the same period of time. In some embodiments, an amount is increased, decreased, or maintained over a period of time, relative to an amount at an earlier point in time for the same organ.

The term "simultaneous administration," as used herein, means that the AAT and the additional treatment are administered with a time separation of no more than about 15 minute(s), such as no more than about any of 10, 5, or 1 minutes.

By administering AAT to an organ transplant prior to the transplantation, development of IRI injury in the organ transplant can be reduced or prevented. As a result, the function of the organ transplant is more rapidly recovered, which is a prerequisite for the success of the organ transplantation. In kidney transplantations, the prevention of renal dysfunction after transplantation decreases the dependence of the patient on hemodialysis. In liver, heart, and lung transplantations, the early proper function of the organ transplant is critical and prevention of graft dysfunction should decrease mortality of the patients. By adding AAT to the preservation solution used during machine perfusion, IRI injury in the organ transplant can be prevented and functional recovery after transplantation promoted.

In some embodiments, in addition to the use of AAT during ex vivo perfusion of the organ, AAT is also administered to the organ recipient before, during, or after the organ is transplanted. In some embodiments, AAT is administered to the organ recipient before the organ transplant. In some embodiments, AAT is administered to the organ recipient during the organ transplant. In some embodiments, AAT is administered to the organ recipient after the organ is transplanted. In some embodiments, the AAT is administered to the organ recipient intravenously or subcutaneously. In some embodiments, the agent is administered to the organ recipient by intravenous injection or subcutaneous injection, etc.).

"Recombinant AAT" as used herein, refers to AAT that is the product of recombinant DNA or transgenic technology. The phrase, "recombinant AAT," also includes functional fragments of AAT, chimeric proteins comprising AAT or functional fragments thereof, fusion proteins or fragments of AAT, homologues obtained by analogous substitution of one or more amino acids of AAT, and species homologues. "Recombinant AAT," also refers to AAT proteins synthesized chemically by methods known in the art such as, e.g., solid-phase peptide synthesis. Amino acid and nucleotide sequences for AAT and/or production of recombinant AAT as described by, e.g., U.S. Pat. Nos. 4,711,848; 4,732,973; 4,931,373; 5,079,336; 5,134,119; 5,218,091; 6,072,029; and Wright et al., Biotechnology 9: 830 (1991); and Archibald et al., Proc. Natl. Acad. Sci. (USA), 87: 5178 (1990), are each herein incorporated by reference for its teaching of AAT sequences, recombinant AAT, and/or recombinant expression of AAT.

Preparation of AAT

According to one aspect of the present invention a purified stable composition of AAT is provided. International application WO 2005/027821, to the applicant of the present invention, provides pharmaceutical compositions comprising a purified, stable, active AAT in the form of a ready to use sterile solution. WO 2005/027821 also provides a process, which combines removal of contaminating substances (i.e., lipids, lipoproteins and other proteins) and separation of active from inactive AAT by sequential chromatography steps. The process disclosed in that invention is highly suitable for a large-scale production of AAT, in the range of tens of kilograms or more. The mixture of proteins from which the AAT is purified is preferably Cohn Fraction IV- 1 paste, but can include other Cohn Fractions, separately or in combination; human blood plasma; plasma fractions; or any protein preparation containing AAT. For instance, the process is applicable to purification of recombinant human AAT from the milk of transgenic animals.

In that application, the mixture of proteins comprising AAT is dispersed in an aqueous medium, preferably water, at a ratio of about 13 to about 35 liters per about 1 kg of source material, preferably Cohn Fraction IV- 1 paste. The pH of the dispersion is adjusted to a pH range between about 8.0 and about 9.5. The pH adjustment stabilizes the AAT and promotes the dissolution of the AAT in the dispersion, thereby increasing the production yield. Dispersion may take place at an elevated temperature of between 30°C and 40°C for further increase in AAT solubility.

A particular advantage of that process is the elimination of contaminants or by- products that otherwise compromise the efficiency of AAT purification processes. In particular, Cohn Fraction IV- 1 paste preparations contain a significant amount of the lipoprotein Apo A-l, which has the effect of compromising column flow and capacity during purification. Other non-desired proteins such as albumin and transferrin are also present in the paste preparation. Removing a portion of such contaminants according to the invention disclosed in WO 2005/-27821 is performed by two steps: (a) removing contaminating lipids and lipoproteins with a lipid removal agent and (b) precipitating a portion of contaminating protein from the AAT-containing aqueous dispersion. The removal of contaminating proteins, without loss of AAT, enables a significant reduction in equipment scale, e.g., column size.

The precipitate that forms can be separated by conventional means such as centrifugation or filtration, and is then discarded. The supernatant is ready for further purification, for example on an anion exchange resin. The AAT is then eluted from the column. The solution is treated to reduce the water content and change the ionic composition by conventional means such as by diafiltration, ultrafiltration, lyophilization, etc., or combinations thereof.

According to one embodiment, the AAT-containing effluent obtained after the first anion exchange chromatography is concentrated by ultrafiltration. The retentate is then diafiltered against pure water to reach conductivity within the range of from about 3.5 to about 4.5 mS/cm.

To further purify the AAT-containing solution obtained after the first anion exchange chromatography, the solution is loaded on a cation exchange resin with the same type of buffer used for the anion-exchange step, having appropriate pH and conductivity to allow the AAT to pass and be washed off with the buffer flow through, while contaminating substances are retained on the cation exchange resin. The AAT-containing solution obtained after the cation exchange chromatography can be treated to reduce its water content. According to one embodiment, the solution is concentrated by ultrafiltration.

The ion-exchange chromatography is also used to separate active AAT from inactive AAT. That invention further comprises methods for separating active AAT from other contaminating substances, including solvent/detergent compounds used for viral inactivation. Such separation is achieved by the second anion exchange chromatography. The AAT eluted from the second anion exchange chromatography step is therefore not only highly active, but also highly pure. Throughout the process of that invention only one type of buffer is used, with adjustment of pH and conductivity as required throughout the various process steps. According to one embodiment, the buffer is any suitable acid/salt combination that provides an acceptable buffer capacity in the ranges of pH required throughout the process. According to preferred embodiments the process uses a buffer other than citrate-based buffer. According to yet other embodiments, the buffer anion is acetate. According to one embodiment, the process of that invention further comprises viral removal and/or viral inactivation steps. Methods for viral removal and inactivation are known in the art.

One method for viral removal is filtration, preferably nanofiltration, removing both enveloped and non-enveloped viruses. According to one embodiment, the viral removal step comprises filtration. According to another embodiment, the virus removal step is performed after the cation exchange chromatography. Typically, the cation exchange flow-through solution containing AAT is concentrated, and then nanofiltered. According to one embodiment, the method of viral inactivation employed comprises a solvent/detergent (S/D) treatment. The viral inactivation step is preferably performed prior to loading the solution on the second anion exchange resin. According to one embodiment, the detergent used is polysorbate and the solvent is Tri-n-Butyl-Phosphate (TnBP). According to another embodiment, the polysorbate is Polysorbate 80. According to one embodiment Polysorbate 80 may be added from about 0.8% to about 1.3% volume per weight (v/w) of the resulting mixture and TnBP may be added from about 0.2% to about 0.4% weight per weight of the resulting mixture. The solution containing active, purified AAT obtained after the second anion exchange chromatography can be further processed to obtain a pharmaceutical composition for therapeutic, diagnostic, or other uses. To prepare the product for therapeutic administration, the process further comprises the steps of changing the ionic composition of the solution containing purified, active AAT to contain a physiologically compatible ion and sterilizing the resulted solution.

The purified AAT obtained by the process of that invention is highly stable. According to one embodiment, the pharmaceutical composition comprises at least 90% pure, preferably 95% pure, more preferably 99% pure AAT. According to another embodiment, at least 90% of the AAT is in its active form.

According to some embodiments, highly dispersible dry powder compositions are used, comprising a high concentration of active AAT and specific excipients, suitable for pulmonary delivery of AAT. The dry powder compositions disclosed herein comprise, according to some embodiments, AAT molecules in their monomeric form, having a low level of aggregation. The AAT dry powder compositions exhibit exceptional stability and low aggregation properties, and thus are highly suitable for use with inhalation devices as well as in other dry-powder dosage forms.

Pharmaceutical Compositions and Methods of Treatment

The term "pharmaceutical composition" is intended to be used herein in its broader sense to include preparations containing a protein composition in accordance with this invention used for therapeutic purposes. The pharmaceutical composition intended for therapeutic use should contain a therapeutic amount of AAT, i.e., that amount necessary for preventative or curative health measures.

As used herein, the term "therapeutically effective amount" refers to an amount of a protein or protein formulation or composition which is effective to treat a condition. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g. by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more acceptable diluents or carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically.

The AAT-containing pharmaceutical compositions disclosed in W02005/027821 to the Applicant of the present invention is advantageous over hitherto known AAT- containing preparations, as the AAT is highly stable even when the composition is kept in a liquid form. Therefore, it is not necessary to lyophilize the AAT preparation for stable storage in the form of a powder. According to certain currently preferred embodiments, AAT in a ready-to-use liquid formulation is used with the methods of the present invention.

Liver transplantation

Chronic liver disease and cirrhosis is the 12^th leading cause of death in the US with over 38,000 deaths attributable to liver disease in 2014. Liver transplantation is the only available cure. According to national data from the OPTN, there are approximately 14,500 patients currently on the liver transplant waiting list in the US. Over 11,600 new patients were added to the list in 2015. The number of transplants performed each year has steadily risen, with 2015 marking the first year that over 7,000 transplants were performed in the US, however there remains a severe shortage of suitable organs. This is evidenced by long wait times prior to transplant, as well as high morbidity and mortality experienced by patients on the waiting list. In 2015 alone, 1,500 patients died while waiting for a transplant and an additional 1,500 were removed from the list because they became too ill to transplant. These numbers likely underestimate the magnitude of the organ shortage, as the indications for liver transplant would certainly increase if more livers were available.

The increased demand for livers has led to increased use of livers from marginal donors including donors after cardiac death (DCD), as well as livers with increased warm and cold ischemia time, and fatty livers. Compared to standard quality livers, these marginal livers are at increased risk of ischemic reperfusion injury (IRI) and subsequent ischemic biliary injury, graft dysfunction, and graft failure following transplantation. Organ preservation with ex situ machine perfusion has emerged as a promising therapy for prevention and treatment of preservation injury and IRI in these marginal grafts prior to transplant. Although beneficial, successful treatment of IRI will require more than simply machine perfusion alone. Ongoing research in liver transplantation is focused on strategies to optimize each aspect of the transplant process, including organ harvest techniques and methods of organ preservation as well as the recipient procedure.

Pathophysiology of hepatic IRI

The challenge in treating IRI lies in its complex pathophysiology, involving an intricate network of molecular pathways and interactions between circulating blood cells, cytokines and chemokines, the complement system, vascular endothelium, and interstitial compartments, as well as a multitude of biochemical mediators of inflammation. In liver transplantation, cessation of blood flow causes ischemia and tissue hypoxia. This disrupts the aerobic metabolism in the hepatic parenchyma, leading to rapid depletion of ATP stores, a build-up of metabolic intermediates, production of reactive oxygen species (ROS), and initiation of hepatocyte apoptosis. The consequences of these actions are cell death and damage to the sinusoidal endothelium, with impairment of its barrier function. This process continues and is exacerbated upon reperfusion of the transplanted liver as the restored supply of oxygen provides the substrates necessary for free radical production and a new wave of inflammatory cells and mediators are released into the hepatic circulation. Injured hepatocytes and Kupffer cells generate an overwhelming inflammatory response by secreting TNF-alpha, IL-l, IL-6, chemokines, and ROS, which in addition to causing direct damage to the endothelium and extracellular matrix, initiate recruitment and activation of neutrophils and other inflammatory cells in the microvasculature.

Recent studies have demonstrated that neutrophils function as the primary effector cells in reperfusion injury, and neutrophilic invasion across the injured sinusoidal epithelium into the interstitial tissue is a key step in this process. Infiltration into injured tissue is mediated by a multitude of biochemical mediators, including the proinflammatory cytokines TNF-alpha, IL-l-beta, IL-6, and IL-8, as well as complement, toll-like receptors (TLRs), and ROS. Once in the parenchyma, activated neutrophils initiate multiple proinflammatory processes. Neutrophil adherence to target cells (hepatocytes) is promoted by increased expression of the adhesion molecules CD l l/CD 18, which triggers cell death from oxidative stress. A positive feedback loop stimulates additional neutrophil recruitment by secretion of proinflammatory chemokines and cytokines, thereby amplifying the inflammatory response. Neutrophils also release granules containing mediators that activate the complement cascade and initiate apoptosis. Neutrophil-derived serine proteases, namely neutrophil elastase (NE), are a critical component of the neutrophilic response and act by degrading components of the ECM and the endothelium. The importance of NE in hepatic IRI was reported by Uchida et al, who demonstrated that following reperfusion in a mouse model of liver IRI, a specific NE inhibitor attenuated hepatocellular damage, reduced inflammatory cell recruitment and invasion, down-regulated secretion of proinflammatory cytokines, and reduced apoptosis ( Transplantation . 20l0;89(9): 1050- 1056).

Unlike DBD organs, which have an intact circulation until the time of cross clamp, DCD organs endure an obligatory period of warm ischemia (WIT) during the interval between circulatory arrest and organ cooling. The WIT is of variable duration and strongly correlates with the degree of IRI and subsequent graft dysfunction following transplant. In fact, the American Society of Transplant Surgeons (ASTS) has issued a practice guideline recommending against the use of DCD organs with a WIT > 30 minutes due to an unacceptably high rate of post-transplant dysfunction. Additional major risk factors for IRI and liver graft dysfunction are CIT, degree of steatosis, and donor age. The ASTS guidelines discourage the use of organs with CIT > 8 hours, macrosteatosis > 30%, and advanced donor age.

Machine perfusion improves the function of marginal donors

The development of the static cold storage (SCS) method of liver graft preservation in the l980's revolutionized liver transplantation and remains the standard of care for preserving liver grafts prior to transplant. SCS dramatically reduces the metabolic activity in the graft resulting in improved graft quality as well as prolonging the duration of acceptable cold ischemia time. Although SCS is adequate for the majority of standard quality donor livers, a major shortcoming of SCS is that it is unable to completely eliminate metabolic activity, resulting in a progressive loss of energy stores and eventual loss of graft viability. This is relevant when SCS is applied to the preservation of marginal livers, where the continued depletion of energy stores in SCS and resultant increased ischemic injury to the already impaired graft, lead to unacceptably high rates of ischemic biliary injury, primary graft failure, and early dysfunction. An additional shortcoming is that SCS precludes a thorough assessment of graft quality and does not provide any opportunity for therapeutic intervention targeted at repair or recovery of compromised grafts.

Recent research has resulted in the emergence of ex situ machine perfusion (MP) as a pioneering innovation in organ preservation, particularly in the preservation of ECD/DCD grafts. During MP, the liver graft is perfused via the portal vein and hepatic artery, providing the graft with nutrients and removing toxic metabolites. The primary benefit of MP compared to SCS is the ability to reinitiate and maintain cellular metabolism during storage by delivery of oxygen and nutrients. This enables a continuation of normal metabolism, natural repair pathways, and reconstitution of energy stores. In this way, ex situ MP has significant potential to reduce preservation injury and treat IRI.

A number of techniques for MP have been developed but the optimal conditions, content of the perfusate, and perfusion technique have yet to be determined. One key parameter is the temperature of the perfusion system, and three categories predominate: hypothermic (0-8°C), subnormothermic (20-33°C) and normothermic (37°C), where each has theoretical advantages and disadvantages. Briefly, hypothermic MP (HMP) is the easiest to apply and therefore has the largest body of evidence, showing efficacy in both animal models as well as human studies, including an impressive report of improved graft function in 31 DCD livers preserved with HMP prior to transplantation. Although HMP has effectively decreased preservation injury and IRI, the reduced metabolic activity limits the evaluation of graft function and thus precludes an accurate assessment of viability. By increasing the temperature, subnormothermic MP (SMP) introduces the theoretical possibility of evaluating graft function and may better predict which ECD organs will be suitable to transplant. Animal studies of SMP compared to HMP and SCS have provided evidence that SMP results in improved liver function compared to both HMP and SCS, and the first report in human liver transplantation has recently appeared, describing the benefit of SMP compared to SCS [Dutkowski et ah, Ann Surg. 20l5;262(5):764-771] . Normothermic machine perfusion (NMP) is the most technically challenging of the three temperatures, but also has the greatest theoretical upside. Minimizing cold storage limits perfusion injury, allows restoration of normal organ function under physiological conditions, and allows for real-time assessment of liver function and determination of graft viability. An important advantage of NMP over HMP or SMP is the ability to perform therapeutic interventions or organ modifications during the preservation period. Mergental et al reported the outcomes of successful human transplantation of five declined liver grafts after a period of NMP [Am J Transplant. 20l6;l6(l l):3235-3245]. Ravikumar et al have demonstrated the safety and feasibility of a novel NMP device for organ preservation in a phase 1 study of 20 human liver transplants [Clinical Trial. Am J Transplant. 2016; 16(6): 1779- 1787]. Interestingly, in a separate study, this group also demonstrated improved postreperfusion hemodynamic parameters during the recipient transplant procedure with grafts that had been preserved with NMP compared to those preserved with SCS, suggesting that the effects of NMP may extend beyond the theoretical benefits already discussed [ Transplant Direct. 2016;2(9) :e97] .

There is experimental evidence for the therapeutic benefit of MP with both subnormothermic and normothermic techniques following conventional cold storage in a rat model of extended criteria liver transplant and an experimental evidence regarding the optimal perfusion solution and treatment duration [Izamis et al, PLoS One. 20l3;8(7):e69758]

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Alpha- 1 -Antitrypsin reduces IRI during Ex- Vivo liver perfusion

Human alpha- 1 antitrypsin (AAT) was administered during a period of normothermic machine perfusion and subsequent ex vivo reperfusion of discarded human donor livers to investigate the effect on ischemic reperfusion injury. The primary objective of the study was to determine whether AAT treatment during organ preservation prior to transplantation can prevent hepatic IRI. This objective was studied with the following aims:

Aim 1: Assess the efficacy of AAT for the treatment of IRI during machine perfusion. The effect of AAT therapy was determined through comprehensive analysis of liver function prior to treatment, at specific intervals throughout machine perfusion, at the end of machine perfusion, and during reperfusion. The results were further stratified according to specific parameters of the donor and graft in order to elucidate the effects of AAT on livers with varied donor characteristics.

Aim 2: Determine the mechanism of AAT in the treatment of hepatic IRI. In order to further understand the mechanism of AAT in the pathophysiology of IRI in liver transplantation, a thorough biochemical and histologic assessment of the inflammatory, apoptotic, and immune-modulating pathways known to be affected by AAT were performed.

Aim 3: Verify the therapeutic range for AAT and the dosing regimen required to maintain target AAT levels. AAT levels in the perfusate were measured and correlated with liver function to determine the optimal therapeutic range for AAT. In addition, different dosing protocols were compared in order to establish the regimen necessary to maintain AAT levels within the therapeutic range.

Study Design:

Acquisition and use of human deceased donor livers for research

Deceased human livers designated for research purposes were obtained from the New England Organ Bank. The livers were obtained from multiple donors and were therefore heterogeneous with regard to donor characteristics and included both DBD and DCD with variable donor age, duration of WIT, CIT, and degree of macrosteatosis. All available livers were used, regardless of donor characteristics, recognizing that the degree of IRI and the mechanisms underlying the IRI may differ significantly from liver to liver. Although this heterogeneity between whole livers may affect the overall results due to potential variable effects of AAT on livers with variable characteristics, including all livers will allow a detailed subgroup analysis of the effects of AAT. This will guide future research and clinical practice to predict which population of livers is most appropriate for AAT therapy.

Development of split liver perfusion approach

To address the problem of variability between organs from different donors, a split liver technique was developed. In this approach, a single donor liver is split into left and right lobes, which are perfused simultaneously on separate pumps, effectively eliminating the variability between the organs in each experiment. Additionally, by perfusing two lobes of the same liver simultaneously, a natural model for experimentation has been developed where one lobe can be manipulated while the other lobe serves as an ideal unmanipulated control. Specific to this study, AAT was administered to one lobe in the perfusate, while the other did not receive any additional treatment. The lobe (right versus left) selected to receive treatment was alternated with each experiment to mitigate any potential effect of laterality. The ex vivo liver splitting technique as described by Vagefi et ah, [Arch Surg. 2011; 146(9): 1052- 1059] was slightly modified to optimize it for the proposed research studies. In brief, after exposing the portal vein (PV) and hepatic artery (HR) to the bifurcation of right and left lobe branches, these structures were divided. The right hepatic vein was separated from the vena cava and then transected from the left bile duct, just above the bifurcation. Parenchymal division was completed by a clamp- fracture clip technique. Middle hepatic vein branches to segment 5 and/or 8 were left on the right lobe side to ensure unimpeded outflow. When aberrant arterial or portal anatomy was encountered, the artery was reconstructed by standard techniques to allow a single lumen inflow to the lobe. The splitting procedure requires only 20-25 minutes of additional liver prep time on ice and does not alter the experimental design since the liver is routinely maintained in cold ischemia over an hour while the perfusion device and graft are prepared.

Normothermic machine perfusion method and administration of AAT

Following the ex vivo split procedure, each lobe was exposed to NMP according to a protocol previously described by Karimian et al., [J Vis Exp. 20l5;(99):e52688]. In brief, the perfusion system consists of two independent circuits for portal and arterial perfusion, each including a roller pump, hollow-fiber oxygenator, and a bubble trap. Sensors allow for continuous measurement of pressure on the inflow vessels. Flow was automatically regulated to achieve a pressure of 4-7 mmHg via the portal vein (typical flow rate is about 700ml/min) and 50-80 mmHg on the artery (typical flow about 200ml/min). Approximately 2L of perfusate, consisting of Williams Medium E (Sigma) supplemented with hydrocortisone (10 mg/l), insulin (2 U/l), heparin (1000U/1), penicillin (40,000 U/l) streptomycin (40 mg/l) was oxygenated and buffered with a carbogen mixture of 95% 02 / 5% C02, to give a maximum partial oxygen pressure of > 700 mmHg and undepleted oxygen outflow (> 200 mmHg). Preliminary data suggests that complete restoration of energy stores occurs with 3 hours of ex situ perfusion, therefore NMP was continued for 3 hours prior to initiating reperfusion.

AAT was administered at the initiation of NMP by infusion into the perfusion system of the treatment lobe. The AAT dosage was designed to maintain a target serum concentration of 3 mg/mL for the duration of perfusion. Ex situ whole-blood reperfusion to induce IRI

In order to simulate the recipient transplant operation, each lobe was reperfused with whole blood following the completion of NMP as previously described by Op Den Dries et ah, [Am J Transplant. 2013 ; 13(5): 1327-1335] . Briefly, each lobe was disconnected from the perfusion machine and flushed with cold UW solution, similar to current clinical practice. Each lobe was then transferred into a bowl of PBS solution at room temperature during which it was rewarmed poikilothermically for 20 minutes, which represents the warm ischemia time during which the anastomoses are sewn. During this time, the perfusion system was flushed with fresh PBS, primed with ABO- matched whole human blood recovered from the donor during the procurement procedure and warmed to physiological temperature. After flushing each lobe with warm Lactated Ringers to clear the UW solution, each lobe was reconnected to the perfusion system and heparinized blood perfusion was started.

The MGH Transplant Center and Center for Engineering in Medicine have described the first pre-clinical model for ex situ whole blood reperfusion of human livers following machine perfusion. In their study, which was conducted with discarded DCD human livers, reperfusion with diluted autologous whole blood acquired from the donor at the time of procurement was performed following a period of SMP. Preliminary results of 3 discarded livers showed that macroscopic reperfusion injury of the liver occurred within 15 minutes of reperfusion and the liver remained well-perfused throughout the 3 hours of reperfusion. Bile production began almost immediately and was sustained throughout. Portal vein and hepatic artery flow as well as oxygen uptake were constant throughout reperfusion, demonstrating the stability of the system. ATP levels at the conclusion of reperfusion were decreased by 45% compared to those measured at the end of SMP. This was probably a consequence of the warm ischemia during the sew-in time. By closely simulating reperfusion as it occurs in clinical practice, this model defines the specific effects of reperfusion and thus improves the accuracy of the current MP models used to study human liver transplant.

Outcome Measurements:

NMP provides the opportunity for continuous assessment of ischemia/reperfusion injury, liver function, and graft quality. It also provides the ability to monitor any change in parameters that may occur throughout the treatment duration in response to NMP, reperfusion and assess the therapeutic efficacy of AAT. Objective and accurate biomarkers that can be easily measured at the time of transplant are essential to determine the effect of the proposed interventions on graft quality and viability.

The first group of tests assessed the graft for markers of ischemic injury. Baseline testing was performed at the initiation of NMP and the complete battery was repeated at 30 minute intervals throughout the duration of NMP as well as at the beginning and end of the reperfusion phase of the experiment. The flow and pressure through the hepatic artery and portal vein were monitored continuously. Laboratory tests of liver injury included a liver function panel (AST/ALT/LDH/GGT/bilirubin/albumin) and chemistry panel

(sodium/potassium/BUN/glucose/calcium), blood gas analysis (p02, pC02, HC03-, pH) and lactate. The p02 of the inflow and outflow cannulas were measured in order to calculate oxygen consumption. Methodology for these tests has been previously described by Bruinsma et ah, [Am J Transplant. 2014; 14(6)].

Additional assessments of IRI as well as the therapeutic efficacy of AAT included measurement of gene expression of inflammatory cytokines and components of the IRI response, including TNF-alpha, IL-lbeta, IL-6, IL-8, and TLR4 by RNA extraction and RT-PCR [Uchida et ah, Transplantation. 20l0;89(9): 1050- 1056]. Neutrophil infiltration into the hepatic parenchyma is a necessary step in the process of IRI. Myeloperoxidase (MPO) is an enzyme specific for neutrophils and an MPO assay was used to provide a measure of hepatic neutrophil accumulation [Uchida et ah, Transplantation. 2010;89(9):1050-1056]. Additional assessments of IRI included an apoptosis assay using the TUNEL method on liver biopsy specimens, as well as assays that measure specific enzyme activity in the perfusate, including neutrophil elastase, as described elsewhere [Uchida et ah, Transplantation. 20l0;89(9): 1050- 1056]. This combination of tests provides further information about the mechanism of IRI in liver transplant and of how AAT modifies various aspects of the ischemic response.

The development of ischemic biliary injury, specifically NAS, is the most common, and perhaps the most consequential effect of IRI. Accurate assessment of the bile ducts is a key component that can be used to determine the efficacy of treatment with AAT as well as to predict future complications. This is a particularly difficult task in the preclinical experimental studies of human livers as the development of NAS occurs months after transplantation, making real-time assessment impractical and requiring a surrogate marker for the risk of developing NAS. A collaborative clinical analysis was performed by the MGH Transplant Center and the Netherlands group, in which histologic predictors of NAS were identified [Op Den Dries et ah, J Hepatol. 20l4;60(6): 1172- 1179]. In this study of 128 transplants, it was found that loss of the biliary epithelium occurred at the time of transplant in most grafts and did not predict future occurrence of NAS. However, injury of deep peribiliary glands and the vascular plexus was significantly more prevalent and more severe in livers that later developed NAS compared to those that did not develop strictures. Of note, injury of the deep peribiliary glands may be remedied by ex situ MP [Op Den Dries et al., Am J Transplant. 2013 ; 13(5): 1327-1335] . This study provided evidence for a strong correlation between specific histologic characteristics of the biliary system at the time of transplant and future development of ischemic biliary injury and thus the histologic appearance of the peribiliary glands and vascular plexus were assessed to determine the risk for long term NAS.

Considering that a central mechanism of NMP is restoration of cellular metabolism, demonstration of energy restoration serves as an important indicator for the successful resuscitation of the graft. Decreased ATP levels have been shown to increase the risk for primary non-function of the graft and grafts that function well show better ATP recovery following reperfusion than poor functioning grafts [Verhoeven et al., J Hepatol. 20l4;6l(3):672-684]. Energy recovery can be measured with an ATP assay. For this purpose a luminescence-based assay (ATP Cell Viability Kit; Biovision) was used to measure the absolute levels of ATP in liver biopsies. Mitochondrial function was evaluated by DNA quantification with PCR and transmission electron microscopy (TEM) where the samples were scored as previously described [Bruinsma et al., Sci Rep. 20l6;6:224l5]

Bile production and quality have been identified as useful indicators for graft viability as they reflect the function of hepatocytes and cholangiocytes [Verhoeven et al., J Hepatol. 20l4;6l(3):672-684]. The rate of bile production was monitored and the quality was determined by measuring pH and bicarbonate concentrations as well as LDH levels. A more acidic pH and higher levels of LDH indicate biliary epithelial injury. In addition, the phospholipid concentration was measured and the bile salt:phospholipid ratio calculated. Bile salts are toxic to the epithelium and a high ratio of bile salts :phospholipid is associated with development of ischemic biliary injury [Verhoeven et al., J Hepatol. 20l4;6l(3):672-684]. An additional test used to assess liver function was the indocyanine green (IGC) clearance test, performed at the conclusion of reperfusion as previously described [Bruinsma et al., Sci Rep. 20l6;6:224l5]

Graft function was evaluated by a metabolomic analysis designed to assess the central energy metabolism during perfusion, as has been previously described [Bruinsma et al., Sci Rep. 20l6;6:224l5]. This method enables the identification of pathways and mechanisms of liver disease (and the biomarkers associated with them) beyond the traditional markers of liver injury in order to obtain a more comprehensive assessment of donor liver function. To perform the analysis, liver biopsy samples were acquired and analyzed for metabolic cofactors: ATP/ADP/AMP, NADH/NAD+, NADPH/NADP, FAD, GSH/GSSG. Additional metabolites were measured by gas chromatography. These techniques are fully described by Bruinisma et al [Sc/ Rep. 20l6;6:224l5].

Results:

Five human liver grafts declined for transplantation were obtained for the ex vivo split procedure. Following the split procedure, NMP was performed on each lobe for 5-6 hours with A AT added to the perfusate of the experimental lobe.

The composition of the perfusate is detailed in Table 1:

The livers acquired for the study were from multiple donors and were therefore heterogeneous with regard to donor characteristics and included both DBD and DCD with variable donor age, duration of WIT, CIT, and degree of macrosteatosis.

5 The heterogeneous liver characteristics of the split liver model are presented in Table 2:

As demonstrated in Figure 1, a stable resistance and improved hemodynamics were measured in the AAT treated lobes compared to the control treated lobes.

AAT-treated lobes had a higher total flow rate (Figure 2), and less severe liver injury as 10 demonstrated by lower levels of liver enzymes (AST and ALT) in the blood (Figures 3- 4), superior energy restoration (ATP: AMP ratio, Figure 5 ), reduced pro-inflammatory cytokine secretion (IL-8, Figure 6), and a reduced apoptotic rate (Figure 7) compared to control treated lobes.

These results emphasize that AAT is an effective treatment for hepatic IRI during organ 15 preservation prior to transplantation. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method of protecting an organ susceptible to ischemic reperfusion injury (IRI), the method comprises administering to said organ, an effective amount of alpha 1 -antitrypsin (AAT) during machine perfusion.

2. The method of claim 1, wherein the administering to said organ occurs before said organ has been transplanted into a subject in need of organ transplantation.

3. The method of claim 1, wherein the administering to the organ occurs before or after said organ has been harvested from the donor.

4. The method of claim 1, wherein the AAT is naturally occurring AAT purified from an unpurified mixture of proteins by a process comprising of chromatography on a plurality of ion exchange resins, comprising a first anion exchange resin followed by a cation and a second anion exchange resins.

5. The method of claim 1, wherein the organ is a solid organ selected from the group consisting of liver, lung, kidney, pancreas, intestine, and heart.

6. The method of claim 1, wherein the effective amount of AAT is sufficient to maintain a target concentration of about 0.2 mg/mL to about 20 mg/ mL for the duration of perfusion.

7. The method of claim 1, wherein the duration of perfusion is from about one hour to about 72 hours.

8. The method of claim 1, wherein the AAT is recombinant or transgenic AAT.

9. The method of claim 1, wherein the method results in reduced frequency of transplant rejection.

10. The method of claim 1, wherein the method results in reduced apoptosis or reduced injury of the organ.

11. The method of claim 1, wherein the method results in a decreased organ discard rate.

12. The method of claim 1, wherein the method results in enablement of increase in organ preservation time.

13. The method of claim 1, wherein said administration is perfusing or flushing the organ with a preservation fluid containing AAT.

14. The method of claim 13, wherein said preservation fluid contains, in addition to AAT, at least one component selected from the group consisting of electrolytes, hormones, steroids, blood substitutes, oxygen carriers, and cell-protecting agents.

15. A method of preserving a harvested organ, the method comprises administering to said organ a preservation fluid for perfusion prior to the implantation of said organ in a patient requiring such implantation, wherein said preservation fluid contains alpha 1 -antitrypsin (AAT) in a concentration sufficient to preserve the organ.

16. The method of claim 15, wherein the AAT is naturally occurring AAT purified from an unpurified mixture of proteins by a process comprising of chromatography on a plurality of ion exchange resins, comprising a first anion exchange resin followed by a cation and a second anion exchange resins.

17. The method of claim 15, wherein the organ is a solid organ selected from the group consisting of liver, lung, kidney, pancreas, intestine, and heart.

18. The method of claim 15, wherein the organ is a liver.

19. The method of claim 15, wherein the effective amount of AAT is sufficient to maintain a target concentration of about 0.2 mg/mL to about 20 mg/ mL for the duration of perfusion.

20. The method of claim 15, wherein the duration of perfusion is from about one hour to about 72 hours.

21. The method of claim 15, wherein the AAT is recombinant or transgenic

AAT.

22. The method of claim 15, wherein the method results in reduced frequency of transplant rejection.

23. The method of claim 15, wherein the method results in reduced apoptosis or reduced injury of the organ.

24. The method of claim 15, wherein the method results in a decreased organ discard rate.

25. The method of claim 15, wherein said preservation fluid contains, in addition to AAT, at least one component selected from the group consisting of electrolytes, hormones, steroids, blood substitutes, oxygen-carriers and cell-protecting agents.

26. A method of prophylaxis of ischemic reperfusion injury (IRI) in an organ at risk of IRI, comprising administering to said organ, an effective amount of alpha 1 -antitrypsin (AAT) during ex-vivo machine perfusion.

27. The method of claim 26, wherein prophylaxis of IRI results in prophylaxis of IRI-associated organ dysfunction.

28. The method of claim 26, wherein the duration of perfusion is from about one hour to about 72 hours.

29. The method of any of claims 1, 15 or 26, wherein the organ originates from a deceased donor.

30. The method of claim 29, wherein the organ is liver.

31. The method of claim 30, wherein the IRI is ischemic biliary injury.