Methods for preparing and maintaining livers and portions thereof for liver transplantation

Abstract

Applicant discloses herein methods and reagents for treating isolated livers, or isolated liver portions, prior to transplantation into a subject in need of a liver transplant, to prepare and maintain these isolated livers and portions for liver transplantation, and to reduce liver fat in these livers. The methods comprise perfusing an isolated liver or liver portion with a solution containing a pyrimidine cyclohexyl compound (e.g., miricorilant) or a pyrimidine cyclohexenyl compound (e.g., CORT125385). The reagents for the uses and methods disclosed herein contain a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound. Perfusion with miricorilant-containing reagents improved lactate clearance. Perfusion of isolated livers with these reagents increased lipid levels in the perfusate as compared to their initial levels. Improved lactate clearance and increased lipid levels in the perfusate are believed to indicate reduced fat levels in the isolated liver, and to indicate improved suitability of these livers for transplantation.

Description

PATENT 085178-1509844-018410WO Methods for Preparing and Maintaining Livers and Portions Thereof for Liver Transplantation BACKGROUND

[0001] Fatty liver disease is a common condition caused by the storage of extra fat in the liver. Fatty liver disease is due to an abnormal retention of lipid (fats) within hepatocytes. Liver disorders can be categorized in different groups of diseases, such as alcohol-induced fatty liver disease (AFLD), metabolic dysfunction-associated fatty liver disease (MAFLD, formerly NAFLD), metabolism associated steatohepatitis (MASH / NASH), drug-or alcohol-related liver disease, viral diseases, immune mediated liver diseases, metabolic liver diseases, and complications associated with hepatic insufficiency and / or liver transplantation.

[0002] In humans, end-stage chronic liver disease or liver failure can be treated with a liver transplantation. Most transplanted livers are obtained from deceased liver donors. Liver disease- related mortality is rising globally, and the demand for suitable organs for transplantation is set to continue to increase. Up to 10,000 liver transplant operations are performed each year in the United States. Thus, there is great need for livers, or portions of livers, that can be used for liver transplantation. However, livers with substantial intra-cellular fat deposition (steatosis), or that exhibit insufficient lactate clearance, are typically not suitable for use in liver transplantation.

[0003] In view of the unmet and growing need for livers and liver portions for transplantation, methods for increasing the liver donor pool are needed. What is needed in the art are methods for maintaining the health of donor livers, and for rendering donor livers with substantial intra- cellular fat deposition or other undesirable characteristics suitable for use in liver transplantation. Accordingly, there is need in the art for improved methods and reagents for maintaining livers in good condition prior to transplantation, for improving the suitability of donor livers for transplantation and for increasing the numbers of suitable donor livers available for transplantation.SUMMARY

[0004] Applicant discloses herein methods and reagents for treating isolated livers, or isolated portions of livers, prior to transplantation into a subject in need of a liver transplant, effective to reduce the levels of fat in the liver, or portion of liver, to be used for a liver transplant. Such a reduction in liver fat is believed to improve the suitability of the liver or liver portion for use in liver transplantation. Such a reduction in liver fat is believed to increase the amount of time, following removal, that a liver or liver portion may be maintained in condition for use prior to its use in liver transplantation. An increase in the level of triglycerides, or of free fatty acids, or of cholesterol in the perfusate perfusing the isolated liver or liver portion is an indication of fat reduction in the liver or liver portion. (In the following, for brevity, “liver” will refer both to an isolated liver and to an isolated portion of a liver.) It is believed that the additional triglycerides found in the perfusate derive from the liver. It is further believed that such increased levels of triglyceride found in the perfusate are indicative of reduced levels of triglycerides in the liver. It is also believed that increased levels of free fatty acids, or of cholesterol, found in the perfusate are indicative of reduced levels of free fatty acids, or of cholesterol, in the liver. The methods comprise perfusing a liver with a solution containing a pyrimidine cyclohexyl compound, such as, e.g., miricorilant (disclosed as Example 6, compound 3b of U.S. Patent 8,685,973), or a pyrimidine cyclohexenyl compound, such as CORT125385 (disclosed as Example 10 of U.S. Patent 11,542,238). The reagents for preparing a liver for transplantation, and for maintaining a liver for later use in liver transplantation contain a pyrimidine cyclohexyl compound, such as miricorilant, or contain a pyrimidine cyclohexenyl compound, such as CORT125385.

[0005] Applicant discloses herein methods and reagents for treating livers prior to transplantation into a subject in need of a liver transplant, effective to reduce liver fat in a donor liver as compared to the initial level of fat in the liver. The methods and reagents for treating livers prior to transplantation are believed to be able to reduce the level of triglycerides, or free fatty acids, or both, in the donor liver as compared to their initial levels. In embodiments, Applicant discloses herein methods and reagents for preparing a liver for transplantation, effective to reduce liver fat in a donor liver as compared to the initial level of fat in the liver. The methods and reagents for preparing a liver for transplantation are believed to be able to reduce the level of triglycerides, or free fatty acids, or both, in the donor liver as compared to their initial levels. The methods for treating livers prior to transplantation, and for preparing a liver fortransplantation, comprise perfusing a liver with a solution (the perfusate) containing a pyrimidine cyclohexyl compound (e.g., miricorilant), or containing a pyrimidine cyclohexenyl compound, such as CORT125385. Reagents for treating livers prior to transplantation and for preparing a liver for transplantation disclosed herein contain a pyrimidine cyclohexyl compound (e.g., miricorilant), or contain a pyrimidine cyclohexenyl compound, such as CORT125385. An increase in cholesterol in the perfusate, or a reduction in cholesterol in a liver prior to transplantation may be an indication of the efficacy of the removal of triglycerides or free fatty acids from that liver.

[0006] The methods and reagents disclosed herein for treating and preparing livers prior to transplantation into a subject in need of a liver transplant may be effective to improve lactate clearance by the liver as compared to the initial level of lactate clearance in the liver. In some cases, improved lactate clearance may indicate improvement in the metabolism of the liver (e.g., by increased use of lactate as a source of energy, thereby reducing lactate in the perfusate), and may be an indication that a treated liver is suitable for use in transplantation into a subject in need of a liver transplant. Methods and reagents disclosed herein may improve lactate clearance by an isolated liver as compared to the initial level of lactate clearance in that liver.

[0007] In embodiments of the methods and reagents disclosed herein, the pyrimidine cyclohexyl compound binds to the glucocorticoid receptor (GR) and is a pyrimidine cyclohexyl glucocorticoid receptor modulator (GRM) or a mixed GR agonist / antagonist. Such pyrimidine cyclohexyl compounds are disclosed, for example, in U.S. Patent 8,685,973. In embodiments of the methods and reagents disclosed herein, the pyrimidine cyclohexenyl compound binds to the GR and is a pyrimidine cyclohexenyl GRM or a mixed GR agonist / antagonist; such pyrimidine cyclohexenyl compounds are disclosed, for example, in U.S. Patent 11,542,238.

[0008] In embodiments, the pyrimidine cyclohexyl compound, or the pyrimidine cyclohexenyl compound is present in the perfusate at initial concentrations of between about 0.1 micromolar( M) and about 100 M, or between about 0.5 M and about 20 M, or between about 1 M andabout 10 M. In embodiments, the initial concentration of the pyrimidine cyclohexyl compound or of the pyrimidine cyclohexenyl compound is about 5 M. In embodiments, the liver is placed in the perfusion machine, and is allowed to stabilize while being perfused with a perfusate solution lacking pyrimidine cyclohexyl or pyrimidine cyclohexenyl compounds. Perfusion of the liver with a perfusion solution containing a pyrimidine cyclohexyl compound or a pyrimidinecyclohexenyl compound, or both (“compound-containing perfusate”) may be initiated at any suitable time after placement of the liver in the perfusion machine. Perfusion of the liver with a compound-containing perfusate is typically begun upon metabolic stabilization of the liver in the perfusion machine. In embodiments, the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound may be included in the perfusate prior to initiating perfusion of the liver; or the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound may be added to the perfusate upon initiation, or soon after initiation, of perfusion of the liver. In embodiments, in addition to including an initial amount of the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound in the perfusate when perfusion of the liver with compound-containing perfusate is initiated (e.g., typically upon metabolic stabilization of the liver in the perfusion machine), further pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound may be added to the perfusate during perfusion of the liver. Such further pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound may be added to the perfusate once, or twice, or more times after initiation of perfusion with compound-containing perfusate. Such further addition of the pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound may be in varying amounts, and at varying time intervals (e.g., at 3 hours, or 3 hour and 6 hours, or 3, 6, and 9 hours) following initiation of perfusion with the compound-containing perfusate, or the further amounts of the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound may be continuously added to the compound- containing perfusate during perfusion of the liver.

[0009] The methods and reagents disclosed herein provide improved methods of maintaining a liver, e.g., between the time of its removal from the donor until it is transplanted into the transplant recipient. The methods and reagents disclosed herein provide improved methods of preparing a liver, for transplantation into a recipient. The methods and reagents disclosed herein provide methods and reagents for improving the usefulness of a donor liver for liver transplantation. The methods and reagents disclosed herein provide improved methods for increasing the suitability of a donor liver for use in liver transplantation. The methods and reagents disclosed herein are believed to be able to reduce triglyceride levels in a liver, where the liver would otherwise not be suitable for use in liver transplantation due to excess triglyceride levels in the liver, thereby improving the suitability of that liver for use in liver transplantation.

[0010] The novel methods and novel reagents disclosed herein include disclosure of novel uses, and novel uses of compounds in the manufacture of novel reagents for use in treating livers for transplantation. It will be understood that all such discussion and disclosure of these novel methods and novel reagents apply equally to uses of the novel compound-containing perfusates, including the use of a perfusate comprising a pyrimidine cyclohexyl compound, the use of a perfusate comprising a pyrimidine cyclohexenyl compound, and the use of a perfusate comprising both a pyrimidine cyclohexyl compound and pyrimidine cyclohexenyl compound, in the treatment of a liver for transplantation. It will be understood that all such discussion and disclosure of these novel methods and novel reagents apply equally to use of a pyrimidine cyclohexyl compound and to use of a pyrimidine cyclohexenyl compound in the manufacture of a perfusate for treating a liver for transplantation.

[0011] The methods and reagents and uses disclosed herein provide other improvements in the characteristics of livers, improving their ability to be used in liver transplantation, and increasing the number of livers available for liver transplantation by reducing the numbers of such livers that otherwise would be rejected and not used in liver transplantation. Thus, the methods and reagents disclosed herein provide benefits to patients in need of liver transplantation. The methods and reagents disclosed herein further provide benefits to medical facilities which prepare, store, and transport livers for transplantation, and provide benefits to medical practitioners who prepare livers for, and transplant livers into, patients in need of liver transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG.1A provides a schematic diagram representing an exemplary machine for normothermic machine perfusion of a liver. Pyrimidine cyclohexyl- and pyrimidine cyclohexenyl-containing perfusates as disclosed herein may be used to maintain livers prior to liver transplantation, and to prepare them for such transplantation into a recipient. Other types of machines, operating at a variety of temperatures (including, e.g., near freezing, cool, room temperature, warmed, and about body temperature) may be used for preparing and maintaining a liver according to the present methods with the present reagents.

[0013] FIG.1B provides an example of a schematic timeline for the treatment of a liver after it has been removed from the donor and prior to transplantation.

[0014] FIG.2A shows pH and CO2measures (pCO2and HCO3levels) in the perfusate perfusing donor liver LD-24, where miricorilant was added at t = 11 hours. This figure illustrates the safe treatment of a donor liver, in which the liver maintains the perfusate pH within the range of about pH 7.3 to about pH 7.5 following perfusion of the liver with the perfusate as disclosed herein.

[0015] FIG.2B shows shows hematocrit (Hct) and chemistry (levels of sodium, potassium,chloride, glucose, and lactose) of the perfusate perfusing donor liver LD-24, and in the bile produced by the liver.

[0016] FIG.2C shows improvement of lactate clearance in 2 out of 4 cases from the perfusate bathing and perfusing a donor liver when miricorilant (5 M) is included in the perfusion reagent. Insufficent lactate clearance may be an indication that a donor liver might not be suitable for transplantation. Improved lactate clearance following exposure to miricorilant indicates that miricorilant caused renewal or increase of metabolic activity in the liver. Beneficial effects were also seen for the other two livers of the four treated.

[0017] FIG.3A1 presents perfusate triglyceride, cholesterol, and free fatty acids for the donor livers LD-24 LD-26, LD29, and LD-30. The legend t=6 indicates measurements taken after 6 hours of perfusion. Triglyceride measurements were based on free glycerol. The amount of free glycerol measured in the perfusate was subtracted from the measurements to obtain the results shown in the figure. Human plasma contains on average between about 0.05 millimoles per liter (mmol / l) and about 0.11 mmol / l free glycerol; the amounts of free glycerol measured in these perfusates were consistent with this average range. (For some very hemolytic samples, no free glycerol could be measured. Thus, for the lipid layer of LD-24, 0.11 mmol / l was therefore subtracted. For LD-26 t=4h, the average free glycerol levels of t=3h and t=5h were subtracted.) Fig.3A1 shows that donor liver perfusion with a perfusion reagent containing 5 M miricorilant increased lipid levels in the perfusate in 2 of 4 livers. It is believed that the lipid source that led to such increased lipid levels in the perfusate is the donor liver. Increased lipid and lipid-soluble analyte levels in the perfusate are believed to indicate decreased lipid and lipid-soluble analyte levels in the liver itself. Lipids and lipid-soluble analytes whose levels increased included triglycerides, cholesterol, and free fatty acids.

[0018] FIG.3A2 presents liver triglyceride, cholesterol, and free fatty acids for the liver LD-30, and includes comparative graphs with data from donor livers LD-24, LD-26, LD-29, and LD-30. Triglyceride measurements were based on free glycerol.

[0019] FIG.3B shows the lipid layer that formed on the top of the miricorilant-containing perfusate during perfusion of donor liver LD-24.

[0020] FIG.3C shows the triglyceride levels measured in the perfusate over time during perfusion of donor liver LD-24 with a miricorilant-containing perfusate. A single measurement of the top layer was made at the end timepoint.

[0021] FIG.3D shows free fatty acid levels measured in the perfusate over time during perfusion of donor liver LD-24 with a miricorilant-containing perfusate. A single measurement of the top layer was made at the end timepoint.

[0022] FIG.3E shows cholesterol measured over time in the perfusate during perfusion of donor liver LD-24 with a miricorilant-containing perfusate. A single measurement of the top layer was made at the end timepoint.

[0023] FIG.4 shows increased lipid levels in the perfusate bathing both livers treated with the pyrimdine cyclohexenyl compound CORT125385 in the perfusate. The left-most graph shows triglyceride, the middle graph shows total cholesterol, and the right-most graph shows free fatty acid levels for the two livers. DETAILED DESCRIPTION INTRODUCTION

[0024] Liver disease can be a serious health threat. Patients suffering from a liver disorder such as, e.g., a fatty liver disease, hepatitis, liver failure, liver cancer, cirrhosis, or other liver disorder may be in need of a liver transplant. A liver transplant requires that a liver, or a portion of a liver, be obtained from a donor, and then maintained in a healthy state from the time it was removed from the donor until the time it is surgically placed in the recipient. (As noted above, “liver” will be understood to refer both to a liver, and to a portion of a liver.) In some cases, livers for transplantation are stored on ice, or in a cold chamber or bath for up to about 12 hours. However, in order to prevent damage that cold temperatures may cause, and to better maintain livers, donor livers may be maintained, prior to transplantation into the recipient, in a machine designed to provide the liver with temperature near body temperature, with sufficient oxygenation to maintain liver health, and other characteristics that those livers require for near-physiologicmetabolic activity. (Although livers may remain metabolically active at lower temperatures, or with low oxygenation, etc., it is better to maintain isolated livers in conditions closely approximating the conditions found in a living body than to maintain them in sub-optimal conditions.) Development and use of such machines, the use of which is termed “normothermic machine perfusion” (NMP) has accelerated efforts in providing a platform for therapeutic intervention to optimize donor livers. (“Normothermic” means normal body temperature, i.e., about 37° Celsius.) NMP protects livers from the damaging effects of traditional (prior) methods of liver preservation, and so affords a greater window of opportunity where the isolated livers can be continuously supported and assessed, and therapeutic measures applied to the livers if necessary, before transplantation.

[0025] Implementation of the normothermic machine perfusion (NMP) has accelerated efforts in providing a platform for therapeutic intervention to optimize donor livers. Higher-risk livers are protected by NMP from the damaging effects of traditional preservation (e.g., cold storage). NMP also provides a greater window of opportunity where the isolated liver can be continuously supported and assessed before transplantation.

[0026] Many livers are found to be “marginal” upon harvest from the liver donor; that is, their condition is such that they may not be suitable for use in liver transplantation surgery. Determination that a liver is marginal and not suitable for use in transplantation depends on multiple factors. For example, livers that have high levels of triglycerides, or high levels of cholesterol, or high levels of fatty acids, where high levels are greater than about 5% – 10% of liver weight, may be considered marginal in quality and may be rejected for use in liver transplant surgery. For further example, a liver having more than 5% fat is considered a fatty liver; in combination with other factors, a total liver percentage of 15%, or 20%, or 25% may lead to rejection of an isolated liver for use in transplantation. Livers with much more fat content than that (e.g., severely fatty livers with fat contents of about 30%, 40%, or greater) may be rejected for use in transplantation on the basis of fat content alone. Steatotic livers, defined as having intrahepatic triglyceride levels of at least 5% to 10% of liver weight, or as having 5% or more hepatocytes that contain lipid vacuoles, are typically not considered suitable for transplantation.

[0027] For example, a liver having more than 5% hepatocytes that contain lipid vacuoles is considered “steatotic”; a steatotic liver with many more than 5% hepatocytes that contain lipidvacuoles (e.g., severely steatotic livers in which the percentage of hepatocytes that contain lipid vacuoles is about 40% or more) may be rejected for use in transplantation on the basis of steatosis alone. Perfusing such marginal livers with reagents (perfusates) as disclosed herein would increase triglyceride and other lipid levels in the perfusate and it is believed would decrease the levels of triglycerides and / or other lipids in the liver. It is believed that such perfusion would improve the condition of such marginal livers so that they would be suitable for use in liver transplantation surgery, and thus to increase the number of livers suitable for use in liver transplantation that are available to treat patients in need of liver transplants. Such increased numbers of suitable livers could be used to treat patients able to benefit from liver transplantation surgery but who might otherwise not have had a liver available to them for transplantation.

[0028] Donor livers are rare and are a scarce yet critical resource in the treatment of patients with advanced liver disease. Such patients typically have few other treatment options for their potentially fatal conditions. Use of the reagents and methods disclosed herein may allow the use of livers that otherwise would not be suitable for transplantation, and so would be discarded and the potential recipient denied the opportunity for transplantation surgery, or the transplant surgery for that potential recipient delayed until a different donor liver would be obtained. However, due to the precarious health of candidates for liver transplantation surgery, delay of such surgery could have dire consequences, and might preclude any surgery if the patient’s health continues to decline. Thus, the present methods and reagents are believed to be useful by increasing the donor pool to meet the needs of patients suffering from liver disease and in need of a liver transplant.

[0029] In embodiments, Applicant discloses herein methods and reagents for preparing a liver for use in a liver transplant. Applicant discloses herein methods and reagents for use in improving the likelihood that a donor liver will be suitable for transplantation into a patient in need of a liver transplant, as compared to the likelihood that the liver would be suitable for transplantation at the time of its removal from the donor. In embodiments, Applicant discloses herein methods and reagents for improving the condition of a liver for use in a liver transplant, as compared to the condition of that liver at the time of its placement in a machine or container for storage (or maintenance) prior to transplantation. In embodiments, Applicant discloses herein methods and reagents for improving the length of time a liver may be maintained prior to its use in a liver transplant, as compared to the length of time that liver could be maintained, without useof the present methods and reagents, in a machine or container for storage (or maintenance) prior to transplantation. In embodiments, Applicant discloses herein methods and reagents for reducing liver fat levels in a liver for use in a liver transplant, as compared to the initial fat levels in the liver at the time of placement of that liver in a machine or container for storage (or maintenance) prior to transplantation. In embodiments of the use of the methods and reagents disclosed herein, lactate clearance may be improved in livers for transplantation, as compared to the initial level of lactate clearance in the liver (see, e.g., Fig.2C).

[0030] Applicant provides improved methods and reagents for use in maintaining livers prior to transplantation, and for use in NMP. The novel reagents disclosed herein include solutions containing a pyrimidine cyclohexyl compound, such as, e.g., miricorilant, or a pyrimidine cyclohexenyl compound, such as, e.g., CORT125385. All reagents disclosed herein are compatible with, and supportive of, the maintenance of livers ex vivo for many hours. In addition to a pyrimidine cyclohexyl or a pyrimidine cyclohexenyl compound, the novel reagents disclosed herein may include other components, such as, e.g., salts, buffers, and red blood cells. The novel methods disclosed herein include perfusing a liver with such a reagent.

[0031] . Applicant discloses herein methods for decreasing levels of liver fat in a liver prior to transplantation of said liver into a recipient, said liver having an initial level of liver fat, the method comprising: Perfusing said liver with a solution comprising a pyrimidine cyclohexyl or a pyrimidine cyclohexenyl compound for a period of time, Whereby the level of liver fat in the liver is decreased as compared to said initial level of liver fat. Liver fat may be, or may include, triglycerides, free fatty acids, and other lipids and lipid-soluble compounds. The liver may be perfused for as long as needed; e.g., for up to a day, or up to two days, or up to a week. Thus, the period of time may be any suitable time, including periods of time of between about one hour and about twelve hours, or about a day, or about two days, or longer. In embodiments of the methods and uses disclosed herein, further pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound may be added to the perfusate after initiation of perfusion with compound-containing perfusate. Such further addition of the pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound to the perfusion solution may be in varying amounts, and at varying time intervals (e.g., at 3 hours, or 3 hour and6 hours, or 3, 6, and 9 hours). In embodiments, such further addition of pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound may be at 2 hour intervals, or 4 hour intervals, or other intervals (such other intervals may be unequal intervals, e.g., addition at 1 hour, at 3 hours, at 6 hours, and at 10 hours after initiation of perfusion). In embodiments, the further amounts of the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound may be continuously added to the perfusate during perfusion of the liver.

[0032] Applicant discloses herein reagents for use in maintaining a liver after removal of that a liver from a liver donor. In embodiments, these reagents may be used to decrease liver fat levels in a liver after removal of that liver from the liver donor, as compared to the fat levels in the liver prior to placement of the liver in a container or machine for maintenance of a liver prior to transplantation. In embodiments, the reagent comprises a pyrimidine cyclohexyl compound such as, e.g., miricorilant, or a pyrimidine cyclohexenyl compound, such as, e.g., CORT125385. Liver fat may be, or may include, triglycerides, free fatty acids, and other lipids and lipid-soluble compounds.

[0033] In embodiments, the reagent includes between about 0.1 micromolar ( M) and about 100M, or between about 0.5 M and about 20 M of the pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound, or between about 1 M and about 10 M of the pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound. In embodiments, the reagent includes about 5 M of the pyrimidine cyclohexyl compound or pyrimidine cyclohexenyl compound.

[0034] In embodiments of the methods and reagents disclosed herein, pyrimidine cyclohexyl and pyrimidine cyclohexenyl compounds suitable for the methods and uses disclosed herein bind to GR, and modulate GR as GRMs or mixed GR agonist / antagonists. Pyrimidine cyclohexyl compounds suitable for the methods and uses disclosed herein are disclosed in U.S. Patent 8,685,973. In embodiments, the pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4- Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has thestructure (Example 6, compound 3b of U.S. Patent 8,685,973, hereby incorporated by reference in its entirety).

[0035] Pyrimidine cyclohexenyl compounds suitable for the methods and uses disclosed herein are disclosed in U.S. Patent 11,542,238. In embodiments, the pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'- biphenyl]-4-yl)pyrimidine-2,4(1H,3H)-dione, which is disclosed as Example 10 of U.S. Patent 11,542,238, and which has the structure. CORT125385 has two enantiomers: (R)-5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, which has the structure5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-yl)pyrimidine- 2,4(1H,3H)-dione, which has theenantiomers may be used in the practice of the methods and uses disclosed herein. U.S. Patent 11,542,238 is hereby incorporated by reference in its entirety.

[0036] Perfusion of the liver with a compound-containing perfusate may be initiated at any suitable time; perfusion with a compound-containing perfusate is typically begun after metabolic stabilization of the liver in the perfusion machine. In embodiments of the methods and reagents disclosed herein, the reagent is used to perfuse a liver, and the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound in the compound-containing perfusate is initially present at concentrations of between about 0.1 M and about 100 M, or between about 0.5 M and about 20 M, or between about 1 M and about 10 M. (As used herein, the term “perfusate” refers to a reagent as described herein when used to perfuse a liver.) In embodiments of the reagents disclosed herein, the initial concentration of the pyrimidine cyclohexyl compound or the pyrimidine cyclohexenyl compound is about 5 M. That is, such compound levels are the initial levels of the pyrimidine cyclohexyl or the pyrimidine cyclohexenyl compound in the reagent; it will be understood that the level of the pyrimidine cyclohexyl compound (e.g., miricorilant) or the pyrimidine cyclohexenyl compound (e.g., CORT125385) may be reduced over time during use (e.g., during perfusion of a liver).

[0037] In embodiments, in order to maintain the pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound levels over time during perfusion of a liver with a compound-containing perfusate, further pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound may be added to the perfusate during perfusion of the liver with a compound-containing perfusate. Such addition may be in varying amounts at varying intervals following initiation of perfusion with a compound-containing perfusate, or the further amounts of the pyrimidine cyclohexyl or the pyrimidine cyclohexenyl compound may be continuously added to the perfusate during perfusion of the liver with compound-containing perfusate. Such further addition of the pyrimidine cyclohexyl or the pyrimidine cyclohexenyl compound may be effective to replace pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound lost or metabolized during perfusion, and may be effective to maintain the concentration of the pyrimidine cyclohexyl or the pyrimidine cyclohexenyl compound in the compound-containing perfusate during perfusion of the liver. Such maintained concentration of the pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound may be, e.g., between about 1 M to about 10 M, and may be about 5 M.

[0038] In embodiments of the reagents disclosed herein, the reagent comprises a pyrimidine cyclohexyl compound, a pyrimidine cyclohexenyl compound, or both, and red blood cells. In embodiments, the reagents as disclosed herein may include leukocytes, may include thrombocytes, and may include both leukocytes (e.g., up to about 1x 106 / L) and thrombocytes (e.g., up to about 10x109 / L). In addition, other compounds and medications may be included in the reagents.

[0039] The perfusing step in the methods disclosed herein may include perfusion of the liver with a compound-containing perfusate for a period of time, where the period of time may be between about 15 minutes to about 12 hours, inclusive, and may continue for longer periods of time. In embodiments of the methods, the perfusing step continues for at least three hours. In embodiments of the methods, the perfusing step continues for at least six hours. In embodiments of the methods, the perfusing step continues for at least nine hours. In embodiments of the methods, the perfusing step continues for at least 12 hours. Thus, the period of time may be about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, or more.

[0040] Perfusion may comprise perfusion of the liver with a compound-containing perfusate at a perfusion rate of between about 0.5 liter per minute (L / min) to about 5 L / min, inclusive. In embodiments, the perfusion rate is selected from 1 L / min, 2 L / min, 3 L / min, 4 L / min, and 5 L / min.

[0041] Liver fat may comprise many components, including triglycerides, free fatty acids, and lipid soluble compounds such as cholesterol. In embodiments of the methods disclosed herein, the amount of liver fat is decreased by perfusion with a reagent disclosed herein by at least about 5% (where % indicates weight percent) as compared to said initial amount of fat in the liver (at the time of placement in a machine or compartment for storage and maintenance prior to transplantation). Such a 5% or greater decrease may be effected after about 3 hours of perfusion, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In embodiments of the methods disclosed herein, the amount of liver fat is decreased by at least about 10%, or by at least about 15% as compared to said initial amount of liver fat, afterabout 3 hours of perfusion, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In further embodiments of the methods disclosed herein, the amount of liver fat is decreased by at least about 20%, or by at least about 30%, as compared to said initial amount of liver fat, after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver. Such reductions in liver fat may be reductions in triglyceride levels, where liver triglyceride levels are decreased by at least about 10%, or by at least about 15%, or by at least about 20%, or by at least about 30%, after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver. Such reductions in liver fat may be reductions in free fatty acid levels, where liver free fatty acid levels are decreased by at least about 10%, or by at least about 15%, or by at least about 20%, or by at least about 30%, after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver. Reductions in cholesterol levels, where liver cholesterol levels are decreased by at least about 10%, or by at least about 15%, or by at least about 20%, or by at least about 30%, after at least about 3 hours of perfusion of the liver may provide an indirect measure of the efficacy of the perfusion in removing liver fat.

[0042] An indication of decreased liver fat, and of decreased liver triglyceride, free fatty acid, and cholesterol levels, is an increase in the level of fat, or of triglyceride, or of free fatty acid, or of cholesterol, in the perfusate which is perfusing the liver. Such levels increase over time during perfusion of a liver with reagents as disclosed herein. Changes of these levels in the perfusate over time during perfusion of a liver with a reagent disclosed herein provide indications of changes over time of liver fat, triglyceride, free fatty acid, and cholesterol levels in the liver, and may be used to track such changes, and to determine when, and if, a particular liver is suitable for use in transplantation.

[0043] In embodiments of the methods disclosed herein, the amount of triglyceride in the perfusate is increased by at least about 10% (where % indicates weight percent) as compared to said initial amount of triglyceride in the perfusate after about 3 hours of perfusion, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In embodiments of the methods disclosed herein, the amount of triglyceride in the perfusate is increased by at least about 15% as compared to said initial amount of triglyceride in the perfusate after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver. In furtherembodiments of the methods disclosed herein, the amount of triglyceride in the perfusate is increased by at least about 20%, or by at least about 30%, as compared to said initial amount of triglyceride in the perfusate after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver.

[0044] In embodiments of the methods disclosed herein, the amounts of fatty acids in the perfusate are increased by at least about 10% (where % indicates weight percent) as compared to said initial amounts of fatty acids in the perfusate after about 3 hours of perfusion, or after about 6 hours , or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In embodiments of the methods disclosed herein, the amounts of fatty acids in the perfusate are increased by at least about 15% as compared to said initial amounts of fatty acids in the perfusate after about 3 hours of perfusion, or after about 6 hours of perfusion of the liver. In further embodiments of the methods disclosed herein, the amounts of fatty acids in the perfusate are increased by at least about 20%, or by at least about 30%, as compared to said initial amounts of fatty acids in the perfusate after about 3 hours of perfusion, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver.

[0045] In embodiments of the methods disclosed herein, where the amount of triglyceride in the perfusate is undetectable or negligible, detectable amounts of triglyceride are observed in the perfusate after about one hour, or after about 2 hours, or after about 3 hours, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In embodiments of the methods disclosed herein, where the amounts of fatty acids in the perfusate are undetectable or negligible, detectable amounts of fatty acids are observed in the perfusate after about one hour, or after about 2 hours, or after about 3 hours, or after about 6 hours, or after about 8 hours, or after about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver. In embodiments of the methods disclosed herein, where the amount of cholesterol in the perfusate is undetectable or negligible, detectable amounts of cholesterol are observed in the perfusate after about one hour, or after about 2 hours, or after about 3 hours, or after about 6 hours, or after about 8 hours, orafter about 10 hours, or after about 12 hours, or after about 15 hours, or after about 18 hours, or after about 24 hours, or more, of perfusion of the liver.

[0046] In embodiments of the reagents disclosed herein, and of the methods using said reagents, the salt composition, pH, and osmolarity of the perfusate are all similar to the corresponding salt composition, pH, and osmolarity of normal human blood, and are all compatible with the physiological maintenance of mammalian tissue. For example, reagents disclosed herein may comprise between about 135 and about 150 millimolar (mM) sodium; between about 3 and about 6 mM potassium; between about 120 and about 160 mM chloride, or other halide; between about 10 and about 50 mM buffer, such as a carbonate, or phosphate, or other buffer (e.g., 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS) or other physiologically acceptable buffer); and may include an antioxidant such as, e.g., butylated hydroxytoluene (BHT), buylated hydroxyanisole (BHA), tert-butyl hydroquinone (TBHQ), a tocopherol, or other physiologically acceptable antioxidant. Such perfusates as described herein, both infra and supra, without added pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound, are standard perfusates.

[0047] In embodiments of the reagents disclosed herein, and of the methods using said reagents, in addition to the pyrimidine cyclohexyl or the pyrimidine cyclohexenyl compound, perfusates for use in perfusing livers may include glucose, for example, may include between about 2 mM to about 50 mM glucose, or about 3 mM to about 8 mM glucose, or between about 4 mM to about 6 mM glucose. For example, a perfusate may include a volume extender such as Gelofusine®(a succinylated gelatin solution 4% for intravenous infusion, available from, e.g., B. Braun Medical, St-Gallen, Switzerland) or Haemaccel®(a gelatin derivative, available from Piramal Enterprises Ltd, Mumbai, India). In embodiments of the reagents disclosed herein, the reagent may contain Gelofusine®, red blood cells, human serum, human serum albumin, heparin, and calcium gluconate. In embodiments, other physiologically acceptable ingredients may also be included. An exemplary perfusate is described in the following Table: TABLE 1 PerfusateSAGM is a solution containing saline, adenine, glucose and mannitol used to resuspend red blood cells (RBCs).

[0048] During perfusion, an infusion solution containing one or more of an anticoagulant such as heparin, a vasodilator such as epoprostenol, and an amino acid or amino acid-containing mixture (such as, e.g., aminoplasmal 10% solution containing multiple amino acids, acetate, and citrate (available from B. Braun)) may be infused in (added to) the perfusate and / or into the liver during perfusion. For example, an infusion solution may be infused into the perfusate at a rate of between about 0.1 milliliter per hour (0.1 ml / hour) and about 20 ml / hour, or at a rate of between about 0.5 ml / hour and about 10 ml / hour. An exemplary infusion solution may contain sodium taurocholate (e.g., 5.6 grams (g) dissolved in 27 ml NaCl, to a final volume 30 ml); may contain Flolan®(available from GlaxoSmithKline, Durham, North Carolina, USA, at a level of, e.g., 0.25 mg in 30 ml); may contain heparin (e.g., 25,000 units); may contain aminoplasmal with 10% glucose (e.g., 375 ml aminoplasmal mixed with 125 ml of 40% glucose; these components may be combined to provide 500 ml aminoplasmal with 10% glucose) for infusion at an infusion rate of about 1 ml / hour into a perfusate bathing a liver.

[0049] In use during perfusion of a liver, the perfusate will typically be provided with, e.g., nitrogen, oxygen, carbon dioxide, and / or other gases, where provision of such gas or gases may be effected by bubbling gas through the liquid perfusate, by using a membrane oxygenator, or by otherwise contacting the liquid perfusate with the gas or gasses. In embodiments of the reagents disclosed herein, and of the methods using said reagents, reagents disclosed herein may have a pH of between about pH 7.0 to about pH 7.8, preferably between about pH 7.3 to about pH 7.5, or between about pH 7.35 to about pH 7.45 when in use in perfusing a liver (where about, as used here, indicates ± 0.1 pH). Typically, the perfusate pH is initially set at pH 7.35 to about 7.45, as a healthy liver should be able to restore the perfusate pH by itself to about pH 7.4.

[0050] In use during perfusion of a liver, the perfusate (and liver perfused by the perfusate) will typically be maintained at a physiological temperature of about 35° C to about 38° C, or preferably about 36° C to about 37° C.B. DEFINITIONS

[0051] The terms “a,” “an," or “a(n)”, when used in reference to a group of substituents or "substituent group" herein, mean at least one. For example, where a compound is substituted with "an" alkyl or aryl, the compound is optionally substituted with at least one alkyl and / or at least one aryl, wherein each alkyl and / or aryl is optionally different. In another example, where a compound is substituted with "a" substituent group, the compound is substituted with at least one substituent group, wherein each substituent group is optionally different.

[0052] As used herein, the term “soon after” refers to a time within fifteen minutes, or within half an hour, or within an hour of the referred-to event. For example, soon after initiation of perfusion refers to a time period of about fifteen minutes, or about half an hour, or about an hour, after perfusion of an isolated liver is begun.

[0053] As used herein, the term “about”, e.g., as used in the phrase “about X” where X is a numerical value, is used to indicate a range of ±10% of the named value of X unless otherwise specified.

[0054] As used herein, the term “patient” refers to a human that is or will be receiving, or has received, medical care for a disease or condition.

[0055] As used herein, the term “liver” refers to a whole liver, and also includes reference to a portion of a liver. References to a liver and to a portion of a liver include reference to isolated livers and to isolated liver portions, e.g., to a liver, or a portion thereof, that has been removed from a donor in preparation for its transplantation to a recipient. Livers for transplantation, and portions of such livers, must be isolated (i.e., removed from the donor) prior to use in a liver transplantation procedure.

[0056] As used herein, the terms “donor” and “liver donor” refer to a mammal, typically a human, that is or will be having their liver, or a portion thereof, removed for use in liver transplantation.

[0057] As used herein, the terms “recipient” and “liver recipient” refers to a mammal, typically a human, that is or will be receiving, or has received, a liver via liver transplantation.

[0058] Fatty liver disease (FLD, also known as hepatosteatosis) is a prevalent liver condition that occurs when lipids accumulate in liver cells, and may be caused by, at least in part, abnormal hepatic lipid deposits. FLD includes, e.g., alcoholic fatty liver disease, and nonalcoholic fattyliver disease. Fatty liver disease may be, e.g., macrovesicular steatosis or microvesicular steatosis.

[0059] The lipid accumulation in FLD causes cellular injury and makes the liver susceptible to further injuries. FLD is characterized by the build-up of excessive fat (lipids) in liver cells, generally caused by abnormal retention of lipids by the liver cells (i.e., steatosis). In addition to fat, proteins and water are retained in the hepatocytes, which can lead to a ballooning of hepatocytes. The accumulation of fat in the liver may be attributed to a perturbation of one of the following steps in the lipid metabolism of hepatocytes and adipocytes: (1) increased free fatty acid delivery to the liver; (2) increased free fatty acid synthesis within the liver; (3) decreased beta-oxidation of fatty acids; and (4) decreased very low-density lipoprotein synthesis or secretion. (Bacon et al., Gastroenterology, 1994, 107:1103-1109). Other fatty liver diseases can develop in a patient with other types of liver diseases, such as but not limited to, chronic viral hepatitis C (HCV), chronic viral hepatitis B (HBV), chronic autoimmune hepatitis (AIH), diabetes and Wilson’s disease.

[0060] As used herein, the term “steatosis” refers to a condition characterized by fat build-up in liver cells, leading to excess fat in the liver (abnormally high levels of liver fat, as compared to normal levels of liver fat). A liver having at least 5-10% weight to weight (w / w; weight of fat deposits as % of total weight of the liver) fatty deposits may be considered to be steatotic ( See, e.g., Clark et al., J. Am. Med. Assoc., 2003, 289:3000-3004 and Adams et al., Can. Med. Assoc. J., 2005, 172:899-905). A liver with fatty deposits comprising up to 25% (w / w) may be considered mildly steatotic, and a liver with fatty deposits comprising greater than 25% (w / w) may be considered severely steatotic.

[0061] Liver fat is typically less than about 5% of liver tissue (where % may be determined by volume, by histology (e.g., % hepatocytes exhibiting macrovesicular steatosis) by MRI-PDFF techniques, by weight (e.g., % of triglyceride of wet liver weight), or by other accepted methods). Components of liver fat include, e.g., triglycerides, and fatty acids. Livers may also contain other lipid soluble components such as cholesterol. Amounts of liver fat greater than about 5% are typically considered pathological.

[0062] As used herein, the term “reagent” refers to a composition for use in one or more of preparing a liver for transplantation into a recipient; maintaining a liver prior to transplantation into a recipient; reducing triglyceride levels, or fatty acid levels, or both in a liver for livertransplantation. Use of a reagent in the perfusion of an isolated liver may result in improving lactate clearance in that liver for liver transplantation; and other uses related to liver transplantation.

[0063] As used herein, the terms “perfusate” and “perfusion solution” refer to a solution for perfusing a liver. A reagent containing a pyrimidine cyclohexyl compound as disclosed herein is a perfusate, and is a perfusion solution, when used to perfuse a liver. A liver may be perfused a) when placed in a perfusate (e.g., a reagent containing miricorilant), b) when a perfusate is pumped into a vein or artery of the liver, or both a) and b). For example, perfusate may be introduced into a liver via the hepatic artery or portal vein (or both) and may exit the liver via the hepatic veins (also termed portal veins, which connect with the inferior vena cava). Perfusate exiting the liver may be recycled back into the liver again, or, in some cases, may be discarded and fresh perfusate introduced into the liver. In embodiments, some of the perfusate exiting the liver may be discarded and some recycled back into the liver again, while some fresh perfusate is also introduced into the liver as needed.

[0064] As used herein, “gelofusine” refers to Gelofusine®and related plasma substitutes and plasma volume expanders. The commercial product gelofusine®is a water-based 4% (w / v) succinylated gelatin solution containing sodium chloride (where w / v means weight per volume); 1 L of gelofusine contains: 40 grams (g) of succinylated gelatin, 154 mmol / L sodium, 120 mmol / L chloride, water for injection, and sodium hydroxide for pH adjustment.

[0065] As used herein, the term “glucocorticoid receptor” (“GR”) refers to the type II GR, an intracellular receptor which specifically binds to cortisol and / or cortisol analogs such as dexamethasone (See, e.g., Turner & Muller, J. Mol. Endocrinol. October 1, 200535283-292). The glucocorticoid receptor is also referred to as the cortisol receptor. The term includes isoforms of GR, recombinant GR and mutated GR.

[0066] The term “glucocorticoid receptor modulator” (GRM) refers to a compound which modulates GC binding to GR, or which modulates any biological response associated with the binding of GR to an agonist. For example, a GRM that acts as an agonist, such as dexamethasone, increases the activity of tyrosine aminotransferase (TAT) in HepG2 cells (a human liver hepatocellular carcinoma cell line; ECACC, UK). A GRM that acts as an antagonist, such as mifepristone, decreases the activity of tyrosine aminotransferase (TAT) in HepG2 cells. TAT activity can be measured as outlined in the literature by A. Ali et al., J. Med. Chem., 2004,47, 2441-2452. GRMs can combine agonist and antagonist characteristics, based on different readouts for GR activity.

[0067] As used herein, the term "compound" is used to denote a molecular moiety of unique, identifiable chemical structure. A molecular moiety ("compound") may exist in a free species form, in which it is not associated with other molecules. A compound may also exist as part of a larger aggregate, in which it is associated with other molecule(s), but nevertheless retains its chemical identity. A solvate, in which the molecular moiety of defined chemical structure ("compound") is associated with a molecule(s) of a solvent, is an example of such an associated form. A hydrate is a solvate in which the associated solvent is water. The recitation of a "compound" refers to the molecular moiety itself (of the recited structure), regardless of whether it exists in a free form or an associated form.

[0068] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients such as the said compounds, their tautomeric forms, their derivatives, their analogues, their stereoisomers, their polymorphs, their deuterated species, their pharmaceutically acceptable salts, esters, ethers, metabolites, mixtures of isomers, their pharmaceutically acceptable solvates and pharmaceutically acceptable compositions in specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient (s), and the inert ingredient (s) that make up the carrier, as well as any product which results, directly or indirectly, in combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

[0069] As used herein, the term “pyrimidine cyclohexyl compound” refers to a compound, and salts thereof, as described and disclosed in U.S. Patent 8,685,973, the entire contents of which patent is hereby incorporated by reference in its entirety.

[0070] Exemplary pyrimidine cyclohexyl compounds for use in the methods and reagents disclosed herein include those described in U.S. Patent No.8,685,973. In embodiments, the pyrimidine cyclohexyl compound is the compound (E)-6-(4-Phenylcyclohexyl)-5-(3- trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione (“miricorilant”, also known as “CORT118335”), which has the structure:(see Example 6, compound 3b of U.S. Patent 8,685,973.

[0071] In embodiments of the methods and reagents disclosed herein, the pyrimidine cyclohexyl compound is selected from the group consisting of:.

[0072] In embodiments of the methods and reagents disclosed herein, the pyrimidine cyclohexenyl compound suitable for use in the methods and uses disclosed herein is selected from the group consisting of the compounds disclosed in US 11,542,238, hereby incorporated by reference in its entirety. For example, the pyrimidine cyclohexenyl compound may be selected from the group consisting of the compounds claimed in any of the claims of US 11,542,238. The pyrimidine cyclohexenyl compound suitable for use in the methods and uses disclosed herein may be, for example, a compound claimed in US 11,542,238. Pyrimidine cyclohexenylcompounds suitable for the methods and uses disclosed herein include, for example, the following compounds:.

[0073] In embodiments, the pyrimidine cyclohexenyl compound is the compound CORT125385 which is claimed in claim 24 of US11,542,238, having the chemical name 5-benzyl-6-(4'-chloro- 2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-yl)pyrimidine-2,4(1H,3H)-dione, and having the structure(the racemic mixture), and its enantiomers claimed in claims 31 and 32 of that patent:.

[0074] “Salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of pharmaceutically-acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that the pharmaceutically- acceptable salts are non-toxic. Additional information on suitable pharmaceutically-acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0075] The term “metabolic stabilization”, e.g., as referring to a liver placed in a perfusion machine, refers to the adjustment of the liver to the conditions in the perfusion machine, while being perfused with a standard perfusate, in which, after a period of time, the conditions of the liver become reasonably constant as indicated by analytes or metabolites measured in the perfusate.

[0076] The term “clinical phase” refers to the initial time period during which an isolated liver or liver portion, placed in a perfusion machine, is considered to be suitable for use for transplantation into a recipient in need of a liver transplant (see Fig. 1B). An isolated liver or liver portion typically achieves metabolic stabilization in the clinical phase during perfusion; although the examples discussed in the present application are from livers that were declined for use in transplantation, perfusion with compound-containing perfusate may begin during the clinical phase, and livers treated with compound-containing perfusate may be used for transplantation. It is believed that perfusion of an isolated liver or liver portion with compound- containing perfusate may prolong the clinical phase, allowing maintenance of livers or liverportions for greater times than would be possible with perfusion using a perfusate lacking pyrimidine cyclohexyl or pyrimidine cyclohexenyl compounds. EXAMPLES

[0077] The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters which could be changed or modified to yield essentially similar results. EXAMPLE 1. PERFUSION OF A HUMAN LIVER FOR TRANSPLANTATION WITH MIRICORILANT Miricorilant-treated Human Donor Liver (Normothermic Machine Perfusion)

[0078] The following is a description of the preparation and maintenance of a liver for later use in a liver transplantation surgical procedure in which the pyrimidine cyclohexyl compound miricorilant was included in the perfusate. The liver was placed on the perfusion device, perfused with a standard perfusate, and was allowed to stabilize. Upon metabolic stabilization of the liver on the perfusion device, perfusion with a perfusion solution containing miricorilant was initiated. Similar procedures were followed when the pyrimidine cyclohexenyl compound CORT125385 was included in the perfusate (as described in Example 2 below). These procedures were followed for the livers discussed in this example.

[0079] Following removal of the liver from the donor, in some cases a liver may be placed on ice or in a cooled container for transport. In other cases, the liver may be placed in a reservoir (e.g., a temperature-controlled bath) containing a perfusate containing a pyrimidine cyclohexyl, or placed in a temperature controlled container or on a temperature controlled surface for supporting a liver and the liver is then perfused with a perfusate containing a pyrimidine cyclohexyl compound such as miricorilant. Machines used for normothermic machine perfusion include, for example, the Organox Metra (OrganOx Ltd., United Kingdom. In either case, the liver is eventually placed in a machine or container and perfused with a reagent as disclosed herein. The liver or portion thereof may be perfused with the perfusate via a vein or artery as well (e.g., tubing containing the perfusate may be connected to the hepatic artery and portal vein for fluid flow into the liver, with perfusate exiting via the hepatic veins (also termed portal veins, which connect with the inferior vena cava). The perfusate includes red blood cells; preferred perfusates do not include, or include only a minimal amount of, white blood cells (WBCs).

[0080] An isolated liver may contain about 0.5 liter (L) to about 1 L of fluid. In perfusing the livers discussed in this example, in addition to the fluid from the isolated liver, about 1 L of perfusate was pumped through the system at a rate of 1.4 liters per minute (L / min). Fig.1A provides a schematic illustration of a system for perfusing a liver according to the methods disclosed herein. The perfusate enters the reservoir from above and leaves at the bottom, and so is in constant movement. For a system such as the exemplary one shown in Fig.1, a tube (that has the blood parameter sensor and the sampling port) connects to tubing that goes from the oxygenator directly to the reservoir. Perfusate enters the liver from the reservoir (e.g., via “arterial tubing” connected to the hepatic artery and portal vein). Since both the reservoir and the arterial tubing draw perfusate from the oxygenator, for all intents and purposes the perfusate is the same in both reservoir and in the arterial tubing. Perfusate is drawn from the reservoir tubing instead of the arterial tubing; this provides protection against possible introduction of air bubbles into the liver, since such bubbles (if any) would go into the air-containing reservoir and not the liver, and so do no harm.

[0081] In this example, the pyrimidine cyclohexyl compound miricorilant was added to the perfusate upon metabolic stabilization of the liver on the perfusion device. (In the following example, the pyrimidine cyclohexenyl compound CORT125385 was added to the of perfusate upon metabolic stabilization of the liver and then every 3 hours thereafter). As disclosed herein, perfusion of livers with perfusates disclosed herein (compound-containing perfusates containing a pyrimidine cyclohexyl compound, a pyrimidine cyclohexenyl compound, or both) may develop a lipid layer on the top of the perfusate in the bath (typically appearing as a foamy layer on the surface of the perfusate). It is believed that such a lipid layer develops best if the flow of the perfusate is stable. It is further believed that such a lipid layer is indicative of increased lipid or lipid soluble components (e.g., triglyceride, cholesterol, and / or fatty acids) in the perfusate, and that such a lipid layer is indicative of decreased amounts of lipids and lipid soluble compounds in the liver.

[0082] The perfusate consisted of Gelofusine®as the perfusate base with packed red blood cells (RBCs; the packed RBCs are similar to the regular RBCs used for transfusions). As noted above, perfusates may also include human serum human serum albumin, and other physiologically acceptable components. Standard RBCs were used in the perfusate; such RBCs contain less than 106 / unit leukocytes (where a unit is about 280 mL) and less than 1010 / L thrombocytes. The RBCsmay be matched packed cells, and need not be obtained from the liver donor. In addition to RBCs, or in place of RBCs, perfluorocarbon synthetic artificial blood formulations may be included in the perfusate to provide or enhance oxygenation.

[0083] At the start of the perfusion of the liver, a bolus of heparin, bicarbonate, calcium gluconate and cefazoline is provided. In the perfusions disclosed herein, the perfusate included the following: 0.5 L + liver volume Gelofusine®, 2.5 RBC bags, 10k U heparin, 10% Calcium gluconate, and 5 M Miricorilant. During perfusion of the liver, following the addition of that bolus, the perfusate included heparin, bile salts such as sodium taurocholate, epoprostanol, insulin and Aminoplasmal with 10% glucose. Such perfusates as described herein, without added pyrimidine cyclohexyl or pyrimidine cyclohexenyl compound, are standard perfusates.

[0084] During perfusion of the liver, perfusion data were measured ‘live’ by the machine on the system as it ran. The blood gas and blood chemistry measurements were done on perfusate drawn directly from the system immediately prior to the measurement with a regular syringe and were not treated before injection into a chemical analysis device, or, alternatively, before they were analyzed by the machine perfusing the liver. In addition to the analyses for lipids and lipid soluble analytes, liver enzymes (e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST)), and other analytes were measured. The perfusate lipid data were derived from the perfusate drawn directly from the system with a regular syringe, centrifuged and the supernatant was snap frozen (samples not centrifuged immediately were kept on ice until this could be done).

[0085] During perfusion of the liver, perfusion data measured included: liver enzyme levels (including alanine aminotransferase (ALT), aspartate aminotransfersase (AST), alkaline phosphatase (AP), and / or glutamyl transpeptidase (GGT)). Other liver proteins (including haptoglobin, total bilirubin, alpha-2-microglobulin, resistin, cleaved or intact cytokeratin-18) were measured in addition to serum glucose and insulin resistance parameters. Since the level of ALT activity is frequently increased in NASH patients (Angulo and Lindor, Best Pract Res Clin Gastroenterol, 2002, 16(5):797-810), this criterion was considered a surrogate marker for assessing liver injury.

[0086] Livers were obtained from livers donated for transplant that were subsequently deemed unsuitable for use in transplantation into a liver transplant recipient. As indicated in Fig.1B, livers for transplantation were placed in a perfusate lacking pyrimidine cyclohexyls, and maintained for several hours, until such time as that liver was deemed unsuitable fortransplantation (e.g., by rising lactate levels rendering the liver unsuitable), at which time the perfusate was changed to one including the pyrimidine cyclohexyl compound miricorilant at a concentration of 5 M. Samples of the perfusate and from the liver itself were obtained at this time. The livers were treated with miricorilant-containing perfusate, and samples of the perfusate and liver were obtained at multiple timepoints during perfusion. Note that the “clinical phase” illustrated in Fig.1B is optional. For example, if a liver has been declined for use in transplantation based on, for example, CT imagery or some other reason, then it may not experience a clinical phase; in such a case, the perfusion of the liver will start at time T=0.

[0087] Characteristics of the donors from whom these livers were obtained are presented in the following Table: TABLE 2

[0088] As illustrated in Fig. 2A, blood pH rose, bile pH fell, while blood and bile gas (CO2and bicarbonate levels) measurements remained fairly constant over time during ten hours of perfusion of a liver (where “blood” indicates the perfusate). These measurements are of interest since the bile pH, lactate and glucose levels provide indications regarding the quality of the bile,and thus the functioning of the liver. While the titles refer to gases, these graphs include measures of the partial pressure of CO2 gas (pCO2 in mmHg) and also include measurements of the compound bicarbonate in solution in the perfusate / bile (HCO3-).

[0089] As illustrated in Fig. 2B, in the donor liver LD-24, blood hematocrit (%) fell slightly, while bile hematocrit remained constant over time during ten hours of perfusion of a liver (where “blood” indicates the perfusate) The constancy of the bile hematocrit level suggests that there was little leakage of red blood cells from vessels into the bile ducts. Blood chemistry and bile chemistry remained fairly constant over time during ten hours of perfusion of a liver, although glucose fell somewhat in blood, and was lower in the bile after one and two hours of perfusion, recovering at later times (where “blood” indicates the perfusate). Such a decrease in glucose was to be expected, since although liver preferentially uses lactate as an energy source, the liver will start metabolizing glucose when lactate levels fall (e.g., to less than about 10 mmol / L).

[0090] As illustrated in Fig. 2C, lactate clearance was improved following inclusion of 5 M miricorilant in the perfusate; in two instances immediate improvement was observed; in two other instances, beneficial effects may have been delayed. Insufficent lactate clearance can be a reason to decline a donor liver. Renewed lactate clearance as shown in the figure is important because it indicates that the liver is utilizing the lactate and turning it into energy to use for its metabolic processes. Renewed lactate generation very shortly after miricorilant administration indicates that miricorilant acts on the liver to stimulate or restore liver defatting processes that need energy. Improved lactate clearance may be an indication that the condition of a prospective donor liver is improving, and may suggest that the prospective donor liver may be suitable for transplantation. As shown in Fig. 2C, lactate levels in the perfusate perfusing the donor liver LD- 24 (which was insufficient at the start of miricorilant treatment) decreased over time during perfusion with a perfusate containing 5 M miricorilant. Lactate levels in the perfusate perfusing the donor liver LD-26 first increased, then decreased over time during perfusion with a perfusate containing 5 M miricorilant. Lactate levels in the perfusate perfusing donor liver LD-29 decreased over time during perfusion with a perfusate containing 5 M miricorilant. Lactate levels in the perfusate perfusing donor liver LD-30 (a steatotic liver) first increased, remained steady for about 3 hours, then decreased during perfusion with a perfusate containing 5 M miricorilant.

[0091] FIG.3A shows that donor liver perfusion with a perfusion reagent containing 5 M miricorilant increased lipid levels in the perfusate in 2 of 4 livers. Lipid levels measured included the levels of triglyceride, cholesterol, and fatty acids shown in the figure. It is believed that the lipid source that led to such increased lipid levels in the perfusate was the donor liver. Increased lipid levels in the perfusate are believed to indicate decreased lipid levels in the liver itself. Lipids whose levels increased included triglycerides, cholesterol, and free fatty acids.

[0092] FIG.3B is an image showing the lipid layer formed at the surface of the miricorilant- containing perfusate perfusing donor liver LD-24. Such lipid layers are not typically seen on the surfaces of perfusates lacking miricorilant during perfusion of livers.

[0093] FIGs.3C, 3D, and 3E show the levels of triglycerides, free fatty acids, and cholesterol levels measured over time during perfusion of donor liver LD-24 with a miricorilant-containing perfusate. Triglyceride, free fatty acids, and cholesterol levels measured in the perfusate are shown.

[0094] Triglyceride levels in the perfusate at six hours of perfusion were enriched as compared to the initial triglyceride levels in the perfusate during perfusion with a perfusate containing 5 M miricorilant. These increased triglyceride levels were found both in the perfusate and in the layer on top of the perfusate (as shown in Fig.3B, this layer was a distinct layer on top of the perfusate in the perfusate reservoir). Free fatty acid levels first decreased, and then increased at six hours, as compared to the minimum level reached at about three hours, of perfusion with the perfusate containing 5 M miricorilant in the perfusate. Cholesterol levels increased steadily during six hours of perfusion with the perfusate containing 5 M miricorilant in the perfusate.

[0095] The results shown in FIGs.3A – 3E show that lipid levels (triglycerides, free fatty acids, and cholesterol) in the perfusate may increase over time during perfusion of a liver with a miricorilant-containing perfusate. In addition to these changes measured in the perfusate, lipids in a distinct layer on top of the perfusate reservoir were enriched when measured after six hours of perfusion. It is believed that the source of the lipids that make up such increased lipid levels was the donor liver itself. Such increases in lipid levels are thus believed to be due to transfer of the lipids from the donor liver to the miricorilant-containing perfusate and to a layer that may form on top of the miricorilant-containing perfusate. (Such a layer may appear thicker, and appear to be more yellow, than the perfusate below the upper layer; it may resemble the sort of separation that may be seen when mixing water and oil.) Thus, it is believed that perfusion of adonor liver with a miricorilant-containing perfusate can be effective to reduce lipid levels in that donor liver, or in portions of that donor liver, and to improve lactate clearance by the liver. EXAMPLE 2. PERFUSION OF A HUMAN LIVER FOR TRANSPLANTATION WITH CORT125385

[0096] This Example discusses results from the preparation and maintenance of a liver for later use in a liver transplantation surgical procedure in which the pyrimidine cyclohexenyl compound CORT125385 was included in the perfusate. This compound was added to the perfusate perfusing the livers as a racemic mixture including both enantiomers of CORT125385. Similar to the perfusate used with miricorilant, the perfusate to which CORT125385 was added was as described in Table 1 above, with 5 M CORT125385 instead of 5 M miricorilant. CORT125385 was added to the perfusate upon metabolic stabilization of the liver and then every 3 hours thereafter. During perfusion, an infusion solution containing bile salts, epoprostenol, heparin, insulin, and aminoplasmal with 10% glucose was continuously added to the perfusate during perfusion. Perfusate samples were taken from oxygenated blood before it entered the hepatic artery.

[0097] The timeline for the procedure followed for normothermic machine perfusion (NMP) of livers with perfusate containing CORT125385 discussed in this example was similar to the timeline discussed with regard to NMP perfusion of livers with perfusate containing miricorilant as discussed in Example 1. CORT125385 was added to the perfusate upon metabolic stabilization of the liver and then every 3 hours thereafter.

[0098] Two livers were treated with CORT125385, from liver donors identified as LD-36 and LD-37. Liver LD-36 weighed 2.48 kilograms (kg) and was obtained from a man who was 67 years old at the time of circulatory death; his body mass index (BMI) was 34. The liver was declined for transfusion due to steatosis. It received 12 hours of CORT125385 perfusion out of a total of 15 hours perfusion time. This liver was maintained on ice for approximately one and one half hours after preparation for perfusion but before starting perfusion.

[0099] Liver LD-37 weighed 2.2 kg and was obtained from a male who was 73 years old at the time of brain death; his body mass index (BMI) was 26. The liver was declined for transfusion due to greater than 50% steatosis; at that time preparation for NMP was begun. The liver received 12 hours of perfusion.

[0100] As illustrated in FIG.4, perfusate lipid levels in both livers treated with CORT 125385 increased over time during perfusion with perfusate including 5 M CORT 125385. Perfusate triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) levels increased in both livers during perfusion. Thus, similarly to the pyrimidine cyclohexyl compound miricorilant, perfusion of isolated livers with the pyrimidine cyclohexenyl compound CORT125385 increased perfusate lipid levels (e.g., TG, TC, and FFA). Thus, it is believed that perfusion of a donor liver with a CORT125385-containing perfusate can be effective to reduce lipid levels in that donor liver, or in portions of that donor liver. SUMMARY

[0101] Thus, in view of the results of Example 1 and of Example 2, it is believed that perfusion of a donor liver with a pyrimidine cylcohexyl compound such as miricorilant, and that perfusion of a donor liver with a pyrimidine cylcohexenyl compound such as CORT125385 can be effective to reduce lipid levels in that donor liver, or in portions of that donor liver. Such reductions in lipid levels are believed to improve the suitability of such livers for transplantation into patients in need of a liver transplant.

[0102] All patents, patent publications, publications, and patent applications cited in this specification are hereby incorporated by reference herein in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In addition, although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WE CLAIM:

1. A method for reducing the level of lipids, or of lipid soluble compounds, in an isolated liver or liver portion, said isolated liver or liver portion having an initial level of lipids, or of lipid soluble compounds, the method comprising: Perfusing said isolated liver or liver portion with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound for at least one hour, Wherein said at least one hour is measured from the time of initiation of said perfusion with said solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound, Whereby the level of lipids, or of lipid soluble compounds in the isolated liver or liver portion is reduced as compared to said initial level of lipids, or of lipid soluble compounds.

2. The method of claim 1, wherein said pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4-Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has the3. The method of claim 1, wherein said pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, and which has the structure.

4. The method of claim 1, wherein said solution comprises between about 0.5 micromolar( M) and about 20 M of said pyrimidine cyclohexyl compound or of said pyrimidinecyclohexenyl compound when prepared prior to use.

5. The method of claim 1, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound or of said pyrimidine cyclohexenyl compound when prepared prior to use.

6. The method of claim 2, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound when prepared prior to use.

7. The method of claim 3, wherein said solution comprises about 5 M of said pyrimidine cyclohexenyl compound when prepared prior to use.

8. The method of claim 1, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound continues for a period of time after said time of initiation, said period of time selected from at least three hours, at least six hours, at least nine hours, and at least twelve hours, and wherein a further amount of said pyrimidine cyclohexyl or said pyrimidine cyclohexenyl compound is added to the perfusion solution after said time of initiation.

9. The method of claim 1, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound comprises perfusion of said isolated liver or liver portion at a perfusion rate of between about 1 liter per minute (L / min) and about 2 L / min.

10. The method of claim 1, wherein said lipid, or lipid soluble compound, is a triglyceride compound, and the triglyceride level in said isolated liver or liver portion is reduced as compared to said initial triglyceride level.

11. The method of claim 10, wherein said level of triglyceride is reduced by at least 10% as compared to said initial level of triglyceride.

12. The method of claim 1, wherein said lipid, or lipid soluble compound, is cholesterol, and the cholesterol level in said isolated liver or liver portion is reduced as compared to said initial cholesterol level.

13. The method of claim 12, wherein said cholesterol level is reduced by at least 10% as compared to said initial cholesterol level.

14. The method of claim 1, wherein said lipid, or lipid soluble compound, is a free fatty acid, and the free fatty acid level in said isolated liver or liver portion is reduced as compared to said initial free fatty acid level.

15. The method of claim 14, wherein said free fatty acid level is reduced by at least 10% as compared to said initial free fatty acid level.

16. A method for reducing liver fat in an isolated liver or liver portion, said isolated liver or liver portion having an initial level of liver fat, the method comprising: Perfusing said liver or liver portion with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound for at least one hour, Wherein said at least one hour is measured from the time of initiation of said perfusion with said solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound, Whereby the level of liver fat in the isolated liver or liver portion is reduced as compared to said initial level of liver fat.

17. The method of claim 16, wherein the pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4-Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has the structure18. The method of claim 16, wherein said pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, and which has the structure.

19. The method of claim 16, wherein said solution comprises between about 0.5 micromolar( M) and about 20 M of said pyrimidine cyclohexyl compound or of said pyrimidinecyclohexenyl compound when prepared prior to use.

20. The method of claim 17, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound when prepared prior to use.

21. The method of claim 18, wherein said solution comprises about 5 M of said pyrimidine cyclohexenyl compound when prepared prior to use.

22. The method of claim 16, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound continues for a period of time after said time of initiation, said period of time selected from at least three hours, at least six hours, at least nine hours, and at least twelve hours, and wherein a further amount of said pyrimidine cyclohexyl or said pyrimidine cyclohexenyl compound is added to the perfusion solution after said time of initiation.

23. The method of claim 18, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound comprises perfusion of said isolated liver or liver portion at a perfusion rate of between about 1 liter per minute (L / min) and about 2 L / min.

24. The method of claim 16, wherein said level of liver fat is reduced by at least 10% as compared to said initial level of liver fat.

25. A method for maintaining an isolated liver or portion thereof in a healthy state for later use in liver transplantation, said isolated liver or liver portion having an initial level of triglyceride, the method comprising: Perfusing said isolated liver or liver portion with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound for at least one hour, Wherein said at least one hour is measured from the time of initiation of said perfusion with said solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound, Whereby the isolated liver or liver portion is maintained in a healthy state in preparation for transplantation.

26. The method of claim 25, wherein said pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4-Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has the structure.

27. The method of claim 25, wherein said pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, and which has the structure.

28. The method of claim 25, wherein said solution comprises between about 0.5 micromolar( M) and about 20 M of said pyrimidine cyclohexyl compound or of said pyrimidinecyclohexenyl compound when prepared prior to use.

29. The method of claim 26, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound when prepared prior to use.

30. The method of claim 27, wherein said solution comprises about 5 M of said pyrimidine cyclohexenyl compound when prepared prior to use.

31. The method of claim 25, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound continues for a period of time after said time of initiation, said period of time selected from at least three hours, at least six hours, at least nine hours, and at least twelve hours, and wherein a further amount of said pyrimidine cyclohexyl or said pyrimidine cyclohexenyl compound is added to the perfusion solution after said time of initiation.

32. The method of claim 25, wherein said perfusing step comprises perfusion of said isolated liver or liver portion at a perfusion rate of between about 1 liter per minute (L / min) and about 2 L / min.

33. A method for treating an isolated liver or portion to prepare said isolated liver or liver portion for use in liver transplantation, said isolated liver or liver portion having an initial level of lactate clearance, the method comprising: Perfusing said isolated liver or liver portion with said perfusion solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl for at least one hour; Wherein said at least one hour is measured from the time of initiation of said perfusion with said solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound, Whereby the level of lactate in the perfusion solution perfusing the isolated liver or liver portion is reduced as compared to said initial level of lactate in the perfusion solution, Wherein said level of lactate clearance is improved as compared to said initial level of lactate clearance, and said isolated liver or portion thereof is prepared for use in liver transplantation.

34. The method of claim 33, wherein said pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4-Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has the structure35. The method of claim 33, wherein said pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, and which has the structure.

36. The method of claim 33, wherein said solution comprises between about 0.5 micromolar( M) and about 20 M of said pyrimidine cyclohexyl compound or of said pyrimidinecyclohexenyl compound when prepared prior to use.

37. The method of claim 34, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound when prepared prior to use.

38. The method of claim 35, wherein said solution comprises about 5 M of said pyrimidine cyclohexenyl compound when prepared prior to use.

39. The method of claim 33, wherein said perfusing with a solution comprising a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound continues for a period of time after said time of initiation, said period of time selected from at least three hours, at least six hours, at least nine hours, and at least twelve hours, and wherein a further amount of said pyrimidine cyclohexyl or said pyrimidine cyclohexenyl compound is added to the perfusion solution after said time of initiation.

40. The method of claim 33, wherein said perfusing step comprises perfusion of said isolated liver or liver portion at a perfusion rate of between about 1 liter per minute (L / min) and about 2 L / min.

41. A perfusion reagent for use in preparing an isolated liver or liver portion for liver transplantation, or for use in maintaining an isolated liver or liver portion for liver transplantation, said perfusion reagent comprising red blood cells, heparin, calcium gluconate, and a pyrimidine cyclohexyl compound or a pyrimidine cyclohexenyl compound.

42. The perfusion reagent of claim 41, wherein said pyrimidine cyclohexyl compound is miricorilant, (E)-6-(4-Phenylcyclohexyl)-5-(3-trifluoromethylbenzyl)-1H-pyrimidine-2,4-dione, which has the structure.

43. The perfusion reagent of claim 41, wherein said pyrimidine cyclohexenyl compound is CORT125385, 5-benzyl-6-(4'-chloro-2'-(trifluoromethyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4- yl)pyrimidine-2,4(1H,3H)-dione, and which has the structure.

44. The perfusion reagent of claim 41, further comprising a succinylated gelatin solution.

45. The perfusion reagent of claim 44, wherein said succinylated gelatin solution is a 4% succinylated gelatin solution suitable for intravenous infusion.

46. The perfusion reagent of claim 41, further comprising saline, adenine, and a sugar.

47. The perfusion reagent of claim 46, wherein said sugar is selected from glucose and mannitol.

48. The perfusion reagent of claim 41, comprising between about 0.5 micromolar ( M) andabout 20 M of said pyrimidine cyclohexyl compound or of said pyrimidine cyclohexenyl compound.

49. The perfusion reagent of claim 42, wherein said solution comprises about 5 M of said pyrimidine cyclohexyl compound.

50. The perfusion reagent of claim 43, wherein said solution comprises about 5 M of said pyrimidine cyclohexenyl compound when prepared prior to use.

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