Methods for removing uremic toxins from the blood

Abstract

The present invention relates to a method for removing protein-bound uremic toxins from the blood of a patient who requires it. The method involves introducing a substitution substance, selected from (C6-C12) fatty acids, salts of (C6-C12) fatty acids, precursors of the said (C6-C12) fatty acids, or mixtures thereof, into the blood. The substitution substance replaces the protein-bound uremic toxins, thereby releasing unbound uremic toxins into the blood. The unbound uremic toxins are then removed from the blood by any method, including dialysis. The method according to the present invention is effective with low concentrations of the substitution substance at plasma concentrations ranging from 0.5 to 3 mM.

Description

[Technical Field]

[0001] The present invention relates to a method for removing uremic toxins from blood, and more particularly to a dialysis method that enables the removal of protein-bound uremic toxins from the blood of patients requiring dialysis. [Background technology]

[0002] The kidneys perform various functions, including the removal of waste products from body fluids, such as blood. Kidney damage is associated with the widespread accumulation of uremic toxins, leading to many harmful biological effects. Removing these toxins is important and is often carried out by dialysis, such as hemodialysis, peritoneal dialysis, hemofiltration, or hemodiafiltration.

[0003] Uremic toxins are retained in patients with kidney impairment and constitute a group of substances involved in the development of uric acid syndrome. The accumulation of uremic toxins interferes with the normal function of many organs and exhibits significant cardiovascular toxicity, thereby contributing to an increased cardiovascular risk in patients with kidney impairment.

[0004] Uremic toxins can be divided into three main groups: firstly, free, water-soluble, low molecular weight molecules (MW less than 500 kDa, its prototype being urea) that are readily removed by standard renal replacement therapy; secondly, medium molecular weight molecules (0.5–60 kDa, its prototype being beta-2 microglobulin) whose removal is improved by high-cutoff dialysis membranes and high-volume convection therapies; however, in recent years, the focus has shifted to a third category consisting of protein-bound uremic toxins (PBUTs). These last ones are characterized by widespread toxicity, but their removal strategies are still limited by their tight binding to serum albumin.

[0005] Indoxyl sulfate (IS) and p-cresyl sulfate (p-CS) are representative prototypes of PBUTs and have therefore been widely studied. They have been associated with overall mortality, cardiovascular disease, and worsening renal function in patients with chronic kidney disease (CKD).

[0006] Thus, the removal of these substances is a major concern in patients with end-stage renal disease (ESRD). The difficulty lies in the fact that only the free fraction of PBUT (i.e., less than 10%) is available for diffusion across the dialysis membrane. The binding of PBUT to albumin involves non-covalent, reversible bonds, such as electrostatic or hydrophobic interactions, van der Waals forces, or hydrogen bonds. Therefore, increasing the free fraction of PBUT by using chemicals that compete with PBUT for binding to the albumin site, i.e., displacement substances, can be a beneficial option for increasing the free fraction and thus improving PBUT clearance in dialysis patients.

[0007] While some of these substitution substances have been identified, their use has often proven problematic.

[0008] For example, unsaturated long-chain fatty acids, such as oleic acid and linoleic acid, have been considered, but due to their lipophilic properties and the fact that they are known to worsen dyslipidemia, they are not practical for use in hemodialysis.

[0009] Shorter-chain fatty acids have also been proposed. Octanoates have been shown to replace the affinity for the Sudlow II site of albumin, but only at high concentrations, e.g., 0.24 M, which makes the use of octanoates unsuitable for in vivo dialysis procedures.

[0010] Other substances, such as ibuprofen or furosemide, have also been considered, but many of these substances either exhibit ineffective substitution properties, require high concentrations to be effective, and / or are toxic to patients, causing hemolysis at relatively low concentrations, thus making their use in hemodialysis unsuitable.

[0011] Therefore, there is a need to develop substitution substances that are compatible with dialysis procedures, exhibit effective substitution properties for uremic toxins at low concentrations, and are non-toxic to patients and easy to administer. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] http: / / www.arguslab.com / arguslab.com / ArgusLab.html [Non-Patent Document 2] https: / / bianca.science.uu.nl / prodigy / [Non-Patent Document 3] J Am Soc Nephrol. 2012, 23(7), pp. 1258~1270 [Non-Patent Document 4] J Am Chem Soc. 1941. 63(12): pp. 3529~30 [Non-Patent Document 5] Clin Chem. January 1992;38(1):132~40 [Non-Patent Document 6] Mondal H, Lotfollahzadeh S. Hematocrit. January 2, 2023. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 [Non-Patent Document 7] https: / / www.r-project.org / [Overview of the Initiative]

[0013] The present invention solves the problems mentioned above. After extensive research, the inventors have identified a replacement substance that effectively excretes and replaces protein-bound uremic toxins in the low plasma concentration range, i.e., 0.5 - 3 mM, while being compatible with the patient's blood, and thus enabling safe use during the dialysis process.

[0014] Thus, a method for removing protein-bound uremic toxins from the blood of patients who require it, a) introducing a replacement substance into the blood under conditions such that the replacement substance replaces the protein-bound uremic toxin, thereby releasing the unbound uremic toxin into the blood; b) removing the unbound uremic toxin from the blood by dialysis; comprising where the replacement substance is selected from (C6 - C12) fatty acids, salts of (C6 - C12) fatty acids, precursors of the (C6 - C12) fatty acids, or mixtures thereof; where the replacement substance is supplied into the blood at a plasma concentration included in the range of 0.5 - 3 mM; is proposed.

[0015] Other characteristics, details and advantages are shown in the following detailed description and figures.

Brief Description of the Drawings

[0016] [Figure 1] It is a diagram showing an experimental setup for an in vitro hemodialysis assay according to Example 5. [Figure 2] It is a diagram showing an in vivo setup 10 that can be used to implement the hemodialysis method according to the present invention. The setup 10 includes a dialysis device 11, an arterial line 12, a venous line 13, a blood flow control device 14, a dialysate flow control device 15, and a syringe 16 containing a solution or emulsion of a replacement substance. [Figure 3]A graph showing the results of the ultrafiltration assay according to Example 2. Figure 3A shows the percentage of free urinary toxin IS as a function of the concentration of the replacement substance sodium hexanoate in mM. Figure 3B shows the percentage of free urinary toxin IS as a function of the concentration of the replacement substance sodium octanoate in mM. Figure 3C shows the percentage of free urinary toxin IS as a function of the concentration of the replacement substance sodium decanoate in mM. Figure 3D shows the percentage of free urinary toxin IS as a function of the concentration of the replacement substance sodium dodecanoate in mM. Asterisks indicate significant differences from the control condition: *P < 0.05, **P < 0.01, ***P < 0.005. [Figure 4] A graph showing the results of the ultrafiltration assay according to Example 2. Figure 4A shows the percentage of free urinary toxin p-CS as a function of the concentration of the replacement substance sodium hexanoate in mM. Figure 4B shows the percentage of free urinary toxin p-CS as a function of the concentration of the replacement substance sodium octanoate in mM. Figure 4C shows the percentage of free urinary toxin p-CS as a function of the concentration of the replacement substance sodium decanoate in mM. Asterisks indicate significant differences from the control condition: *P < 0.05, **P < 0.01, ***P < 0.005. [Figure 5] A graph showing the results of a competition test between two medium-chain fatty acids (octanoate and decanoate) and p-cresyl sulfate (p-CS) for binding to human serum albumin according to Example 3. Figure 5A shows the percentage of free urinary toxin p-CS as a function of the concentration of the replacement substance octanoate in mM. Figure 5B shows the percentage of free urinary toxin p-CS as a function of the concentration of the replacement substance decanoate in mM. Asterisks indicate significant differences from the control condition: *P < 0.05, **P < 0.01, ***P < 0.005. [Figure 6]This graph shows the results of the in vitro hemolysis assay according to Example 4. Figure 6A shows the percentage of hemolysis as a function of the concentration of the substitution agent sodium hexanoate. Figure 6B shows the percentage of hemolysis as a function of the concentration of the substitution agent sodium octanoate. Figure 6C shows the percentage of hemolysis as a function of the concentration of the substitution agent sodium decanoate. Figure 6D shows the percentage of hemolysis as a function of the concentration of the substitution agent sodium dodecanoate. Asterisks indicate a significant difference from the control condition: *P<0.05, **P<0.01, ***P<0.005. [Figure 7] This graph shows the removal of uremic toxins by the substitution substance according to the present invention, as determined in Example 5. Figure 7A shows the concentration of the uremic toxin indoxyl sulfate (IS) in μmol / L as a function of perfusion time expressed in minutes for the sodium octanoate substitution substance. Figure 7B shows the concentration of the uremic toxin p-cresyl sulfate (p-CS) in μmol / L as a function of perfusion time expressed in minutes for the sodium octanoate substitution substance. [Figure 8] This graph shows the removal of IS and p-CS by dialysis during a simulated hemodialysis session according to Example 5. Figure 8A shows the fractional removal of uremic toxins p-CS and IS. Figure 8B shows the amount of uremic toxins p-CS and IS removed from the blood, expressed in μmol / min. [Figure 9] These graphs show hematocrit (Figures 9A-9B), hemolysis (Figures 9C-9D), and plasma protein levels (Figures 9E-9F) measured before and after the simulated hemodialysis session according to Example 5. [Modes for carrying out the invention]

[0017] The present invention is a method for removing protein-bound uremic toxins from the blood of a patient who requires it, a) A step of introducing a substitution substance into the blood under conditions in which the substitution substance replaces uremic toxins bound to proteins, thereby releasing unbound uremic toxins into the blood. b) The process of removing unbound uremic toxins from the blood by dialysis. Includes, The substitution substance is selected from (C6-C12) fatty acids, salts of (C6-C12) fatty acids, precursors of the (C6-C12) fatty acids, or mixtures thereof. The substitution substance is supplied into the blood at a plasma concentration in the range of 0.5 to 3 mM. Regarding the method.

[0018] In particular, patients who require it are those with kidney disease, especially acute or chronic kidney disease, and more specifically, end-stage renal disease or uremic syndrome.

[0019] In other words, the present invention relates to (C6-C12) fatty acids, salts of (C6-C12) fatty acids, precursors of the (C6-C12) fatty acids, or mixtures thereof, for use as substitution substances for removing protein-bound uremic toxins from a patient's blood, wherein the substitution substances used are supplied into the blood at plasma concentrations in the range of 0.5-3 mM.

[0020] In particular, the present invention relates to compounds for the use described above, for the treatment of kidney diseases, especially acute or chronic kidney diseases, and more specifically, end-stage renal disease or uremic syndrome.

[0021] As used herein, the expression "conditions under which the substitution substance replaces the uremic toxin bound to the protein" refers to the conditions under which the substitution substance comes into contact with the protein to which the uremic toxin is bound. In particular, the substitution substance may be perfused into the patient's blood.

[0022] The protein to which the uremic toxin is bound may be any plasma protein, particularly albumin, and more specifically, human serum albumin.

[0023] Step b) of removing unbound uremic toxins from the blood is carried out by a dialysis technique, which may be selected from hemodialysis, peritoneal dialysis, hemofiltration, or hemodiafiltration.

[0024] Step b) is performed specifically by hemodialysis.

[0025] Steps a) and b) may be carried out simultaneously, thereby preferably supplying the substitution substance continuously throughout the dialysis process.

[0026] replacement substance Uremic toxins can be present in a patient's blood in equilibrium as free, unbound uremic toxins, or in the form of complexes with proteins present in the blood.

[0027] The term “substitution agent” as used herein refers to a compound capable of binding to proteins in the blood, particularly albumin, in such a way as to replace uremic toxins bound to said proteins by excreting them. In other words, substitution agents can alter the equilibrium state of “free uremic toxin-protein-bound uremic toxin” to result in an accumulation of free uremic toxins and a decrease in protein-bound uremic toxins. In particular, substitution agents can excrete and replace uremic toxins from Sudlow site I or II of human serum albumin, more specifically from Sudlow site II of human serum albumin.

[0028] The binding affinity of the substitution substance to the protein can be greater than the binding affinity of the uremic toxin to the protein. In particular, the binding affinity of the substitution substance

[0029] Alternatively, the binding affinity of the substitution substance to the protein may be less than the binding affinity of the uremic toxin to the protein, for example, it may be 10% of the binding affinity of the uremic toxin.

[0030] The lower the binding affinity of the substitution substance to the protein, the higher the concentration of the substitution substance that may need to be used.

[0031] The binding affinity of substitution substances and / or uremic toxins may be determined by molecular modeling. In particular, the binding affinity of human serum albumin to Sudlow site II may be determined. Molecular modeling may be performed using Arguslab (http: / / www.arguslab.com / arguslab.com / ArgusLab.html) software and the PRODIGY web server (https: / / bianca.science.uu.nl / prodigy / ).

[0032] The substitution substance used in the method according to the present invention is a medium-chain fatty acid selected from (C6-C12) fatty acids, salts of (C6-C12) fatty acids, precursors of the (C6-C12) fatty acids, or mixtures thereof.

[0033] These substances were unexpectedly identified by the inventors as having sufficient affinity for blood proteins, particularly albumin, to effectively excrete and replace uremic toxins at low plasma concentrations. Shorter fatty acids, such as butanoic acid, may not have sufficient affinity for proteins to effectively excrete and replace uremic toxins. Longer fatty acids may cause toxicity-related problems, such as hemolysis, i.e., destruction of red blood cells, or dyslipidemia.

[0034] The term <<(C6~C12) fatty acids>> refers to fatty acids having a carboxylic acid group attached to an alkyl chain. (C6~C12) fatty acids consist of 6 to 12 carbon atoms, one of which is included in the carboxylic acid group. The remaining carbon atoms constitute the alkyl chain. (C6~C12) fatty acids may be linear or branched. (C6~C12) fatty acids also refer to (C6~C8) fatty acids, (C6~C10) fatty acids, (C8~C10) fatty acids, or (C8~C12) fatty acids.

[0035] Linear (C6-C12) fatty acids are fatty acids in which an alkyl chain to which a carboxylic acid group is attached is linear, and the carboxylic acid group is attached to one of the two -CH3 terminal groups of the alkyl chain, and the carboxylic acid is a primary carboxylic acid. Thus, linear (C6-C12) fatty acids are intended to be compounds selected from hexanoic acid (caproic acid), heptanoic acid (enanthic acid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), decanoic acid (capric acid), undecanoic acid (undecylic acid), and dodecanoic acid (lauric acid).

[0036] Branched (C6-C12) fatty acids are intended to be linear fatty acids as defined above, in which the alkyl chain is further substituted by one or more alkyl groups as defined above. Alternatively, branched (C6-C12) fatty acids may also refer to fatty acids in which a carboxylic acid is bonded to one of the -CH2- groups in the alkyl chain, and the carboxylic acid is a secondary carboxylic acid. Examples of branched (C6-C12) fatty acids include isobutyric acid or isovaleric acid.

[0037] According to the present invention, (C6-C12) fatty acids may be saturated or unsaturated. Saturated fatty acids are intended to be fatty acids that do not have carbon-carbon double bonds in the alkyl chain. Unsaturated fatty acids are fatty acids that have one or more carbon-carbon double bonds in the alkyl chain.

[0038] "(C6-C12) fatty acid salts" refer to the previously defined (C6-C12) fatty acids in which the carboxylic acid group exists in the form of a salt. Examples of fatty acid salts are sodium salts, potassium salts, lithium salts, or ammonium salts. Salts are particularly sodium salts.

[0039] In the context of this invention, "(C6-C12) fatty acid derivatives" refers to fatty acid precursors that can be converted in the bloodstream to (C6-C12) fatty acids as defined herein.

[0040] In particular, reference may be made to glyceryl esters of fatty acids, such as triglyceryl esters, diglyceryl esters, or monoglyceryl esters of fatty acids, and more specifically, triglyceryl esters of fatty acids, such as glyceryl trioctanoate (tricaprylin) or glyceryl tridecanoate (tricaprin).

[0041] Phospholipids and lysophospholipids may also be mentioned. These compounds each contain two or one fatty acid bonded to a glycerol moiety by an ester bond. They further contain a phosphate group bonded to one of the alcohol groups of the glycerol moiety via an OP bond. In the case of lysophospholipids, one of the hydroxyl groups of the glycerol moiety is vacant, meaning it is neither esterified nor bonded to a phosphate group.

[0042] Among phospholipids, those particularly useful in the context of the present invention include, for example, 1,2-diocanoyl-sn-glycero-3-phosphocholine or 1,2-didecanoyl-sn-glycero-3-phosphocholine.

[0043] Among lysophospholipids, those particularly useful in the context of the present invention include, for example, 1-octanoyl-sn-glycero-3-phosphocholine or 2-octanoyl-sn-glycero-3-phosphocholine.

[0044] The use of such (C6-C12) fatty acid precursors is advantageous because it allows for the gradual release of (C6-C12) fatty acids during the dialysis session, particularly in the presence of heparin (used as an anticoagulant in the bloodstream), which impairs the activity of lipoprotein lipase, an enzyme that catalyzes the hydrolysis of triglycerides into fatty acids, thus allowing the derivatives to be gradually hydrolyzed in the bloodstream. These precursors, used as emulsions, have been further shown to be safe for use in humans and to comply with regulatory requirements. Fatty acid precursors can also be used as predialysis boluses instead of continuous predialysis perfusions.

[0045] The substitution material according to the present invention is, in particular, the unsaturated linear fatty acids described above, or salts thereof described above.

[0046] According to one embodiment of the present invention, the substitution material is selected from hexanoic acid, octanoic acid, decanoic acid and dodecanoic acid, or mixtures thereof, particularly octanoic acid or decanoic acid.

[0047] According to one embodiment of the present invention, the substitution substance is a salt of a (C6-C12) fatty acid, more specifically a sodium salt.

[0048] In particular, the substitution substances are sodium octanoate, sodium decanoate, or mixtures thereof, more specifically sodium octanoate.

[0049] According to one embodiment of the present invention, the substitution substance is a precursor of a (C6-C12) fatty acid, and the precursor of the (C6-C12) fatty acid is selected from glyceryl esters of (C6-C12) fatty acids, phospholipids of (C6-C12) fatty acids, or lysophospholipids of (C6-C12) fatty acids, and in particular triglyceryl esters of (C6-C12) fatty acids.

[0050] In particular, the glyceryl ester exists as a composition containing triglycerides of octanoic acid and decanoic acid, and the glyceryl ester contains about 54 mol% octanoic acid and about 40 mol% decanoic acid relative to the total amount of fatty acids constituting the ester, and more specifically, the composition is in the form of an oil-in-water emulsion containing 10% by mass of the glyceryl ester and 10% by mass of soybean oil.

[0051] A useful compound in this embodiment is the commercially available mixture "Medialipid® 20%" (B. Braun Medical), which is a blend of medium-chain triglycerides and refined soybean oil. This commercially available mixture mainly consists of triglyceride esters of octanoic acid (54%), decanoic acid (40%), and small amounts of other acids, such as hexanoic acid (2%) and dodecanoic acid (4%).

[0052] According to certain embodiments, the substitution substance described herein may be the only substitution substance used in the method according to the present invention, i.e., supplied in step a). In other words, the substitution substance may not be used in combination with other substitution substances, such as salicylic acid in particular.

[0053] According to this embodiment, the substitution substance can efficiently remove and replace uremic toxins from proteins without needing to be combined with other substitution substances.

[0054] Uremic toxins Any uremic toxins that bind to plasma proteins, particularly albumin, may be eliminated or replaced by the substitution substances used in the method of the present invention. Examples of such uremic toxins are disclosed by Duranton et al. (J Am Soc Nephrol. 2012, 23(7), pp. 1258-1270).

[0055] In one embodiment, the uremic toxin comprises p-cresyl sulfate (p-CS), p-cresyl glucuronide (p-CG), indoxyl sulfate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid, indole-3-acetic acid, phenylacetic acid, or a mixture thereof.

[0056] The substitution substance according to the present invention is particularly useful for replacing albumin by eliminating indoxyl sulfate and p-cresil sulfate uremic toxin.

[0057] [ka]

[0058] In this invention, the substitution substance is supplied into the blood to achieve a plasma concentration in the range of 0.5 to 3 mM. Within the scope of this invention, this concentration relates to the initial plasma concentration of (C6-C12) fatty acids or their salts. In practice, the concentration of the substitution substance may decrease over time due to metabolism or dialysis clearance of the substitution substance in the blood.

[0059] Furthermore, this concentration relates to the amount of fatty acid equivalent administered. Therefore, when a precursor is used, the plasma concentration relates to the amount of (C6-C12) fatty acids that can be released from the precursor. Thus, triglyceryl esters of (C6-C12) fatty acids supplied at a plasma concentration of 1 mM are considered to be equivalent to (C6-C12) fatty acids at a concentration of approximately 3 mM.

[0060] When a mixture of substitution substances is used, the plasma concentration is the sum of the concentrations of each substitution substance.

[0061] In certain embodiments, the substitution substance is supplied into the blood to achieve a plasma concentration in the range of 0.5–2 mM, more specifically, in the range of 0.5–1 mM.

[0062] When using an excessively low concentration of the substitution substance, especially when the binding affinity of the substitution substance to the protein is lower than the binding affinity of the uremic toxin to the protein, the substitution substance does not need to be present at a concentration sufficient to effectively remove and replace the uremic toxin from the protein.

[0063] At excessively high concentrations, when the substitution substance is supplied into the patient's bloodstream, there is a risk of adverse reactions, such as hemolysis.

[0064] Therefore, according to one embodiment, triglyceryl esters of (C6-C12) fatty acids may be supplied to the patient's blood at a concentration in the range of approximately 0.167 mM to approximately 1 mM, particularly in the range of approximately 0.167 to approximately 0.67 mM, and more specifically in the range of approximately 0.33 to approximately 0.5 mM.

[0065] According to one embodiment, phospholipids of (C6-C12) fatty acids may be supplied to the patient's blood at a concentration in the range of approximately 0.25 mM to approximately 1.5 mM, particularly in the range of approximately 0.25 to approximately 1 mM, and more specifically in the range of approximately 0.25 to approximately 0.5 mM.

[0066] According to one embodiment, lysophospholipids of (C6-C12) fatty acids may be supplied to the patient's blood at a concentration in the range of approximately 0.5 mM to approximately 3 mM, particularly in the range of approximately 0.5 to approximately 2 mM, and more specifically in the range of approximately 0.5 to approximately 1 mM.

[0067] In certain embodiments, the substitution compound is sodium octanoate, supplied at plasma concentrations ranging from 0.5 to 3 mM.

[0068] In certain embodiments, the substitution compound is sodium decanoate, supplied at plasma concentrations in the range of 1–2 mM.

[0069] In one embodiment, the substitution substance is a mixture of dodecanoic acid or a salt thereof and one or more (C6-C10) fatty acids or salts thereof, in particular the dodecanoic acid or a salt thereof is supplied at a plasma concentration of 0.25 mM or less, and one or more (C6-C10) fatty acids or salts thereof is supplied at a concentration in the range of 0.25 to 2.75 mM, particularly in the range of 0.5 to 2.75 mM.

[0070] The substitution substance may be supplied to the patient's blood in the form of a solution or emulsion, particularly a solution.

[0071] The substitution substance may be supplied as an aqueous solution of the substitution substance. In particular, in this embodiment, the substitution substance may be selected from the previously defined (C6-C12) fatty acids or salts thereof.

[0072] Alternatively, the substitution substance may be supplied in the form of an emulsion containing a vegetable oil, such as soybean oil, water, and optionally an emulsifier, such as soybean lecithin. In particular in this embodiment, the substitution substance may be a precursor of a (C6-C12) fatty acid, such as the glyceryl derivatives described above.

[0073] In certain embodiments, the aqueous solution to which the substitution substance is supplied is a buffered aqueous solution, particularly phosphate-buffered saline (PBS), or an isotonic sodium chloride solution (0.9% w / v).

[0074] The aqueous solution in which the substitution substance is supplied is advantageously suited to physiological conditions, i.e., has a pH in the range of 7 to 7.5, particularly in the range of 7.3 to 7.4.

[0075] In certain embodiments, the substitution substance exists as an aqueous solution or emulsion with a concentration of 75 mM to 300 mM.

[0076] The range "75 mM to 300 mM" should also be understood as encompassing the following ranges: 100 mM to 300 mM, 200 mM to 300 mM, 75 mM to 200 mM, and 150 mM to 200 mM. In particular, an aqueous solution of a substitution substance is a saturated aqueous solution of the substitution substance.

[0077] The plasma concentration of the substitution substance can be achieved by controlling its perfusion rate during dialysis, controlling the amount of substitution substance supplied to the blood, adjusting the concentration of the substitution substance in the solution supplied to the patient, and adjusting the flow rate of the solution into the blood.

[0078] The solution or emulsion containing the substitution substance may be supplied to the patient's blood at a flow rate of 75 μL / min to 450 μL / min.

[0079] The dialysate may be supplied to the dialysis machine at a flow rate of 0 ml / min to 1000 ml / min. A flow rate of 0 ml / min is usable in hemofiltration. In hemodialysis or hemodiafiltration, flow rates of, for example, 10 ml / min to 1000 ml / min, particularly 100 ml / min to 1000 ml / min, and more specifically 100 ml / min to 500 ml / min are particularly usable.

[0080] The dialysate may be any commercially available dialysate, for example, a bicarbonate or acetate-based dialysate that contains or does not contain citrate, lactate, or chloride.

[0081] A blood flow rate of 20 ml / min to 500 ml / min is acceptable.

[0082] Dialysis may be performed at temperatures within the range of 35°C to 40°C, particularly at human body temperature.

[0083] The solution or emulsion containing the substitution substance may be supplied extracorporeally. In one embodiment, the substitution substance may be supplied in a line, i.e., an “arterial line” through which blood flows from the patient to the device for the removal of unbound uremic toxins. In an alternative embodiment, the substitution substance may be supplied in a line, i.e., an “intravenous line” through which blood flows from the device for the removal of unbound uremic toxins to the patient.

[0084] The equipment for removing unbound uremic toxins may be a dialysis machine in the case of hemodialysis, a filter in the case of hemodiafiltration, or a combination of the two in the case of hemodiafiltration.

[0085] In other words, the present invention relates to the previously described method, wherein the substitution substance is supplied extracorporeally as a solution or emulsion of the substitution substance in a line in which blood flows from a patient to a device for removing unbound uremic toxins.

[0086] In one embodiment, the substitution substance is supplied by perfusion.

[0087] Alternatively, a solution or emulsion containing the substitution substance may be supplied into the patient's body, for example, by perfusion. This embodiment may be implemented in the case of peritoneal dialysis.

[0088] Referring now to Figure 2, as a non-limiting example for the description of the method according to the present invention, the hemodialysis method described herein earlier may be performed using a setup 10 which includes a dialysis machine 11 connected to the patient by arterial lines 12 and venous lines 13. In this setup, blood flows from the patient to the dialysis machine 10 via arterial lines 12, and the blood returns from the dialysis machine 10 to the patient via venous lines 13. The setup may further include a blood flow control device 14 and a dialysate flow control device 15.

[0089] The dialysis fluid is supplied to the dialysis machine 10. The substitution substance is supplied, for example, by perfusion of a solution or emulsion containing the substitution substance in the arterial line 12 using a syringe 16 or a peristaltic pump.

[0090] The dialysate flow control device 15 is capable of controlling the flow rate of dialysate to the dialysate machine.

[0091] The blood flow control device 14 is capable of controlling the blood flow velocity in the arterial line 12.

[0092] The dialysate flow control device (15) is capable of controlling the flow rate of the dialysate.

[0093] The method according to the present invention has been found to offer several advantages over prior art methods. In fact, the substitution substances used in the present invention can effectively remove and replace uremic toxins from proteins to which they are bound, even at low concentrations. The substitution substances used in the present invention do not exhibit adverse effects, such as widespread hemolysis at the concentrations used, and are also easily handled, as they can be administered, for example, in the form of a solution or a safe, biocompatible emulsion.

[0094] The following embodiments are useful in illustrating the present invention without intending to limit its scope. [Examples]

[0095] chemicals Medium-chain fatty acids (hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid), human serum albumin (reference A9511), and indoxyl sulfate (reference I3875) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). p-Cresyl sulfate was synthesized according to the method described by Feigenbaum & Neuberg (J Am Chem Soc. 1941. 63(12): pp. 3529-30). All solvents were purchased from Carlo-Erba Reagents (Val-de-Reuil, France) and were HPLC grade.

[0096] Competitive assay: Verification of IS substitution by each substitution agent. Competitive interactions of substitution agents were confirmed by ultrafiltration assay under in vitro CKD conditions. Physiologically concentrated HSA (500 μM) was incubated with the maximum achievable concentration of indoxyl sulfate (IS) (250 μmol / L) in phosphate-buffered saline (pH=7.40) at room temperature for 2 hours. Then, 1–3 mM of the substitution agent was added to the solution and incubated in a water bath at 37°C for 1 hour. A control experiment was performed by adding the same volume of PBS instead of the substitution agent. Next, 600 μL of the sample was transferred to a filtration device (Corning®, Spin-X® UF concentrator, Wiesbaden, Germany, MWCO: 5kDa) and centrifuged at room temperature at 10,000 g for 5 minutes and 13,000 g for 15 minutes. The ultrafiltrate was collected, and the free fraction of IS concentration was assayed fluorescently as described below.

[0097] Competitive assay: Verification of p-CS replacement by each replacement agent using Rapid equilibrium dialysis (RED). Rapid equilibrium dialysis is an accurate and reliable method for determining the degree of protein binding. A solution of human serum albumin (500 μM) was mixed with p-CS (250 μM) and incubated at 37°C for 2 hours. A substitution compound was added, and the sample was further incubated at 37°C for 1 hour. The sample (200 μL) was added to the left chamber of a commercially available plate-based rapid equilibrium dialysis machine (RED, Thermo Fisher, Illkirch, France), and 400 μL of PBS was added to the outer chamber of the RED machine. The plate was incubated at room temperature (25°C) for 6 hours with agitation (400 rpm). The concentration of free p-CS was determined by fluorescence quantitative analysis as described below.

[0098] Fluorescence Quantification Assay for Uremic Toxins The samples were diluted with PBS, and fluorescence was measured using a Jasco FP-8300iRM spectrofluorometer (Jasco, Tokyo, Japan). The excitation and emission wavelengths were set to 280 nm / 383 nm for indoxyl sulfate and 260 nm / 295 nm for p-cresil sulfate. Parallel standard curves were drawn for indoxyl sulfate (0-2.5 μmol / L) and p-cresil sulfate (0-200 μmol / L).

[0099] In vitro hemolysis assay One milliliter of fresh bovine blood was incubated at 37°C for 4 hours with a medium-chain fatty acid solution (final concentration: 0.25, 0.5, 1, 2, or 3 mmol / L) or 50 μL of PBS as a control. A positive control for hemolysis (defined as 100% hemolysis) was prepared by incubating one milliliter of bovine blood with 0.1% (v / v) TRITON X100, followed by two freeze-thaw cycles. The samples were centrifuged at 9000xg for 2 minutes, and the plasma was stored at -20°C until the free hemoglobin assay. Hemolysis was estimated by free hemoglobin concentration. Hemoglobin concentration was determined spectrophotometrically as described by Fairbanks et al. (Clin Chem. January 1992; 38(1): pp. 132-40). In short, plasma samples were diluted 1:10 with 9.4 mmol / L Na2CO3, and absorbances were read at 415 nm, 450 nm, and 700 nm using a microplate reader (Tecan, Lyon, France). The free hemoglobin concentration is calculated using the following formula: fHb = 0, 01017 × [(154, 7 × A 415nm )-(130,7×A 450nm )-(123,9×A 700nm )]×d [In the formula, fHb is the concentration of free hemoglobin in g / L. A is the absorbance at the specified wavelength, [d is the dilution ratio] It was calculated using [the specified method].

[0100] The results were expressed as a percentage of complete hemolysis.

[0101] Closed-loop system using an HD generator: Hemodialysis of fresh bovine blood To evaluate the replacement status and increased removal of PBUT during hemodialysis sessions, 20-liter batches of fresh bovine blood were collected from the slaughterhouse under authorization VC18731 (October 4, 2018) issued by the Direction Departmentale de la Protection des Populations-Direction des Services Veterinaires (DDPP-DSV, Lyon, France). The blood was heparinized with heparin sodium (Choay, Cheplapharm, France) at 3,750 UI / L and filtered through a nylon mesh. 200 μmol / L of IS was added to 2 liters (2 L) of blood and gently agitated at 37°C for 2 hours. A Fresenius 5008 CorDiax hemodialysis generator (Fresenius, Sevres, France) and a polysulfone-based hemodialysis membrane (FXHDF1000, 2.2 m²) were used. 2A 2-hour hemodialysis session was performed using Fresenius Medical Care (Bad Homburg, Germany). A closed-loop circuit was implemented by placing arterial and venous ports in the blood batch (see Figure 1 for illustration of the experimental equipment). The blood and dialysate flow rates were set to 200 and 300 mL / min, respectively, and the dialysate temperature was set to 38°C. Octanoate solution (224 mmol / L) as a replacement agent was infused into the arterial line at a flow rate of 150 μL / min (i.e., 33.6 μmol / min). Blood samples were collected from the arterial line at 0, 15, 30, 45, 60, 75, 90, and 120 minutes. The blood was centrifuged at 9,000 g for 2 minutes, and the plasma was stored at -20°C. The total concentration of indoxyl sulfate was assayed by HPLC linked to fluorescence detection as described below. At the start and end of the experiment, hemolysis was estimated by measuring free hemoglobin in plasma as described above. Total protein concentration was measured using the Lowry method with bovine serum albumin as a standard. Hematocrit was measured using the micromethod (Mondal H, Lotfollahzadeh S. Hematocrit. January 2, 2023 In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023). Briefly, 70 μL of blood was collected in a heparinized capillary tube. The end of the capillary tube was sealed with a clay sealant and centrifuged at 9000 rpm for 5 minutes. Hematocrit levels were read using a microhematocrit card reader.

[0102] HPLC assay of protein-bound uremic toxins 5-Hydroxyindole-2-carboxylic acid (5-HICA) was used as an internal standard in all experiments. Plasma samples were centrifuged at 17,000 rpm for 5 minutes. Subsequently, 125 μL of the supernatant was pretreated using an Ostro pass-through sample preparation instrument (Waters, Saint-Quentin-en-Yvelines, France), which included in-well protein precipitation with acetonitrile (375 μL) combined with a pass-through method that allowed for good extraction. The eluate was evaporated to a dry state under a gentle flow of nitrogen gas. The dry extract was resuspended in 125 μL of mobile phase (consisting of 10% (v / v) methanol, 73.1 mmol / L sodium acetate, and 0.3 mmol / L ethylenediaminetetraacetic acid-EDTA-Na2). After further centrifugation at 25,000 rpm for 10 minutes, 5 μL of the supernatant was injected into an Agilent 1,100 system (Agilent Technologies, Dover, DE, USA) equipped with a C18 column (C18 Synergi 2.5 μm Polar-RP 100A-100x2 mm, Phenomenex, Torrance, CA, USA). The total PBUT concentration was determined by reverse-phase HPLC with a uniform flow rate of 0.2 ml / min. Fluorescence analysis (excitation λ) was performed. ex :270nm; emission λ em p-CS and IS were determined by (320 nm). The retention times for 5-HICA, p-CS, and IS were 2.3 minutes, 4.1 minutes, and 6.3 minutes, respectively. The measured values ​​were calibrated using standard curves for all analytes and were linear across the experimental concentration range. The dynamic range of calibration was 0.31–40 μmol / L for IS and 1.56–200 μmol / L for p-CS.

[0103] statistical analysis All data were compared using nonparametric statistics with a small number of replicates (n=5-8). Data are presented as medians (interquartile range). The statistical significance of ultrafiltration experiments and hemolysis measurements was studied using the Kruskal-Wallis test, and subsequently the Dunn test where appropriate. Curves for IS and p-CS reduction were constructed using all datasets and nonlinear models. For each toxin, the Mann-Whitney U test was used to compare the amount removed and the fraction removed. Hematocrit, hemolysis, and protein concentration before and after hemodialysis sessions were compared with controls or octanoates (paired data) using the signed-rank Wilcoxon test. Differences were considered significant at a p level of less than 0.05. All statistical analyses were performed using Graphpad Prism (Graphpad Software, La Jolla, CA) and the open-source R software (https: / / www.r-project.org / ).

[0104] Molecular modeling To estimate the equilibrium constant (Kd) for the binding of human serum albumin to Sudlow site II, XRD structures were obtained from the Protein Data Bank (PDB) for the ligands indoxyl sulfate (PDB code 2BXH), dodecanoate (PDB code 1E7F), decanoate (PDB code 1E7E), and octanoate (PDB code 5X52). For p-cresyl sulfate for which no PDB structure was available, docking simulations were performed using the software Arguslab (http: / / www.arguslab.com / arguslab.com / ArgusLab.html) and the PDB structure of HSA-indoxyl sulfate (PDB code 2BXH). The docking box was placed at the center of indoxyl sulfate, and docking simulations were achieved by a genetic algorithm with default parameters. For butyrate and hexanoate, these complexes were obtained by creating both ligand butyrate and hexanoate from octanoate and deriving the corresponding HSA complexes from the complex of HSA-octanoate (PDB code 5X52). Subsequently, ΔG0 was estimated using the PRODIGY server by submitting the PDB files of the HSA complexes.

[0105] Results (Example 1) Molecular modeling The binding affinity of human serum albumin for Sudlow site II was determined using molecular modeling for the uremic toxins indoxyl sulfate and p-cresyl sulfate, and the substituted substances hexanoate, octanoate, and decanoate. The binding affinity (ΔG0) was predicted from crystallographic and modeling data using the PRODIGY (Protein Binding Energy Prediction) website (https: / / bianca.science.uu.nl / prodigy / ). The following equation was used to calculate K a as follows: ΔG0 = -RT ln K a . K d was calculated as 1 / K a .

[0106] [Table 1]

[0107] (Example 2) Replacement of p-CS and IS by ultrafiltration assay The free fraction of IS was measured using the ultrafiltration experiment described earlier. Under baseline conditions, the free fraction of IS was 10.0% (9.3%–14.2%), indicating strong binding to HSA.

[0108] Incubation with hexanoate increased the free fraction of IS only at the maximum concentration tested (3 mM), reaching 36% (34%–39%) (P<0.05) (Figure 3A).

[0109] Incubation with octanoate dose-dependently increased the free fraction of IS, reaching 55% (50% to 59%) at a concentration of 3 mM (P<0.001) (Figure 3B).

[0110] Similar results were obtained in incubation with decanoate, where the free fraction of IS increased to 54% (52%~58%) at a concentration of 3 mM (P<0,01) (Figure 3C).

[0111] Incubation with dodecanoate dose-dependently increased the free fraction of IS, reaching 42% (41%–44%) of IS at a concentration of 3 mM (P<0.001) (Figure 3D).

[0112] The free fraction of p-CS was measured using the ultrafiltration experiment described earlier. Under baseline conditions, the free fraction of p-CS was 14% (11%–16%), indicating strong binding to HSA.

[0113] Incubation with hexanoate increased the free fraction of p-CS only at the maximum concentration tested (3 mM), reaching 36% (34%–41%) (P<0.05) (Figure 4A).

[0114] Incubation with octanoate dose-dependently increased the free fraction of p-CS, reaching 57% (45% to 63%) at a concentration of 3 mM (P<0.001) (Figure 4B).

[0115] Similar results were obtained in incubation with decanoate, where the free fraction of p-CS increased to 59% (57%~63%) at a concentration of 3 mM (P<0,01) (Figure 4C).

[0116] (Example 3) Replacement of p-CS by rapid equilibrium dialysis The binding of p-CS (250 μM) to human serum albumin (500 μM) was measured at pH 7.4 and 25°C in the presence of octane (A) or decanoate (B) at concentrations of 62.5 μmol / L to 2 mmol / L. Free fractions were isolated using rapid equilibrium dialysis (RED instrument, Thermo-Scientific), and p-CS was assayed by fluorescence spectroscopy (Figure 5). It was shown that octane at a concentration of 0.5 mM effectively eliminated and replaced p-CS.

[0117] (Example 4) In vitro hemolysis assay Fresh bovine blood was incubated at 37°C for 4 hours (the entire duration of a normal hemodialysis session) in the presence of MCFA (1-3 mmol / L), and the amount of free hemoglobin in the plasma was used as an indicator of hemolysis (Figures 6A-6D).

[0118] Incubation of blood with hexanoic acid (3.6% (2.8%~4.4%), P=0.210) or octanoic acid (2.3% (2.1%~2.5%), P=0.789) did not cause any significant hemolysis compared to the control (1.5% (1.3%~3.0%)). Incubation of bovine blood with decanoic acid caused hemolysis at a concentration of 3 mmol / L (21.9% (20.9%~22.0%), P<0.001). In contrast, incubation of bovine blood with dodecanoic acid caused hemolysis at concentrations greater than 0.25 mmol / L (15.3%~24.9% at 0.5 and 3 mmol / L, P<0.05). In summary, the data indicate that the substitution substances promote hemolysis at a level acceptable for clinical use.

[0119] (Example 5) Hemodialysis using fresh bovine blood To conduct a realistic-scale proof of concept, bovine blood was supplemented with uremic toxins at concentrations observed in patients with ESKD and dialyzed for 2 hours using a clinically used HDF generator (i.e., Fresenius 5008 CorDiax) and HD membrane (FXHDF 1000, Fresenius). A sodium octanoate solution was continuously infused into the arterial line and compared to physiological saline as a control. The mean temperature of the blood batches (throughout the protocol) was 35.0 ± 0.8°C and 35.2 ± 1.3°C for the control and octanoate infusion, respectively (U=9, P=0.548). As shown in Figure 7, inline perfusion of octanoate significantly increased the dialysis-induced removal of IS and p-CS. The p-CS concentration decreased from 48 μmol / L to 30 μmol / L over a 2-hour HD session in the control experiment, but decreased from 58 μmol / L to 6 μmol / L in the octanoate-treated samples. In a well-coordinated manner, IS concentration decreased from 220 μmol / L to 115 μmol / L in the control experiment, but decreased from 214 μmol / L to 14 μmol / L in the experiment with continuous octanoate infusion. The half-life of IS in plasma was 36.4 minutes under control conditions and 15.2 minutes after octanoate infusion. The half-life of p-CS in plasma decreased from 20.6 to 12.8 minutes after octanoate infusion. Toxin removal increased from 0.3 μmol / min to 1 μmol / min for pCS and from 1.5 μmol / min to 3.5 μmol / min for IS after predialysis infusion (Figure 8). Fraction removal of IS and pCS increased significantly from 36% to 91% (2.5-fold, p<0.001) in IS and from 38% to 88% (2.3-fold, p<0.001) in pCS (Figure 8). Continuous infusion of octane for 2 hours did not result in any significant change in hematocrit or hemolysis levels (Figure 9). These results clearly demonstrate the absence of significant adverse effects of octane infusion on bovine red blood cells. No difference was observed in plasma protein concentration, indicating no net hemofiltration level and ruling out hemoconcentration.

Claims

1. A method for removing protein-bound uremic toxins from the blood of patients who require it, a) A step of introducing a substitution substance into the blood under conditions in which the substitution substance replaces uremic toxins bound to proteins, thereby releasing unbound uremic toxins into the blood. b) The process of removing unbound uremic toxins from the blood by dialysis. Includes, The substitution substance is selected from (C6-C12) fatty acids, salts of (C6-C12) fatty acids, precursors of the said (C6-C12) fatty acids, or mixtures thereof. The substitution substance is supplied into the blood at a plasma concentration in the range of 0.5 to 3 mM. method.

2. The method according to claim 1, wherein dialysis is selected from hemodialysis, peritoneal dialysis, hemofiltration, or hemodiafiltration, particularly hemodialysis.

3. The method according to claim 1 or 2, wherein the substitution substance is selected from hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid, or a mixture thereof.

4. The method according to claim 1 or 2, wherein the substitution substance is a salt of a (C6-C12) fatty acid, particularly a sodium salt.

5. The method according to claim 1 or 2, wherein the substitution substance is sodium octanoate, sodium decanoate, or a mixture thereof, particularly sodium octanoate.

6. The method according to claim 1 or 2, wherein the precursor of a (C6-C12) fatty acid is selected from glyceryl esters of (C6-C12) fatty acids, phospholipids, or lysophospholipids.

7. The method according to claim 6, wherein the precursor of (C6-C12) fatty acids is selected from glyceryl esters of (C6-C12) fatty acids, and the glyceryl ester exists as a composition comprising triglycerides of octanoic acid and decanoic acid, in particular the glyceryl ester comprising about 54 mol% octanoic acid and about 40 mol% decanoic acid relative to the total amount of fatty acids constituting the ester, and more specifically the composition is in the form of an oil-in-water emulsion comprising 10% by mass of the glyceryl ester and 10% by mass of soybean oil.

8. The method according to any one of claims 1 to 7, wherein the substitution substance is the only substitution substance introduced in step a).

9. The method according to any one of claims 1 to 8, wherein the uremic toxin is selected from p-cresyl sulfate (p-CS), p-cresyl glucuronide (p-CG), indoxyl sulfate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid, indole-3-acetic acid, and phenylacetic acid, particularly from p-cresyl sulfate or p-indoxyl sulfate.

10. The method according to any one of claims 1 to 9, wherein the protein is albumin, in particular human serum albumin.

11. The method according to any one of claims 1 to 10, wherein the substitution substance is supplied into the blood to achieve a plasma concentration in the range of 0.5 mM to 2 mM, particularly in the range of 0.5 mM to 1 mM.

12. The method according to any one of claims 1 to 11, wherein the substitution substance is supplied as an aqueous solution containing the substitution substance, and the substitution substance is particularly selected from (C6-C12) fatty acids or salts thereof.

13. The method according to claim 12, wherein the aqueous solution is a phosphate-buffered saline solution or an isotonic solution of sodium chloride.

14. The method according to any one of claims 1 to 11, wherein the substitution substance is supplied as an emulsion containing the substitution substance, and the substitution substance is particularly a precursor of (C6-C12) fatty acids.

15. The method according to any one of claims 12 to 14, wherein the substitution substance is present in an aqueous solution or emulsion at a concentration of 75 mM to 300 mM, and in particular the aqueous solution is a saturated solution of the substitution substance.

16. The method according to any one of claims 1 to 15, wherein a solution or emulsion containing a substitution substance is supplied to the blood at a flow rate of 75 μL / min to 450 μL / min.

17. The method according to any one of claims 1 to 16, wherein dialysis is performed at a temperature within 35 to 40°C.

18. The method according to any one of claims 1 to 17, wherein the substitution substance is supplied extracorporeally as a solution or emulsion of the substitution substance in a line in which blood flows from a patient to equipment for the removal of unbound uremic toxins.

19. The method according to claim 18, wherein the device for removing unbound uremic toxins is a dialysis machine, a filter, or a combination thereof.

20. The method according to any one of claims 1 to 19, wherein the replacement substance is supplied into the patient's blood by perfusion.

21. The procedure is performed using a dialysis machine (10) connected to the patient by arterial lines (11) and venous lines (12). Blood flows from the patient to the dialysis machine (10) via the arterial line (11), and blood flows from the dialysis machine (10) to the patient via the venous line (12). Dialysis fluid is supplied to the dialysis machine (10), The replacement substance is supplied to the arterial line (12) by perfusion. The method according to any one of claims 1 to 20.

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