Lipid-containing compositions and their application in clearing protein-bound toxins in liver failure

Abstract

A lipid-containing composition, by weight, includes phospholipids, and one or more of vegetable oil, medium-chain triglycerides, antioxidants, and sodium oleate. It has been verified that the various compositions provided by this invention competitively capture protein-bound toxoids in the blood, thereby achieving the purpose of clearing protein-bound toxoids from the blood, especially for patients with liver failure, and also alleviating oxidative stress in patients during dialysis treatment.

Description

Technical Field

[0001] This invention relates to a composition for removing toxins from the body, and more particularly to a composition for removing protein-bound toxins from the blood, used as a reagent or dialysis solution in the treatment of liver failure. Background Technology

[0002] Acute and chronic liver failure is often caused by a variety of factors (such as viral hepatitis, drug poisoning, poisoning by toxic substances, and autoimmune diseases). It has a rapid onset and a high mortality rate (Am J Gastroenterol, 2023.118(7):p.1128-1153). Currently, apart from artificial liver and liver transplantation, there are no other effective treatment methods. Finding new methods and strategies to effectively treat patients with liver failure remains an urgent problem to be solved in clinical research. In liver failure, the liver's metabolic and clearance capacity decreases, leading to the accumulation of toxins such as unconjugated bilirubin, tryptophan, nitric oxide, and bile acids in the body, which promotes the development of serious complications such as hepatorenal syndrome, hepatic encephalopathy, and circulatory disorders (Eur J Gastroenterol Hepatol, 2002.14(2):p.195-203). These toxins are mostly bound to serum proteins (mainly albumin) and are hydrophobic (Biophys Chem, 2013.180-181:p.55-65). Recent studies have found that the aforementioned protein-bound toxins are one of the key factors inducing high mortality rates in patients with liver failure (J Hepatol, 2014.60(2):p.275-81).

[0003] Diffusion is the main mechanism by which hemodialysis removes protein-bound toxoids from the blood, and the efficiency of hemodialysis in removing protein-bound toxoids depends on the level of free toxoids. Due to the hydrophobicity of protein-bound toxoids, their free levels in the blood are usually low. When free toxoids diffuse through the dialyzer, the levels of free toxoids on the blood side and the dialysate side often reach equilibrium rapidly, making it difficult to effectively remove protein-bound toxoids. Even high-flux hemodialysis does not have a significant advantage in its removal effect (Aging Clin Exp Res, 2012.24(1):p.74-8). Therefore, how to remove protein-bound liver failure toxoids from the blood of patients with liver failure in a low-cost and efficient manner, thereby improving patient prognosis, has become a research hotspot in the field of blood purification.

[0004] To reduce the free levels of protein-bound liver failure toxins on the dialysate side, enabling these toxins to continuously diffuse into the dialysate side and be cleared from the blood during hemodialysis, some researchers have demonstrated that albumin dialysis can significantly improve the clearance of liver failure toxins from the blood (J Hepatol, 1999, 31(6): p. 1080-5). However, due to its high cost, it cannot be widely used in clinical practice.

[0005] In recent years, with the rapid development of artificial liver support technology, the MARS system based on albumin dialysis, the DPMAS system based on plasma separation and adsorption, and the Prometheus system have been gradually applied in clinical practice. All of these methods have been proven to effectively improve the clearance efficiency of protein-bound liver failure toxins. The MARS system increases the diffusion of protein-bound toxins by using albumin dialysate and regenerates the albumin on the dialysate side using activated carbon adsorption columns and anion exchange resin adsorption columns for recycling. Studies have found that the therapeutic effect of the MARS system is related to its treatment dose; patients with acute liver failure who receive more than three MARS treatments have significantly higher survival rates than those who receive fewer than three treatments (AnnInternMed, 2013.159(8):p.522-31; JGH Open, 2020.4(4):p.757-763). This also means that MARS treatment for acute liver failure requires repeated administration, significantly increasing treatment costs. At the same time, the MARS system also has limitations such as complex operation and toxin clearance efficiency limited by the adsorption capacity of activated carbon and resin, as well as the circulating albumin solution.

[0006] The Prometheus system removes protein-bound toxoids from component plasma through a plasma separation and adsorption system. The purified plasma is then returned to the body via a high-flux hemodialysis system. Studies have found that the Prometheus system is superior to the MARS system in clearing protein-bound toxoids (Blood Purif, 2012, 34(2): p.158-63). However, because the Prometheus system uses endogenous albumin, it leads to albumin loss in patients. Furthermore, the Prometheus system suffers from drawbacks such as insufficient biocompatibility of the adsorption system, high cost, and complex operation.

[0007] The DPMAS system sequentially passes the separated plasma through an anion exchange resin plasma bilirubin adsorption column and a neutral macroporous resin adsorption column, after which it combines with blood cells and other formed elements and returns to the body. However, the DPMAS system has drawbacks, such as the potential loss of some albumin and clotting factors from the plasma. These limitations restrict the widespread clinical application of this artificial liver system.

[0008] On the other hand, during hemodialysis, the types of reactive oxygen species in the blood increase due to contact between the blood and the dialysis circuit, dialysis membrane, and dialysate. Because antioxidants in the blood are removed, dialysis patients are chronically under oxidative stress. This oxidative stress exacerbates the inflammatory response in dialysis patients and is associated with the onset and aggravation of various complications.

[0009] Regarding the clearance of protein-bound toxins, CN114712307B provides a fat emulsion dialysis solution comprising long-chain fat emulsion oil, medium-chain triglycerides, antioxidants, sodium oleate, glycerol, phospholipids, and solvents, which has been proven to be a solution with good stability and safety. This solution improves the clearance rate of PBUTs (including homocysteine, indole sulfate, and p-thiosulfate) in uremic patients, and the effect has been clinically validated. However, its effectiveness in clearing protein-bound liver failure toxins and reducing oxidative stress levels during artificial liver support therapy in patients with acute and chronic liver failure remains unclear.

[0010] Therefore, developing a new, efficient, safe, and inexpensive artificial liver support treatment plan remains an urgent need for the clinical treatment of patients with liver failure. Summary of the Invention

[0011] One object of the present invention is to provide a lipid-containing composition to facilitate the removal of protein-bound toxins from the blood and to treat patients with liver failure.

[0012] Another object of the present invention is to provide a reagent containing a lipid composition for removing protein-bound toxoids from the blood.

[0013] Another object of the present invention is to provide the use of a lipid-containing composition in the preparation of a medical device (e.g., dialysis fluid) for improving the clearance level of protein-bound toxins in patients with liver failure, in order to solve the problem of poor toxin clearance effect in the prior art for liver failure.

[0014] Another object of the present invention is to provide a medical device, such as a dialysis solution, for hemodialysis of patients with liver failure to remove protein-bound toxins from the blood.

[0015] In this invention, the term "vegetable oil" refers to oil used to prepare compositions containing long-chain lipids, such as, but not limited to, one or more of soybean oil and olive oil.

[0016] In this invention, the term "phospholipid" should be understood as one or a mixture containing multiple phospholipids, such as soybean phospholipids and egg yolk phospholipids, wherein lecithin accounts for 75wt% to 95wt%, and also contains one or more functional phospholipids such as, but not limited to, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and sphingomyelin, accounting for 1wt% to 15wt% of the total phospholipids.

[0017] In this invention, the antioxidants referred to include, but are not limited to, tocopherol and squalene. These substances are used alone or in combination in amounts such as 0.05% to 0.3%.

[0018] In this invention, the medium-chain triglycerides contain fatty acid chains selected from C6-C12 saturated fatty acids, especially C8 and C10 fatty acid chains, for example: caprylic acid content of 55.4-61.4 wt%, 55.4-56.4 wt%, 56.4-57.4 wt%, 57.4-58.4 wt%, 58.4-59.4 wt%, 59.4-60.4 wt%, or 60.4- The content of caprylic acid is 61.4 wt%, and the content of decanoic acid can be 38.5–44.5 wt%, 38.5–39.5 wt%, 39.5–40.5 wt%, 40.5–41.5 wt%, 41.5–42.5 wt%, 42.5–43.5 wt%, or 43.5–44.5 wt%. The total content of caprylic acid and decanoic acid is usually ≥99 wt%, especially ≥99.5 wt%, or even ≥99.9 wt%.

[0019] A lipid-containing composition comprising 2% to 6% phospholipids by weight.

[0020] One specific form of the lipid-containing composition provided by the present invention is an emulsion.

[0021] Another lipid-containing composition, in emulsion form, includes 2% to 6% phospholipids.

[0022] The lipid-containing composition of the present invention also includes medium-chain triglycerides, antioxidants, and sodium oleate. These substances are used alone or in combination in the present invention.

[0023] Another lipid-containing composition, by weight, comprises:

[0024] Vegetable oil 3%–15%,

[0025] Medium-chain triglycerides 1.5%–9%,

[0026] Antioxidant 0.05%–0.3%,

[0027] Sodium oleate 0.05%–0.3%,

[0028] Phospholipids 2%–6%, and

[0029] The remainder is water.

[0030] Verification has shown that the various compositions provided by this invention have the effect of competitively capturing protein-bound toxoids in the blood, thereby achieving the purpose of clearing protein-bound toxoids from the blood, especially for patients with liver failure.

[0031] It exhibits adsorption properties for toxins leading to liver failure and demonstrates good biocompatibility. Applying the lipid-containing composition to the dialysate can continuously adsorb free toxins in the dialysate, attracting more protein-bound toxins from the blood side that cause liver failure to the dialysate side, where they are encapsulated within the lipid-containing composition. This enhances the toxin removal efficiency of dialysis. This method is simple to apply, requires no new equipment, is compatible with existing dialysis treatments, and has the potential to significantly improve dialysis efficacy, increase toxin removal efficiency, reduce mortality in patients with liver failure, and improve their prognosis.

[0032] The lipid-containing composition of the present invention also contains vitamin E to improve the oxidative stress state of dialysis patients, at a content of 0.6% to 2%, especially 0.8% to 1.6%.

[0033] Lipid-containing compositions are used as reagents to remove protein-bound toxoids from the blood. When applied clinically, for example, in the preparation of dialysate, glucose is added, for example, at 10 mM.

[0034] When the composition of the present invention is used clinically, electrolytes such as sodium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, and potassium chloride are also added. These compounds are used alone or in combination in the present invention.

[0035] When the composition of the present invention is used clinically, electrolytes are added, for example, to make the sodium ion concentration 141 mM.

[0036] When the composition of the present invention is used clinically, electrolytes are added, for example, to make the magnesium ion concentration 0.75mM.

[0037] When the composition of the present invention is used clinically, electrolytes are added, for example, to make the calcium ion concentration 1.5 mM.

[0038] When the composition of the present invention is used in clinical practice, electrolytes are added, for example, the potassium ion concentration is in the range of 0-4 mM according to the patient's blood potassium ion level.

[0039] When the composition of the present invention is used clinically, electrolytes are added, for example, to make the bicarbonate ion concentration 35mM.

[0040] When the composition of the present invention is used clinically, electrolytes are added to make the sodium ion concentration 141 mM, the magnesium ion concentration 0.75 mM, the calcium ion concentration 1.5 mM, the potassium ion concentration range 0-4 mM, and the bicarbonate concentration 35 mM.

[0041] This invention provides various lipid-containing compositions, which, when combined with glucose, electrolytes, or physiological saline, are formulated into dialysis solutions or packaged as dialysis kits and other medical devices for removing protein-bound toxoids from the blood of patients with liver failure. The dialysis solution contains the lipid-containing composition of this invention at a concentration of 30 g / L to 50 g / L. Attached Figure Description

[0042] Figure 1 The graph shows the verification results of the clearance of liver failure-associated toxins cholic acid (CA) and glycocholic acid (GCA) by dialysate containing the composition of the present invention using the rapid balance plate method.

[0043] Figure 2 This is a statistical chart showing the toxin clearance rate of test animals after dialysis. Detailed Implementation

[0044] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0045] The following embodiments of the present invention use hemodialysis filtration replacement fluid as a control.

[0046] In the following embodiments of the present invention, the concentration of liver failure toxin was detected using the Chemray 800 fully automated biochemical analyzer from Shenzhen Raydu Life Science & Technology Co., Ltd.

[0047] In the following embodiments of the present invention, SPSS 21.0 statistical software was used for data processing, and the data are expressed as mean ± standard deviation. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001.

[0048] Example 1: Preparation of lipid-containing composition solution

[0049] Weigh out 5.8g soybean oil, 4.3g medium-chain triglycerides, 0.1g α-tocopherol, 0.1g sodium oleate, and 2% phospholipids (by weight, soybean phospholipids: phosphatidylserine = 7:1), mix and stir until fully dissolved to form the oil phase. Pre-treat the organic phase by high-speed shearing. Slowly inject the organic phase into water (75℃) and shear at high speed (e.g., 5000rpm~35000rpm) for 15min. Homogenize the sheared solution using a high-pressure homogenizer at 800-1200bar pressure in an equal increment (e.g., 800bar, 1000bar, and 1200bar, 3 times per gradient) to obtain a lipid-containing composition solution.

[0050] Example 2: Preparation of dialysis solution containing lipid composition

[0051] Glucose and electrolytes were added to the lipid-containing composition solution obtained in Example 1 to prepare a lipid-containing composition dialysate.

[0052] The glucose concentration in the dialysate is 10 mM.

[0053] The dialysate contained sodium ions at a concentration of 141 mM, magnesium ions at a concentration of 0.75 mM, calcium ions at a concentration of 1.5 mM, potassium ions in the range of 0–4 mM, and bicarbonate at a concentration of 35 mM.

[0054] Example 3: Rapid Balanced Plate Dialysis Method for Evaluating the Toxin Clearance Effect in Liver Failure

[0055] Add bile acids at liver failure levels, including 200 μmol / L cholic acid (CA) and 200 μmol / L glycocholic acid (GCA), to a 40 g / L bovine serum albumin (BSA) solution and incubate for 30 min. Add 300 μL of BSA solution containing liver failure toxin to the sample compartment of a rapid equilibration plate (12 kD, Thermo Scientific). Add 500 μL of ordinary dialysate or dialysate containing the lipid composition of this invention (oil phase: 12%, i.e., total amount of vegetable oil and medium-chain triglycerides) to the dialysate compartment. Place the rapid equilibration plate on a shaker and equilibrate and dialyze for 4 hours at 37°C and 250 rpm. Detect the toxin concentration in the sample compartments before and after equilibration using high-performance liquid chromatography (HPLC). Calculate the clearance rate of protein-bound toxins for each peritoneal dialysis solution using the following formula:

[0056] Toxin clearance rate (%) = (Toxin concentration in sample before dialysis - Toxin concentration in sample after dialysis) / Toxin concentration in sample before dialysis × 100%.

[0057] Toxin clearance rate test results are as follows Figure 1As shown in the figure, the clearance rate of bile acids by the dialysate containing the lipid composition was 16.74±0.53%, significantly higher than that of the blank control group (8.53±0.76%) (***p<0.001). The clearance rate of glycocholic acid was 37.78±0.34%, significantly higher than that of the blank control group (24.76±0.76%) (***p<0.001), indicating that the dialysate containing the lipid composition can significantly improve the in vitro clearance rate of liver failure toxins.

[0058] Example 4: Evaluation of liver failure toxin clearance effect by rat hemodialysis

[0059] An acute cholestatic liver failure model was induced using α-nephthylisothiocyanate (ANIT). ANIT was dissolved in 2 mL of olive oil and administered by gavage at a dose of 100 mg / kg once. After 48 hours, rats were randomly divided into a conventional dialysate group and a dialysate group containing the lipid-containing composition (12%) of the present invention.

[0060] A PE50 (polyethylene, PE) catheter pre-filled with heparinized saline (62.5 IU / mL heparin) was used as the vascular cannula, connected to a three-way valve. The rat was placed in a supine position on a heating pad to maintain its body temperature during the procedure. After shaving the neck, the skin was disinfected with povidone-iodine. The neck skin was incised along the midline, and the neck muscles were bluntly separated to locate the right carotid nerve sheath. The right carotid artery, approximately 1.2 cm in length, was bluntly dissected. The distal end of the carotid artery was ligated, and the proximal end of the right carotid artery was temporarily clamped with an arterial clamp. A small incision was made in the artery between the ligation and clamping points using microscissors. The PE50 catheter was slowly inserted into the carotid artery proximal to the heart, and the suture was tied to secure it. The arterial clamp was released, and the three-way valve was opened. Arterial blood pulsation was observed in the vascular cannula. A blood sample was collected before dialysis, taking care to avoid collecting saline. Subsequently, 0.5 mL of heparinized saline was slowly injected. Similarly, the left jugular vein was dissected, cannulated, and heparinized saline was injected.

[0061] A rat hemodialysis platform was constructed using a mini-dialyzer and extracorporeal circulation tubing, with the right carotid artery as the outflow pathway and the left jugular vein as the inflow pathway. A small peristaltic pump controlled the blood flow rate and dialysate flow rate during extracorporeal circulation. The blood flow rate was maintained at 1.5 ml / min, and the dialysate flow rate at 5 ml / min, ensuring a net ultrafiltration rate of 0 ml / min during dialysis. Before hemodialysis, the blood side of the mini-dialyzer was pre-flushed with heparinized saline, and the dialyzer was pre-flushed with standard bicarbonate hemodialysis solution. After weighing, the rats were anesthetized with isoflurane, and heparin sodium was administered for anticoagulation during hemodialysis. A closed-loop circulation mode was used for the dialysate in each rat. Arterial blood and dialysis samples were collected at 10 min, 40 min, 70 min, and 100 min of dialysis. Total bile acids in the blood and dialysate samples were detected using biochemical methods, while indole and tryptophan were detected using high-performance liquid chromatography (HPLC). The clearance rate of toxins in the test animals by each dialysis solution was calculated using the following formula:

[0062] Toxin clearance rate (%) in test animals = Toxin concentration in dialysate / Toxin concentration in blood

[0063] Test results as follows Figure 2 As shown in the figure. Except for 10 min, the clearance rates of the lipid dialysate for the three representative liver failure toxins, namely total bile acids, indole, and tryptophan, were significantly higher than those of the ordinary dialysate at all other time points. This indicates that the lipid dialysate prepared using the lipid-containing composition of this embodiment can significantly improve the dialysis clearance rate of liver failure toxins.

Claims

1. The use of a lipid-containing composition in the preparation of a dialysis fluid for removing liver failure-related toxins, characterized in that... The lipid-containing composition is in emulsion form and, by weight, includes 2%–6% phospholipids, of which 75 wt%–95 wt% lecithin and 1 wt%–15 wt% functional phospholipids. The dialysate contains 30 g / L–50 g / L of the lipid-containing composition and also includes 3%–15% vegetable oil, 1.5%–9% medium-chain triglycerides, 0.05%–0.3% antioxidants, and 0.05%–0.3% sodium oleate. The lecithin is soybean lecithin, and the functional phospholipid is phosphatidylserine.

2. The application according to claim 1, characterized in that... It also contains vitamin E, at a concentration of 0.6% to 2%.

3. The application according to claim 1, characterized in that... The dialysate also includes one or more of glucose, sodium ions, magnesium ions, calcium ions, potassium ions, and carbonate ions.

4. The use of a reagent in the preparation of dialysis fluid for removing liver failure-related toxins, characterized in that... The reagents include the lipid-containing composition of claim 1.

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