Protective aminoacid agents for use in peritoneal dialysis

Abstract

The present invention relates to a protective agent selected from the group consisting of L-glutamine, L-alanyl-L-glutamine, L-glutamyl-L-alanine, L-glutamyl-L-glycine, L-glycyl-L-glutamine, or a mixture thereof, and to a method for preparing the protective agent, the present invention relates to a protective agent for use in the prevention and / or treatment of diseases associated with end-stage nephropathy (ESKD) mediated by liver dysfunction induced by peritoneal dialysis treatment, specifically for the prevention and / or treatment during peritoneal dialysis treatment: (A) liver diseases induced by said peritoneal dialysis treatment and / or (B) diseases associated with ESKD mediated by liver dysfunction induced by said peritoneal dialysis treatment, wherein said protective agent is administered by intraperitoneal administration.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480016703.5 (22) Application Date 2024.03.01 (30) Priority Data 23159875.6 2023.03.03 EP (85) PCT International Application Entering National Phase Date 2025.09.03 (86) PCT International Application Application Data PCT / EP2024 / 055347 2024.03.01 (87) PCT International Application Publication Data WO2024 / 184220 EN 2024.09.12 (71) Applicant: Jeto Protect Flexible Capital Company, Address: Vienna, Austria (72) Inventors: Christoph Overchter, Fabian Ebenstein, Nare, Rebecca Herzog, Klaus Crotchwell (74) Patent Agency: Beijing Longan Law Firm, 11323 Patent Attorney: Quan Xianzhi (51) Int.Cl. A61K 31 / 198 (2006.01) A61K 38 / 05 (2006.01) A61P 1 / 16 (2006.01) A61P 13 / 12 (2006.01) A61K 9 / 00 (2006.01) A61M 1 / 28 (2006.01) A61P 9 / 00 (2006.01) (54) Invention Title Protective Amino Acid Agent for Peritoneal Dialysis (57) Abstract This invention relates to a protective agent selected from the group consisting of L-glutamine, L-alanyl-L-glutamine, L-glutyl-L-alanine, L-glutyl-L-glycine, L-glycyl-L-glutamine, or mixtures thereof, specifically for the prevention and / or treatment during peritoneal dialysis treatment of: (A) liver disease induced by said peritoneal dialysis treatment and / or (B) liver dysfunction mediated by said peritoneal dialysis treatment associated with end-stage renal disease (ESKD), wherein said protective agent is administered via intraperitoneal administration. Claims 1 page, Description 11 pages, Drawings 4 pages, CN 120957713 A 2025.11.14 CN 1 20 95 77 13 A 1. A protective agent selected from the group consisting of L-glutamine, L-alanyl-L-glutamine, L-glutyl-L-alanine, L-glutyl-L-glycine, L-glycyl-L-glutamine, or mixtures thereof, specifically for the prevention and / or treatment during peritoneal dialysis treatment of: (A) liver disease induced by said peritoneal dialysis treatment and / or (B) liver dysfunction mediated by said peritoneal dialysis treatment and associated with end-stage renal disease, i.e., ESKD, wherein said protective agent is administered via intraperitoneal administration.2. The protective agent of claim 1, wherein the liver disease (A) caused by the peritoneal dialysis treatment is a chronic liver disease, particularly non-infectious liver inflammation. 3. The protective agent of claim 2, wherein the non-infectious liver inflammation includes non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and metabolic (functional)-associated fatty liver disease (MAFLD). 4. The protective agent of any of the preceding claims, wherein the disease (B) mediated by liver dysfunction associated with ESKD is a cardiovascular disease, particularly atherosclerotic cardiovascular disease, including inflammation-driven cardiovascular disease and metabolic inflammation-driven cardiovascular disease. 5. The protective agent of any of the preceding claims, wherein administration of the protective agent begins at the start of the peritoneal dialysis treatment. 6. The protective agent of any of claims 1 to 4, wherein administration of the protective agent begins some time after the start of the peritoneal dialysis treatment. 7. The protective agent of claim 6, wherein administration of the protective agent begins once indicators of liver disease or liver-mediated systemic inflammation, particularly metabolic inflammation or cardiovascular disease, are detected. 8. The protective agent according to claim 7, wherein the indicator is selected from the group consisting of biomarkers, such as laboratory markers and / or imaging results. 9. The protective agent according to any one of the preceding claims, wherein the protective agent is administered as a component of peritoneal dialysis fluid (PDF). 10. The protective agent according to claim 9, wherein the peritoneal dialysis fluid is administered as the sole peritoneal dialysis fluid during peritoneal dialysis treatment. 11. The protective agent according to claim 9, wherein the peritoneal dialysis fluid is administered in combination with another peritoneal dialysis fluid. 12. The protective agent according to any one of claims 9 to 11, wherein the peritoneal dialysis fluid is based on glucose as an osmotic agent. 13. The protective agent according to any one of the preceding claims, wherein the protective agent is L-alanyl-L-glutamine, optionally mixed with one or more other protective agents. Claims 1 / 1 page 2 CN 120957713 A Protective Amino Acid Agent for Peritoneal Dialysis Technical Field

[0001] The present invention relates to protective agents for peritoneal dialysis treatment, their specific therapeutic uses, and treatment methods using said protective agents. Background Art

[0002] Chronic kidney disease (CKD) and loss of kidney function lead to uremic toxin retention and fluid excess, most notably in patients with end-stage renal disease (ESKD) (1). ESKD is also closely associated with an increased risk of systemic inflammation, including metabolic inflammation, which is defined as a chronic low-grade inflammatory state induced by metabolic changes. In addition, ESKD is associated with atherosclerosis and cardiovascular disease (CVD) (2).

[0003] Peritoneal dialysis (PD) is a home renal replacement therapy for ESKD with similar survival rates compared to hemodialysis (HD) (3). Peritoneal dialysate (PDF) is injected into the peritoneal cavity using a sterile procedure and removed after several hours. PD is primarily driven by PDF pressure gradients based on hypertonic glucose or colloids, reducing uremic toxins and fluid excess through small solute diffusion and ultrafiltration of water across the peritoneum. While PD provides a life-saving renal replacement, it also introduces typical surgery-related complications, increasing the incidence of uremia in ESKD (3). Prolonged exposure to PDF can lead to a high load of glucose and its degradation products (GDP) in the peritoneum, which is associated with inflammation and vascular changes (4, 5) and may enhance metabolic and cardiovascular risk profiles (6).

[0004] Most commercially available PDFs contain glucose monohydrate as their primary osmotic agent. Alternatively, PDFs may contain glucose polymers (i.e., icodextrin) or colloidal osmotic substances (such as amino acids, peptides).

[0005] Some clinical and experimental observations have shown that PDF is cytotoxic and is associated with a technical failure risk of up to 30% in long-term PD treatment (7).

[0006] WO2008 / 106702A1 provides a glucose-based peritoneal fluid containing a protectant in the form of L-glutamine and a dipeptide capable of releasing L-glutamine, said dipeptide being L-glutyl-L-glycine, L-glycine-L-glutamine, L-glutyl-L-alanine, or L-alanyl-L-glutamine, or a mixture of two or more said dipeptides, wherein said dipeptide is present in a concentration of 2 mM to 25 mM in the dialysate.

[0007] According to this patent application, these dipeptides, particularly L-alanyl-L-glutamine (hereinafter referred to as alanyl-glutamine or "Ala / Gln"), have been found to help prevent said technical failure.

[0008] The common idea behind these protectants is their ability to release L-glutamine. Therefore, although alanyl-glutamine is mentioned primarily below, it is expected that the reported effects will also be achieved when other protective agents from the above group are used.

[0009] Some publications have further investigated the role of Ala / Gln in glucose-based PDF (8-26).

[0010] The liver is increasingly considered a central hub connecting metabolism and CVD (27). To enable the liver to tolerate exposure to gut-derived microbes and dietary molecules, metabolic activity and associated inflammatory processes are tightly controlled. Stimulation occurs when there is a need to clear hepatotropic pathogens or toxic products of metabolic activity (28). Several studies have shown an epidemiological association between CKD and chronic liver disease, with recent focus on metabolic stress leading to renal-hepatic crosstalk (29, 30).

[0011] In CKD, the liver may represent the victim, developing fatty liver disease associated with metabolic (functional impairment).Specific conditions such as MAFLD may also represent the perpetrator, mediating an environment that causes CVD associated with CKD. The liver plays a central role in detecting and responding to extrahepatic and intrahepatic signals through an acute-phase response, which leads to an amplification of inflammatory stimuli by several orders of magnitude. Hepatocytes produce acute-phase proteins (such as complement proteins, C-reactive protein [CRP]), which then promote systemic inflammation through direct effector functions (27, 28).

[0012] Although these pathological mechanisms are very common in ESKD patients receiving PD treatment, the inventors of the present invention are unaware of any mechanistic studies linking PD to liver disease or elucidating the role of PD-induced liver dysfunction in PD-related conditions (such as CVD).

[0013] Clinical reports linking PD to the liver are few and inconclusive. Historical reports have shown the presence of subcapsular hepatic steatosis in patients with PD and its occurrence has been associated with the co-administration of intraperitoneal insulin and glucosyl PDF, which is associated with high rates of peritoneal metastasis and high body weight (31, 32). A small cross-sectional study reported that the high prevalence of chronic liver disease in patients with PD was associated with a high risk of atherosclerosis, and therefore with CVD (33). In some studies, patients with PD have been reported to have higher liver enzymes compared to patients with HD (34), but this has not been observed in other studies (35). Liberato et al. discussed the possibility that the elevated levels of gamma-glutamyl transferase (GGT) observed in two dialysis groups may be associated with malnutrition-inflammation-atherosclerosis syndrome (34), and thus closely associated with CVD. Therefore, it remains unclear whether PD has a specific effect on the liver in clinical PD, and the role of PD-induced liver dysfunction in the inflammatory response.

[0014] Further background information is available from Ferrantelli Evelina et al., Kidney International 89(3)2016, 625-635 and Mikolasevic et al., Medical Hypotheses 82(2)2013, 205-208. Summary of the Invention

[0015] The object of the present invention is to provide an improvement in the side effects associated with PD.

[0016] This object is achieved by the subject matter of claim 1.

[0017] Other preferred embodiments of the invention are disclosed in the dependent claims. Brief Description of the Drawings

[0018] Figure 1: Peritoneal dialysis-induced liver inflammation response. Control and uremic mice with and without PD.

[0019] Inflammation score and non-alcoholic fatty liver disease (NAFLD) activity score (NAS). P-values ​​calculated by the Mann-Whitney U test, without multiplicity correction.

[0020] a. Changes in healthy animals

[0021] b. Changes in uremic animal models

[0022] Figure 2: Effect of alanyl-glutamine supplementation to PDF on PD-induced liver inflammatory response. Control mice, PD-treated uremic mice, and PD and AlaGln-treated uremic mice. P-values ​​calculated by Mann-Whitney U test, without multiplicity correction.

[0023] a. Inflammation score and NAFLD activity score (NAS);

[0024] b. Percentage of area of ​​Oil Red O stained tissue (=fatty degeneration) on liver sections

[0025] c. Percentage of area of ​​Sirius red stained tissue (=fibrosis) on liver sections.

[0026] Figure 3: Effect of alanyl-glutamine administration on specific biomarkers related to cardiovascular disease. Control mice, PD-treated uremic mice, and PD and AlaGln-treated uremic mice.

[0027] The abundance of APOH, CFB, and CRP in liver tissue was assessed by mass spectrometry proteomics analysis.

[0028] Figure 4: Intercorrelation of different liver parameter changes induced by alanyl-glutamine treatment in patients with long-term PD, and correlation with changes in systemic inflammation parameters. A secondary analysis of a prospective multicenter phase II trial on the effects of alanyl-glutamine on clinical parameters of liver injury in patients with long-term PD is supplemented in the PDF.

[0029] a. Spearman rank correlation, black blocks indicate positive correlation, p < 0.1 (without multiplicity correction). White blocks indicate non-significant positive correlation (p ≥ 0.1).

[0030] b. Spearman rank correlation between changes in plasma interleukin-6 and changes in GGT levels (rho = 0.35, p = 0.03, without multiplicity correction). Detailed Description

[0031] The inventors have for the first time analyzed the effects of PD itself on the liver of uremic and non-uremic animals and surprisingly found that PD, particularly PD utilizing glucosyl PDF, itself induces liver disease, thereby increasing the likelihood of disease mediated by liver dysfunction.

[0032] In the inventors' study, uremic and non-uremic animals receiving PD were allowed to conduct mechanistic studies to elucidate the specific effects of PD on the molecular response of the liver in a tightly controlled experimental model of CKD. Experimental data showed that PD treatment was associated with increased liver inflammation and acute-phase reactions as a novel treatment-specific adverse reaction, which may diminish its beneficial effects in reducing uremic toxemia and hyperhydration in ESKD patients. Furthermore, in the inventors' study, the experimental setting of PD in non-uremic animals allowed for unique insights into the pathological mechanisms associated with specific PD surgery, which otherwise do notThese insights were obtained through experiments. Interestingly, the results of these experiments clearly confirmed the differential expression of liver proteins associated with acute-phase response signals in the liver, without CKD as a confounding factor.

[0033] These data do indeed suggest a direct causal role for specific PD factors in inducing liver inflammation and acute-phase response signals, as well as subsequent systemic inflammation (particularly metabolic inflammation) and CVD in patients undergoing PD. Excessive energy and nutrients directly transferred to the liver by PDF-induced (portal venous flow through the peritoneum) may lead to metabolic stress, triggering altered metabolic inflammation and immune responses, thereby promoting liver disease and CVD.

[0034] In summary, the inventors have found direct evidence of PD-induced liver symptoms and dysfunction. By applying established long-term PD exposure models to uremic and non-uremic mice, their histological and proteomic findings directly linked chronic intraperitoneal PDF exposure, via the portal peritoneum-liver axis, to liver inflammation and liver-mediated systemic inflammation (particularly metabolic inflammation), independent of CKD.

[0035] The inventors have found that the use of the protective agent of claim 1, particularly alanyl-glutamine, during intraperitoneal administration during peritoneal dialysis treatment can improve the negative effects of PDF on the liver.

[0036] The inventors investigated the potential effects of alanyl-glutamine supplementation on the liver in a long-term rat PD model. This treatment significantly reduced PD-induced liver symptoms and liver-mediated metabolic inflammation. Histologically, the improvement in NAFLD activity score (NAS) primarily due to reduced inflammatory infiltration provides evidence of alanyl-glutamine-mediated reduction in PD-induced morphological liver pathology. This morphological improvement is correlated with corresponding molecular responses shown in the rat liver proteome.

[0037] Alanyl-glutamine has been shown to reduce liver reperfusion injury in rat models, protect mice from LPS-induced acute liver injury, and significantly reduce steatohepatitis and fibrosis (36-38) in rat NAFLD models.

[0038] However, to date, no one has proposed that the use of alanyl-glutamine directly affects liver disease induced by PD and / or further liver-related negative consequences of PD.

[0039] Therefore, in a first aspect, the present invention provides a protective agent selected from L-glutamine, L-alanyl-L-glutamine, L-glutyl-L-alanine, L-glutyl-L-glycine, L-glycyl-L-glutamine or mixtures thereof, comprising the group specified in the specification 3 / 11 page 5 CN 120957713 A, particularly for the prevention and / or treatment of the following diseases during peritoneal dialysis treatment:

[0040] (A) liver disease induced by said peritoneal dialysis treatment and / or

[0041] (B) liver dysfunction mediated by said peritoneal dialysis treatment and associated with end-stage renal disease (ESKD).

[0042] The protective agent is administered via intraperitoneal administration.

[0043] Another aspect of the invention relates to the preventive and / or therapeutic use of the protective agent during peritoneal dialysis treatment of said disease (A) and / or (B).

[0044] Another aspect of the invention relates to the use of the protective agent in the preparation of medicaments for the treatment or prevention of said disease (A) and / or (B) during peritoneal dialysis treatment.

[0045] Another aspect of the invention relates to a method of preventing and / or treating said disease (A) and / or (B) by administering the protective agent to a subject in need via intraperitoneal administration during peritoneal dialysis treatment.

[0046] The following discussion relates to any of the foregoing aspects with necessary modifications.

[0047] In a preferred embodiment of the invention, the liver disease (A) induced by peritoneal dialysis treatment is a chronic liver disease, particularly non-infectious liver inflammation.

[0048] Non-infectious liver inflammation includes diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and metabolic (functional)-associated fatty liver disease (MAFLD).

[0049] In patients with ESKD, the pathophysiological mechanisms linking PD to the liver cannot be separated from the effects of renal-hepatic crosstalk in CKD and its chronic low-grade inflammatory factors and metabolic factors (e.g., associated with atherosclerotic dyslipidemia and dysglucose abnormalities (30)). CKD and fatty liver disease share many common risk factors (e.g., obesity, hypertension, insulin resistance, type 2 diabetes, atherogenic dyslipidemia), therefore the prevalence of CKD in the NAFLD population is approximately 20-55%, significantly higher than the 5-35% in the non-NAFLD population (39). In addition, several studies have shown a correlation between CKD and NAFLD, describing a broader phenotype of fatty liver disease associated with metabolic (functional impairment) (MAFLD), further emphasizing the pathogenic role of metabolic dysfunction, and the increased risk of CVD from CKD, MAFLD, and their common and non-common risk factors (29, 30, 40–43). In a population-based study, the positive correlation between the fatty liver index (a marker of hepatic steatosis) and CKD incidence was entirely mediated by the combined effects of the most important cardiometabolic risk factors (44). Liver pathological markers were significantly associated with serum CRP, ALT, AST, and lipid markers (i.e., cholesterol, LDL, triglycerides) and CVD in dialysis patients (45). Furthermore, liver function parameters were positively correlated with poor prognosis, CVD, and all-cause incidence in patients with ESKD and PD (46).

[0050] To date, there are no reported data on the effects of alanine-glutamine on liver injury and liver-mediated systemic metabolic inflammation (which may cause CVD) in patients with PD.

[0051] Therefore, in addition to the experiments in animal models described above, the inventors also conducted a secondary analysis of the results of a recent clinical PD trial (in human patients, 8) to validate their experimental findings.

[0052] As a result of this new analysis, the inventors were able to determine that the addition of alanyl-glutamine to PDF significantly reduced clinical parameters of liver injury, reaching statistical significance for LDH and liver-specific ALT.

[0053] In the aforementioned trial (8), the addition of alanyl-glutamine to PDF was also reported to reduce systemic inflammation, which was reflected in a decreasing trend of biomarkers such as hs-CRP and IL-6, reaching statistical significance for IL-8 (8, Table 3).

[0054] The inventors' new secondary analysis now also reveals a significant association between changes in markers of systemic inflammation (interleukin-6) and changes in liver enzymes (GGT) and clinical markers of cell lysis (AST, LDH).

[0055] These findings also confirm the inventors' experimental findings in a mouse model that alanyl-glutamine has therapeutic effects on regulated cell death (e.g., necrotizing apoptosis signaling) and systemic inflammation, particularly metabolic inflammation, indicating that alanyl-glutamine works on the liver of PD patients as both a victim (sentinel) and a perpetrator (mediator).

[0056] In summary, the experimental findings from human clinical trials and new secondary data analysis show that, in experimental systems, intraperitoneal treatment with alanyl-glutamine not only alleviates PD-induced liver inflammatory damage and liver-mediated systemic inflammation, especially metabolic inflammation, but also leads to a reduction in clinical parameters related to liver damage and systemic inflammation in patients with long-term PD.

[0057] In another embodiment of the invention, the disease (B) associated with ESKD mediated by liver dysfunction is a cardiovascular disease, particularly atherosclerotic cardiovascular disease, including cardiovascular disease caused by inflammation and cardiovascular disease caused by metabolic inflammation.

[0058] As mentioned above, the liver is increasingly considered a central hub connecting metabolism and CVD.

[0059] As discussed above, in experimental systems, intraperitoneal treatment with alanyl-glutamine not only alleviated PD-induced liver inflammatory damage and liver-mediated systemic inflammation, particularly metabolic inflammation, but also led to a reduction in clinically relevant parameters of liver damage and systemic inflammation in patients with long-term PD.

[0060] The inventors' discovery of a reduction in novel PD side effects by adding alanyl-glutamine to the PDF provides a new targeted therapeutic option for PD patients to improve systemic inflammation, particularly metabolic inflammation, thereby improving CVD prognosis.

[0061] This new therapeutic option could provide a suitable alternative for the administration of anti-inflammatory biologics such as IL-6-specific antibodies.Alternative approach.

[0062] In one embodiment of the invention, administration of the protective agent begins at the start of the peritoneal dialysis treatment.

[0063] In another embodiment of the invention, administration of the protective agent begins some time after the start of the peritoneal dialysis treatment.

[0064] In this embodiment, administration can begin once indicators of liver disease or liver-mediated systemic inflammation, particularly metabolic inflammation, associated with peritoneal dialysis treatment are detected. These indicators can be biomarkers, such as laboratory markers and / or imaging findings.

[0065] As indicators of liver disease, serum markers of hepatobiliary injury (e.g., AST, ALT, GGT, ALP, bilirubin) and / or imaging findings indicating liver injury and / or hepatitis and / or steatosis / deposition and / or fibrosis (e.g., MRI, CT, PET, liver elastography, ultrasound) can be observed.

[0066] As indicators of inflammation / metabolic inflammation, serum biomarkers (e.g., CRP, hs-CRP, IL-6, IL-8, albumin, fibrinogen, SAA) and / or erythrocyte sedimentation rate and / or imaging findings (e.g., PET) can still be observed.

[0067] As indicators of cardiovascular disease, particularly cardiovascular disease caused by inflammation / metabolic inflammation, biomarkers (e.g., CRP, hs-CRP, IL-6, IL-8, fibrinogen, SAA) and / or imaging findings (e.g., angiography, MRI, CT, PET, ultrasound) can still be observed.

[0068] According to the invention, the protective agent is administered intraperitoneally to reach those sites in the body directly affected by PD treatment.

[0069] Intraperitoneal administration can be performed by intraperitoneal injection or infusion of the protective agent in a suitable drug carrier known to those skilled in the art.

[0070] Administration can be performed intermittently or continuously.

[0071] In a particularly preferred embodiment, the protective agent is administered as a component of the peritoneal dialysis fluid (PDF).

[0072] The PDF containing the protective agent can preferably be exactly the same as the PDF administered to the patient for PD treatment.

[0073] In this case, the body that has already undergone PD treatment does not need to receive another different treatment regimen.

[0074] In this embodiment, the PDF containing the protective agent can be administered from the start of the PD treatment (as the sole PDF specified on page 5 / 11 of the specification, CN 120957713 A).

[0075] Alternatively, if treatment is started with another peritoneal dialysis fluid, the other PDF can be completely replaced by the liquid containing the protective agent, especially after indicators of liver disease or liver-mediated systemic inflammation, particularly metabolic inflammation, are detected.

[0076] Preferably, during PD treatment, the liquid containing the protective agent is administered as the sole PDF. This meansThe patient is treated with only PDF containing the protectant.

[0077] In another embodiment, the fluid may be administered in combination with another peritoneal dialysis fluid. The other peritoneal dialysis fluid may be selected from any available PDF compatible with the PDF containing the protectant.

[0078] In another embodiment of the invention, the PDF containing the protectant is based on glucose as an osmotic agent.

[0079] Preferably, the protectant used according to the invention or in the method of the invention is L-alanyl-L-glutamine, optionally mixed with one or more other protectants.

[0080] Examples

[0081] Examples 1 and 2—Rat Experimental Models

[0082] Methods

[0083] The inventors conducted a mouse PD exposure experiment similar to long-term PD in healthy and uremic mice:

[0084] The experiment consisted of n=34 animals divided into six different groups.

[0085] The animals were divided into the following groups:

[0086] 1) Healthy control animals (“Control”)

[0087] 2) Healthy animals receiving PD treatment (via indwelling catheter) (“PD”)

[0088] 3) Healthy animals receiving PD treatment supplemented with alanyl-glutamine (via indwelling catheter) (“PD+AG”)

[0089] 4) Uremic animals (“Uremia”)

[0090] 5) Uremic animals receiving PD treatment (via indwelling catheter) (“Uremia PD”)

[0091] 6) Uremic animals receiving PD treatment supplemented with alanyl-glutamine (via indwelling catheter) (“Uremia PD+AG”)

[0092] All animals in the uremia group (groups 4, 5, and 6) underwent left 2 / 3 nephrectomy on day 0 of the study and right nephrectomy on day 7 of the study, thereby inducing chronic kidney disease.

[0093] All animals receiving PD treatment (groups 2, 3, 5, and 6) had an indwelling catheter implanted on day 7 of the study.

[0094] Starting on day 14, animals in groups 2, 3, 5, and 6 received daily injections of PDF (DianealPD4 3.86% [Baxter, Deerfield, IL, USA]) via their indwelling catheters for six weeks to induce long-term PDF exposure.

[0095] For animals in groups 3 and 6, the daily PDF injections were supplemented with alanine-glutamine (8 mM) during these six weeks.

[0096] At sacrifice (day 56 of the study), histological analysis of the livers of the animals was performed.

[0097] As described above (47), hematoxylin and eosin (H&E) stained liver tissue was assessed using the NAFLD activity score (NAS) on a facial region image of each mouse.

[0098] Fatty liver was assessed by semi-quantitative measurement of Oil Red O-stained liver tissue using one facial region image per mouse.

[0099] Liver fibrosis was assessed by semi-quantitative measurement of Sirius red-stained liver tissue using one facial region image per mouse.

[0100] Example 1—PD induces liver damage regardless of uremia status—alanyl-glutamine attenuates this effect of PD (6 / 11 pages, CN 120957713 A)

[0101] At the end of the experiment, H&E-stained liver histology showed a significant increase in the number of infiltrating inflammatory immune cells, especially near the interlobular veins (peritoneal flow) of the portal venous system, and an increase in the area of ​​hepatocyte ballooning degeneration in PD-treated animals.

[0102] As shown in Figure 1, these findings were manifested as a significant increase in the NAFLD activity score (NAS) of mice treated with PD.

[0103] Figure 1a shows these changes in healthy animals, while Figure 1b shows these changes in uremia animal models. In both groups, mice treated with PD showed a significant increase in the NAFLD activity score (NAS) compared to mice not treated with PD (p < 0.05).

[0104] Therefore, this effect of PD was observed in both healthy and uremic animals, that is, regardless of the uremic state. These increases in NAS were mainly caused by a higher number of inflammatory infiltrates.

[0105] On the other hand, the addition of alanyl-glutamine to PDF resulted in a significant reduction in the number of inflammatory immune cell infiltrates and a reduction in the area of ​​hepatocyte ballooning degeneration in H&E liver sections, with a significant reduction in NAS semi-quantitatively expressed in uremic mice (p<0.05). Again, this was mainly caused by the presence of inflammatory infiltrates (Fig. 2a). Furthermore, by adding alanyl-glutamine to PDF, a trend toward improvement in PD-induced hepatic steatosis (as shown in the percentage area of ​​Oil Red O stained tissue in Fig. 2b) and fibrosis (as shown in the percentage area of ​​Sirius Red stained tissue in Fig. 2c) was observed.

[0106] Example 2—PD treatment induces liver inflammatory response and acute phase response; similarly, alanyl-glutamine attenuates this effect.

[0107] To investigate the effects of PD-induced or related liver molecular inflammatory responses and to discover potential new therapeutic interventions, mass spectrometry proteomics analysis was performed on the livers of all animals in the rat experiment in Example 1.

[0108] The inventors used Ingenuity pathway Analysis (EMEA Qiagen, Aarhus, Denmark) to extract all identified proteins that are either part of the acute phase response signaling pathway or part of the pathway associated with liver inflammation. In the subset of proteins most important for the differences in livers between PD-treated and non-PD-treated animals identified by PLS-DA (partial least squares discriminant analysis), 57 proteins were significantly differentiated by PD compared to control animals.Significantly abnormal regulation (p<0.05, variance-stable linear model using empirical Bayesian contraction).

[0109] Supplementation with alanyl-glutamine in PDF significantly altered the abundance of 11 acute-phase response and inflammation-related proteins in the liver of uremic animals receiving PD treatment (p<0.05, variance-stable linear model using empirical Bayesian contraction). These 11 proteins are part of acute-phase response signaling (e.g., CFB, APOH, and RELA), glucocorticoid receptor signaling (e.g., HSPA5, NCOR1, NDUFA4, NDUFB3, SDHB, RELA, and IRF3), and necroptosis signaling (e.g., TIMM13, PPID, and IRF3) pathways.

[0110] Figure 3 graphically illustrates this effect on three selected biomarkers (APOH, CFB, and CRP), which are known to be clinically relevant to cardiovascular risk.

[0111] Therefore, the inventors have demonstrated the well-described effects of PD on liver inflammation and liver-mediated systemic inflammation, particularly metabolic inflammation, and the improvement of liver protein levels by supplementing PDF with alanyl-glutamine. The effects of PDF supplementation on PD-induced liver inflammation and liver-mediated systemic inflammation, particularly metabolic inflammation, are primarily achieved by modulating proteins in the acute phase response signaling pathway, the glucocorticoid receptor signaling pathway (regulating NFκB and their respective downstream effects), and the necroptosis signaling pathway.

[0112] Example 3—Alanyl-Glutamine Supplementation in PDF Improves Liver Function (Dysfunction) in Human PD Patients

[0113] To further investigate the effect of alanyl-glutamine supplementation in PDF on liver (dysfunction) induced by human PD, the inventors conducted a secondary analysis of a prospective, multicenter, dual-instruction-database (7 / 11 pages, 9 CN 120957713 A) blinded, controlled, randomized, dual-phase, dual-treatment, crossover, phase II, proof-of-concept study in patients with long-term PD, including eight Austrian centers.

[0114] For this purpose, the inventors included liver tests (GOT / AST, GPT / ALT, GGT, LDH) obtained as safety data in all patients in the complete analysis set, as the protocol-compliant analysis excluded patients with confounding correlations only with local peritoneal effects. This resulted in n=41 patients with long-term PD being randomly assigned to either the placebo group I (arm) or the alanyl-glutamine group I in this crossover study for the purpose of secondary analysis.

[0115] Vychytil et al. have previously described this study cohort and its baseline characteristics (8).

[0116] Supplementation with alanyl-glutamine in the PDF significantly reduced clinical markers of liver injury. In patients with long-term PDIn human trials, these effects of alanyl-glutamine were highly consistent with the effects of alanyl-glutamine at the protein level on regulating cell death (e.g., necroptotic signaling) in mouse experiments described in Example 2.

[0117] Table 1 below shows the effects of alanyl-glutamine supplementation in PDF on functional markers of liver function (disorder).

[0118]

[0119]

[0120] *Log10 transformation due to skewness

[0121] Furthermore, changes induced by alanyl-glutamine supplementation in PDF were observed to be positively correlated with different clinical diagnostic markers of hepatobiliary injury. This is shown in Figure 4a. Since each marker exhibits a different pattern of cell damage within the hepatobiliary system, different significance levels were observed for each correlation pair.

[0122] Figure 4b shows that changes induced by alanyl-glutamine supplementation in PDF were positively correlated with one of the clinical diagnostic markers of hepatobiliary injury (GGT) and a clinical diagnostic marker of inflammation (IL-6). As mentioned above, systemic administration of anti-inflammatory biologics (such as IL-6-specific antibodies) has been developed as a treatment option for metabolic inflammation and reducing CVD risk. Therefore, Figure 4 correlates the observed reduction in clinical diagnostic markers of hepatobiliary injury induced by alanyl-glutamine supplementation in PDF (Table 1, Figure 4a) with the observed reduction in clinical diagnostic markers of inflammation, particularly / including / and metabolic inflammation (Figure 4b). This validates the mechanistic role of alanyl-glutamine observed in the mouse PD model in Examples 1 and 2 in a human randomized, placebo-controlled phase II clinical trial.

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Claims

1. A protective agent selected from the group consisting of L-glutamine, L-alanyl-L-glutamine, L-glutyl-L-alanine, L-glutyl-L-glycine, L-glycyl-L-glutamine, or mixtures thereof, specifically for the prevention and / or treatment of peritoneal dialysis: (A) Liver disease induced by the peritoneal dialysis treatment and / or (B) Diseases related to end-stage renal disease, i.e., ESKD, mediated by liver dysfunction induced by the aforementioned peritoneal dialysis treatment. The protective agent is administered via intraperitoneal administration.

2. The protective agent according to claim 1, wherein the liver disease (A) caused by the peritoneal dialysis treatment is a chronic liver disease, particularly non-infectious liver inflammation.

3. The protective agent according to claim 2, wherein the non-infectious liver inflammation includes non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and metabolic (functional)-associated fatty liver disease (MAFLD).

4. The protective agent according to any one of the preceding claims, wherein the ESKD-related disease (B) mediated by liver dysfunction is a cardiovascular disease, particularly atherosclerotic cardiovascular disease, including inflammation-driven cardiovascular disease and metabolic inflammation-driven cardiovascular disease.

5. The protective agent according to any one of the preceding claims, wherein administration of the protective agent begins at the start of the peritoneal dialysis treatment.

6. The protective agent according to any one of claims 1 to 4, wherein administration of the protective agent begins some time after the start of the peritoneal dialysis treatment.

7. The protective agent according to claim 6, wherein administration of the protective agent is initiated once an indicator of liver disease or liver-mediated systemic inflammation, particularly metabolic inflammation or cardiovascular disease, is detected.

8. The protective agent according to claim 7, wherein the indicator is selected from the group consisting of biomarkers, such as laboratory markers and / or imaging results.

9. The protective agent according to any one of the preceding claims, wherein the protective agent is administered as a component of peritoneal dialysis fluid (PDF).

10. The protective agent according to claim 9, wherein the peritoneal dialysis fluid is administered as the sole peritoneal dialysis fluid during peritoneal dialysis treatment.

11. The protective agent according to claim 9, wherein the peritoneal dialysis fluid is administered in combination with another peritoneal dialysis fluid.

12. The protective agent according to any one of claims 9 to 11, wherein the peritoneal dialysis fluid is based on glucose as an osmotic agent.

13. The protective agent according to any one of the preceding claims, wherein the protective agent is L-alanyl-L-glutamine, optionally mixed with one or more other protective agents.

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