New dialysis fluid

Abstract

The present disclosure provides dialysis solutions comprising ketone bodies, such as acetoacetate, beta-hydroxybutyrate or pharma-ceutically acceptable derivatives, esters and salts thereof, for use in peritoneal dialysis therapy to treat cancer.

Description

[Technical field]

[0001] The present disclosure relates to extracorporeal blood treatment. More specifically, the disclosure relates to the use of peritoneal dialysis in the treatment of cancer. Most specifically, the disclosure relates to a dialysis solution for use in peritoneal dialysis therapy treatment of cancer. [Background technology]

[0002] Most human cancer cells exhibit an altered energy metabolism that distinguishes them from normal cells. Normal cells obtain most of their energy through mitochondrial oxidative phosphorylation, an aerobic process in which glucose is first oxidized by glycolysis and then produces adenosine triphosphate via the tricarboxylic acid (TCA) cycle. Conversely, this pathway is only secondary in cancer cells. This was first observed in the 1920s by Wartburg, who noted that lactate is produced from pyruvate after cancer cells metabolize glucose by glycolysis. In normal cells, this occurs only under anaerobic conditions, but in cancer cells, this alternative pathway is increased even in the presence of abundant oxygen. This phenomenon was named "aerobic glycolysis" or the "Wartburg effect" (Wartburg et al, 1927, Gen Physiol 8:519-530). The presence of a characteristic glycolytic phenotype in cancer cells was also confirmed in subsequent studies, where overexpression of enzymes involved in glycolysis in most cancer cells was observed.

[0003] The metabolic transformations mentioned above give cancer cells a selective growth advantage and contribute to their ability to resist hypoxia and apoptosis. Many tumors grow in hypoxic environments because the rate of tumor cell proliferation exceeds the rate of new blood vessel formation. There are various metabolic changes in cancer cells, the most common and best known being the tendency to generate energy by aerobic glycolysis. In addition, many of the intermediates of glycolysis, such as ribose, glycerol and serine, are also intermediates of biosynthetic and anabolic pathways essential during the growth and proliferation of cancer cells. Glycolysis also produces ATP from ADP, which allows cell proliferation in tumors to be sustained. However, glycolysis is much less efficient than oxidative phosphorylation and therefore requires large amounts of glucose to produce sufficient amounts of ATP. This metabolic pathway therefore requires large amounts of glucose. Many cancer cells become dependent on glucose as their main source of energy. For multiple reasons, glycolytic tumor cells become vulnerable when their glucose supply is targeted.

[0004] Furthermore, many cancer cells also exhibit a dependency on glutamine: not only does the high rate of glutamine uptake exhibited by glutamine-dependent cells result from its role as a nitrogen source in the biosynthesis of nucleotides and amino acids, but glutamine is also the major mitochondrial substrate in cancer, required to generate NADPH for redox regulation and macromolecular synthesis.

[0005] There are other metabolic changes in cancer that play an important role in survival, and importantly, many cancers show a remarkable ability to change their metabolic profile. This plasticity to withstand environmental challenges such as reduced glucose, glutamine or oxygen is crucial for the survival of cancer cells.

[0006] Many metabolic alterations exist in cancer, and various amino acids such as glutamine have important roles in cancer metabolism to control redox balance and generate building blocks for continued proliferation, and moreover, many cancers show a surprisingly good ability to utilize these alternative pathways to change their metabolic profile when they need to adopt new metabolic limitations. This quality is an important feature for cancers and their ability to withstand environmental challenges when metabolic energy resources such as glucose, glutamine or oxygen are scarce.

[0007] Therefore, to effectively affect cancer through metabolic approaches, it is important to affect their metabolic systems from more than one direction. Although glucose reduction can be easily handled by most cancer cells, when several metabolic possibilities are affected simultaneously (such as glucose and glutamine reduction), the sum of these changes becomes much more impactful than the individual parts.

[0008] Besides glucose and glutamine, which are global energy sources for many cancers, serine and glycine fulfill important specific needs to sustain cell growth and proliferation in cancer, for example through one-carbon metabolism. In addition to the large energy requirements, cancer cells must also accumulate building blocks for the construction of new cellular components, including nucleic acids, proteins and lipids, as well as equally important cofactors for the maintenance of their cellular redox state (Amelio et al.: Trends. Biochem. Sci. (2014), vol. 39 (4): 191-198)). Studies have demonstrated that arginine is required for cell proliferation and can be limited in conditions of rapid proliferation, and that depriving cancer cells can affect their survival (Albaugh et al., J.Surg.Oncol.(2017),vol.115(3),273-280). Considering the above, it has been suggested that blood glucose reduction may serve as a strategy to target a wide range of glycolysis-dependent tumors. In hypoglycemic conditions, fat, especially ketone bodies, can replace glucose as the primary metabolic fuel for normal cells. However, many tumors have abnormalities in the genes and enzymes required to metabolize lipids and ketone bodies for energy. Thus, shifting energy from carbohydrates to ketones specifically targets energy metabolism in glycolysis-dependent tumor cells (Seyfried et al, 2010, Nutrition and Metabolism, 7:7).

[0009] According to this approach, for example, WO 2011 / 070527 discloses a method for treating cancerous or non-cancerous proliferative disorders in an individual in which a hemodialysis machine is used to reduce blood glucose concentrations.

[0010] The use of a hemodialysis machine to reduce blood glucose has the advantage that the glucose concentration in the blood can be reduced, and thereby in a more controlled and effective manner, compared to dietary glucose deprivation. However, the method and device disclosed in WO 2011 / 070527 requires a blood glucose sensor and a blood glutamine sensor, which are all connected to the blood intake flow, blood return flow and dialysate, which are all connected to the central control unit of the hemodialysis machine. Furthermore, the central control unit of WO 2011 / 070527 is also connected to an electroencephalograph (EEG) to provide the central unit with information about spontaneous brain electrical activity to initiate the increase in glucose and glutamine levels. Such a large number of sensors and equipment leads to a high level of complexity and associated high costs. Furthermore, the patient undergoing this procedure must consume only a glucose-restricted diet for several days before the procedure is performed. This is not a small burden for the patient.

[0011] There is a continuing need for improved methods of cancer treatment. Most specifically, there is a need for dialysis fluids that can be used in peritoneal dialysis therapy procedures for the treatment of cancer. Summary of the Invention

[0012] The present invention provides a dialysis solution for use in peritoneal dialysis therapy to treat cancer, comprising ketone bodies such as acetoacetate, β-hydroxybutyric acid or pharma-ceutically acceptable derivatives, esters and salts thereof.

[0013] Preferably, the dialysis fluid also contains bicarbonate ions.

[0014] Preferably, the concentration of the ketone body is 1 to 15 mM.

[0015] More preferably, the concentration of the ketone body is 2 to 12 mM.

[0016] Preferably, the concentration of bicarbonate ions is 15 to 40 mM.

[0017] More preferably, the concentration of bicarbonate ions is 20 to 35 mM.

[0018] In a preferred embodiment, the sum of the hydrolysis equivalent concentrations of glucose, pyruvate, and amino acids selected from the group of serine, cysteine, glycine, alanine, glutamic acid, glutamine, proline, aspartic acid, asparagine, threonine, and derivatives thereof in the dialysis solution is up to 3.3 mM.

[0019] The dialysis solution may contain one or more compounds selected from the group of glucose, pyruvate, and amino acids selected from the group of serine, cysteine, glycine, alanine, glutamic acid, glutamine, proline, aspartic acid, asparagine, threonine, or pharma- ceutically acceptable derivatives thereof. Such derivatives may be salts or oligopeptides. When the dialysis solution contains a particular concentration of an oligopeptide, the concentration of the amino acid involved after hydrolysis of the peptide is given (this concentration is referred to herein as the "hydrolysis equivalent concentration"). Examples of such oligopeptides, typically dipeptides in which at least one of the amino acid residues is glutamine, are L-alanyl-L-glutamine and L-glycyl-L-glutamine. When the concentration of the dipeptide L-alanyl-L-glutamine is 1 mM, the hydrolysis equivalent concentration of the alanine and glutamine moieties of this oligopeptide, respectively, is 1 mM alanine and 1 mM glutamine. The hydrolytic equivalent concentration of an amino acid that is not part of an oligopeptide is simply the concentration of the amino acid or amino acid derivative.

[0020] Glutamine-containing oligopeptides, also referred to herein as glutamine-containing compounds, are typically used in place of glutamine in liquid compositions to promote stability and solubility.

[0021] Preferably, the dialysate does not contain pyruvate, serine, cysteine, glutamic acid, proline, aspartic acid, asparagine, threonine, or derivatives thereof.

[0022] Preferably, the dialysate contains a hydrolytic equivalent concentration of glutamine or a derivative thereof of up to 0.3 mM.

[0023] In one embodiment, the dialysis solution may contain an osmotic agent selected from the group of polyethylene glycol and albumin. The osmotic agent is added to make the dialysis solution suitable for peritoneal dialysis.

[0024] In the present disclosure, the term "subject" relates to a human or animal patient in need of treatment.

[0025] The term "ketone bodies" relates to water-soluble molecules containing a ketone group that can be produced by the liver from fatty acids. Typically, the ketone bodies according to the invention are β-hydroxybutyric acid or its pharma- ceutically acceptable derivatives, such as its enantiomers (R)-β-hydroxybutyric acid, (S)-β-hydroxybutyric acid or a mixture of enantiomers or its pharma- ceutically acceptable salts or its pharma- ceutically acceptable esters, as well as acetoacetate. Medium chain fatty acids are also considered to be derivatives of ketone bodies according to the invention. The term "medium chain fatty acids" or "MCT oil" refers to neutral fats with two or three fatty acids with an aliphatic end of 6 to 12 carbon atoms. Such medium chain fatty acids or MCT oils can be converted into ketone bodies in the human body. Examples of infusion solutions containing ketone bodies or ketone body derivatives are Lipofundin® MCT / LCT 20% (B.Braun) or SMOFlipid® 20% (Fresenius Kabi). Further examples can be found in WO 2018 / 114309.

[0026] Preferably, the cancer is a cancer with metabolic changes that render it dependent on glucose and / or glutamine.Typically, the cancer is selected from human colon cancer and glioblastoma, as well as prostate cancer, breast cancer and liver cancer.

[0027] In one embodiment, the dialysis fluid comprises a pharma- ceutically acceptable amount of a pharma- ceutically acceptable cytostatic agent.

[0028] Preferably, the dialysis solution contains an additional osmotic agent selected from the group of pharma-ceutically acceptable polyethylene glycol and albumin.

[0029] The above summary of the present disclosure is not intended to describe each embodiment or every implementation thereof. Advantages, together with a more complete understanding of the present disclosure, will become apparent and appreciated by reference to the following detailed description and claims in conjunction with the accompanying drawings. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic block diagram of an exemplary peritoneal dialysis system that may be used to treat cancer with a dialysate according to the present invention. [Figure 2A] , [Figure 2B] , [Figure 2C] 2A, 2B and 2C show treatment targets according to the present invention. [Figure 2D] , [Figure 2E] 2D and 2E show possible pH shifts in the vicinity of normal and cancer cells associated with dialysis using a dialysate according to the present application. [Figure 2F] FIG. 2F illustrates the hypothesis that peritoneal dialysis using a dialysis fluid according to the present invention renders cancer cells more sensitive to cytostatic drugs. [Figure 3a] , [Figure 3b] , [Figure 3c] , [Figure 3d] , [Figure 3e] , [Figure 3f] , [Figure 3g] , [Figure 3h] , [Figure 3i] , [Figure 3j] , [Figure 3k] Figures 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j and 3k show charts of the results of Test 1. [Figure 4a] , [Figure 4b] , [Figure 4c] , [Figure 4d] 4a, 4b, 4c and 4d show charts of the results of test 2. [Diagram 5] FIG. 5 shows the proliferation rates of A549 and RCC4 cells in media A to C, as indicated. [Figure 6] Figure 6 shows the amounts of lung cancer A549, renal cancer RCC4 and primary RCC cells after 3 days of culture in media A-C at 21 or 5% oxygen. Asterisks indicate significantly different values ​​as determined by two-way ANOVA with Tukey's multiple comparison test. [Figure 7] FIG. 7 shows the results of culturing the human glioma cell line A172 under normoxia and hypoxia in media A-C supplemented with 8 mM Acac, 16 mM BOHB, or a combination of 4 mM Acac / 8 mM BOHB. 4 mM and 8 mM LiCl are used as controls for Acac. Significantly different values, as determined by one-way ANOVA with Sidak's multiple comparison test, are marked with an asterisk. [Figure 8] FIG. 8 shows the results of culturing the glioma cell line U118MG under normoxia and hypoxia in media A-C supplemented with 8 mM Acac, 16 mM BOHB, or a combination of 4 mM Acac / 8 mM BOHB. 4 mM and 8 mM LiCl are used as controls for Acac. Significantly different values, as determined by one-way ANOVA with Sidak's multiple comparison test, are marked with an asterisk. [Figure 9] Figure 9 shows the results of culturing the glioma cell line A172 under normoxia and hypoxia in media B-C supplemented with 8 mM Acac, 16 mM BOHB, or a combination of 4 mM Acac and 8 mM BOHB. 4 mM and 8 mM LiCl are used as controls for 4 mM Acac / 8 mM BOHB or 8 mM Acac, respectively. Significantly different values, as determined by one-way ANOVA with Sidak's multiple comparison test, are marked with an asterisk. [Figure 10]Figure 10 shows the results of culturing the glioma cell line U118MG under normoxia and hypoxia in media B-C supplemented with 8 mM Acac, 16 mM BOHB, or a combination of 4 mM Acac and 8 mM BOHB. 4 mM and 8 mM LiCl are used as controls for 4 mM Acac / 8 mM BOHB or 8 mM Acac, respectively. Significantly different values, as determined by one-way ANOVA with Sidak's multiple comparison test, are marked with an asterisk. [Figure 11] FIG. 11 shows the results of culturing renal cancer cell line RCC4 under normoxia and hypoxia in media B-C supplemented with 8 mM Acac, 16 mM BOHB, or a combination of 4 mM Acac and 8 mM BOHB. 4 mM and 8 mM LiCl are used as controls for 4 mM Acac / 8 mM BOHB or 8 mM Acac, respectively. Significantly different values, determined by one-way ANOVA with Sidak's multiple comparison test, are marked with an asterisk. [Figure 12] Figure 12 shows a schematic diagram of the experimental setup of Example 3. Hemodialysis was performed in anesthetized ketotic Sprague-Dawley rats with a blood flow rate of 5 ml / min. Blood samples were obtained before and after dialysis and at 60, 90, 120, 180 min. Dialysate samples were taken at 10, 20, 40, 60, 90, 120, 150 and 180 min. [Figure 13A] , [Figure 13B] , [Figure 13C] , [Figure 13D] 13A, 13B, 13C and 13D disclose the time course of plasma concentrations of ketones, glucose, urea and plasma base excess during dialysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention relates to a new dialysis fluid for use in peritoneal dialysis therapy to treat cancer.

[0032] Peritoneal dialysis for treating end stage renal disease typically utilizes a dialysis solution or "dialysate" that is infused into a patient's peritoneal cavity through a catheter implanted within the cavity. Figure 1 is a simplified schematic of such a system. The dialysate 1 contacts the patient's peritoneal membrane in the peritoneal cavity 2. Waste, toxins, and excess water flow from the patient's bloodstream through the peritoneal membrane and into the dialysate. The movement of waste, toxins, and water from the bloodstream into the dialysate occurs due to diffusion and osmotic pressure, i.e., an osmotic gradient occurs across the membrane. The spent dialysate is drained from the patient's peritoneal cavity, removing the waste, toxins, and excess water to a waste container 3.

[0033] The cycle then repeats.

[0034] An exemplary system for peritoneal dialysis that may be used with the dialysis fluids disclosed herein is described in EP 1 509 261 A1.

[0035] As already mentioned, the present invention provides a dialysate for use in peritoneal dialysis. The basic idea of ​​dialysis treatment is to change the composition of a body fluid by using a semipermeable membrane and a dialysate. The term "body fluid" typically relates to blood, but may also be the intracellular fluid of the subject being treated. The body fluid is separated from the dialysate by a membrane. The membrane is permeable to small molecules but impermeable to larger molecules. Small body fluid components can therefore pass through the membrane, while larger components are retained where the membrane is. As a result, the concentrations of the body fluid and the dialysate change. The driving forces for these changes are diffusion and osmotic pressure.

[0036] Since aerobic glycolysis or the Wartburg effect is common in many cancer cells, the following treatment goals may be set: a) reducing the concentrations of glucose and components of the citric acid cycle and amino acids derived from these components, such as pyruvate, serine, cysteine, glycine, alanine, glutamic acid, glutamine, proline, aspartic acid, asparagine, threonine and their derivatives, in body fluids; b) maintaining physiological pH in body fluids (pH within the range of 7.2 to 7.6) because lactic acid formed during aerobic glycolysis can locally lower the pH; and c) The addition of ketone bodies to provide an energy source that tumors dependent on aerobic glycolysis cannot rely on.

[0037] Figures 2A, 2B and 2C show some examples of treatment goals and principles of the present invention. Each figure discloses an example of a normal state with respect to the concentration of a particular blood component. Figure 8A shows that at the start of treatment, a patient will typically have glutamine GLN in the range of 0.20-0.8 mM. a During treatment with a dialysate that does not contain glutamine or has a very low glutamine content, the actual blood concentration of glutamine is within the desired range of 0.1 to 0.5 mM, for example within the range of 0.15 to 0.3 mM GLN. b FIG. 2B shows that at the start of treatment, patients typically have a glucose level in the range of 4-8 mmol / l. a During treatment, with no glucose or with a small amount of glucose in the dialysate, the actual blood concentration of glucose is typically within the desired range of 2-4 mmol / l. b Finally, FIG. 2C shows that the blood initially contains very little ketone bodies, such as β-hydroxybutyric acid or its physiologically acceptable salts or esters, e.g., the sodium salt. Thus, the actual value of the concentration of ketone bodies in the patient's blood, KETONE ais about 0. Typically, the first unused dialysate used does not contain ketone bodies or contains only small amounts of ketone bodies. During treatment, the blood concentration of such ketones can be increased to a desired value in the range of 1 to 15 mmol / l, for example in the range of 2 to 12 mM, by the use of a second dialysate containing ketone bodies. b The dialysate should contain a buffering component, such as bicarbonate, to facilitate the maintenance of a physiological pH in the vicinity of the cancer tumor.

[0038] Figure 2D shows the pH in the vicinity of normal healthy cells and cancer cells under normal conditions. It should be noted that normal healthy cells are surrounded by a neutral to slightly alkaline pH, while cancer cells are surrounded by a slightly acidic pH due to the production of lactic acid as a result of aerobic glycolysis. Such a pH may have an immunosuppressive effect that protects cancer cells. Such cancer acidity is described in Huber et al, Seminars in Cancer Biology, vol. 43 (2017), pp. 7489.

[0039] FIG. 2E shows the possible effect of dialysis using the dialysate on such normal healthy cells and cancer cells. In both cases, the pH value in the vicinity of the cells is elevated. This increase is caused by two different mechanisms. First, the dialysate contains no or very little fuel for aerobic glycolysis. Therefore, less lactic acid is produced. Second, the dialysate also contains bicarbonate ions, which have a buffering effect and are weakly alkaline, which also increases the pH. Bicarbonate is a buffer substance commonly used in dialysates for the treatment of end-stage renal disease. As a result of the treatment, the cancer cells in FIG. 2E are not surrounded by an acidic environment and are therefore not protected by any immunosuppressive layer.

[0040] Certain cancer cells in an environment with a small amount of fuel for aerobic glycolysis and a non-immunosuppressive pH should be weaker than the corresponding cancer cells under normal conditions. It is therefore assumed that dialysis treatment with the present dialysis fluid should weaken such cancer cells, and cancer cells treated accordingly should be more sensitive to cytostatic agents. Figure 2F illustrates this assumption. The two figures show the mortality rates of cancer cells as well as different non-cancer cells. Because cancer cells grow rapidly, they are more sensitive to cytostatic agents than typical non-cancer cells. The figure on the left outlines the situation without dialysis, and the figure on the right shows the situation when dialysis treatment is performed using the dialysis fluid according to the present invention.

[0041] The dialysis fluid for peritoneal dialysis must also contain an osmotic agent to ensure that the appropriate osmotic pressure is obtained. However, ketone bodies are a suitable osmotic agent, and additional such agents are typically not required. If an additional osmotic agent is indeed required, it can be selected from the group of polyethylene glycol and albumin.

[0042] Reference Example 1 The sensitivity of various human cancer cell lines to the presence of glucose, glutamine and ketones in the cell culture medium, together with depletion of selected nutrients, was tested to mimic the conditions obtained in cancer dialysis.

[0043] Study 1 was performed on a selection of established human cancer cell lines from renal cell carcinoma, colon carcinoma and glioblastoma. The first study investigated the effect on cell viability of proliferation in the presence of increasing concentrations of ketones and beta-hydroxybutyrate, with limiting levels of glucose and glutamine. The addition of citrate to the cell culture medium was also tested. Cells were cultured under these conditions for 3 days, after which cell viability was determined. In the first study, a major effect on cell viability was found when glutamine was depleted from the culture medium.

[0044] material and method Cell culture conditions Established cell lines from human colon cancer (HCT15, NCI-H508 and COLO205), renal cell carcinoma (769-P, 786-O and RCC4) and glioblastoma (LN-18, A-172 and U-118MG) were selected for analysis. All cell lines were obtained from the American Type Culture Collection (ATCC, LGC standards, UK), except for RCC4 and HCT15, which were purchased from Sigma-Aldrich (Merck, Germany). In addition, human primary renal cell carcinoma (RCC) cells isolated from nephrectomies of patients were included in the study. Culture conditions were as recommended by the American Type Culture Collection (ATCC), i.e. cells were grown in DMEM medium supplemented with 1 mM sodium pyruvate, but sodium pyruvate was also added to RPMI-1640 medium to make the conditions more similar.

[0045] 769-P, RCC4, LN-18, A-172, U-118MG and primary RCC cells were cultured in DMEM high glucose medium, whereas 786-O, HCT15, NCI-H508 and COLO205 were cultured in RPMI-1640 medium as recommended by ATCC. Both media were supplemented with 1% penicillin-streptomycin and 10% fetal calf serum. Cells were expanded and aliquots were frozen according to standard procedures.

[0046] The optimal seeding density was determined for each cell line in 96-well plates according to the "Cell Seeding Density Optimization Protocol to Ensure Log-Phase Growth". The protocol is as follows: -Prepare single cell suspensions and determine cell count / viability. Dilute cells to approximately 160,000 cells / mL in complete medium. Add 200 μL of cells to the top row of a 96-well plate. Dispense 100 μL of complete medium into every other well. A few medium-only control wells are needed on each plate to act as blanks. Using a 12-well channel pipette, dilute the cell preparation 1:2 iteratively down the plate, i.e., add 100 μL of cells to 100 μL of medium in the row below. Then add 50 μL of complete medium to all wells. Cover the plate. Incubate the plates overnight at 37°C and 5% CO2. Add 50 μL of fresh medium to the plate wells to bring the final volume to 200 μL and incubate at 37°C, 5% CO2 for 72 hours. Measure viability using the Cell Titer Glo Assay according to the manufacturer's protocol. Plot log cell number against luminescence intensity to identify the concentration of cells at which logarithmic growth is achieved. The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used as a viability readout.

[0047] Test 1. For each cell line, the optimal number of cells determined above was seeded into 96-well plates on day 0. The next day, cells were washed in PBS and the medium was changed to DMEM (Fisher Scientific) or RPMI-1640 medium (Saveen Werner) without glucose or L-glutamine, and nutrients were added as outlined in Table 3 and in the file "Plate schematic." Three wells were treated for each condition. After 3 days of culture in test condition medium, medium was changed daily and cell viability was determined using the CellTiter-Glo viability test. For each cell line, the test was repeated three times.

[0048] [Table 1]

[0049] [Table 2]

[0050] [Table 3]

[0051] Matrix showing the combination of different growth conditions used in Study 1. * The amount of glucose is shown as a percentage of the concentration present in the standard culture medium for each cell line. Where indicated, 1 mM citrate was added to the medium. The indicated nutrients were added to DMEM (Fisher Scientific) or RPMI-1640 medium (Saveen Werner) without glucose or L-glutamine.

[0052] [Table 4] JPEG2024541652000006.jpg190162

[0053] The results of Test 1 are shown in the chart shown in Figure 6. A clear correlation between the reduction in glutamine and cell culture medium present and the reduction in cell proliferation is shown.

[0054] Test 2. As mentioned above, in test 1, the culture conditions recommended by ATCC were followed. However, pyruvate is a potential energy source that may affect the results. Therefore, in test 2, the same culture conditions as in test 1 were tested on two cell lines, A172 (glioblastoma) and RCC4 (renal cell carcinoma), in DMEM medium without the addition of sodium pyruvate. The results are shown in Figure 7. As in test 1, a clear association is shown between the reduction in the amount of glutamine and cell culture medium present and the reduction in cell proliferation. However, the results are much more pronounced when pyruvate is not present in the cell culture medium. Also, the increase in the concentration of BOHB ketone seems to suppress cell proliferation as well.

[0055] All patents, patent documents and references cited herein are incorporated in their entirety as if each were individually incorporated. The present disclosure is provided with reference to exemplary embodiments and is not meant to be construed in a limiting sense. As previously mentioned, those skilled in the art will recognize that various other exemplary applications may use the techniques described herein to take advantage of the beneficial features of the apparatus and methods described herein. Various modifications of the exemplary embodiments, as well as additional embodiments of the present disclosure, will be apparent by reference to this description.

[0056] Example 2 Study design

[0057] Growth medium To investigate the effect of a nutrient-limited ketogenic environment on cancer cell growth in vitro, three different cell culture media were prepared. Medium A was the complete RPMI1640 medium in which cell lines are normally cultured. Medium B was used as an approximation of the conditions found in normal human serum. The levels of glucose, glutamine, serine, glycine and arginine were adjusted to match the normal physiological levels found in human serum. These nutrients were selected based on their reported use as an energy source and their effect on the metabolic state of cancer cells. Medium C was used to mimic nutrient-limited ketone body-producing cancer dialysis conditions. Here, the levels of selected nutrients were reduced to half of the physiological levels in medium B and the ketone body BOHB was added. The composition of each medium is described in Materials and Methods and listed in Tables 5-6.

[0058] Oxygen levels Human cancer cell lines are typically established and cultured at atmospheric oxygen levels (21% O2), whereas physiological oxygen levels in tissues are considerably lower, varying between 3 and 13% [Ward, Biochim Biophys Acta 2008;1777:1-14]. Within the tumor microenvironment, the rapid proliferation rate of cancer cells combined with an often malformed and defective vasculature often results in hypoxic regions with oxygen levels ranging from 0 to 5%. Considering the effect of oxygen levels on energy metabolism [Xie et al, J Biol Chem 2017;292:16825-16832], and to further mimic physiological conditions in vivo, the growth of cancer cell lines in media A, B and C was tested both at ambient 21% O2 and at the more physiological 5% O2.

[0059] Ketones The ketone bodies acetoacetate (Acac), BOHB and acetone are produced by the liver during fasting or starvation. BOHB is the predominant ketone body in mammals, with Acac accounting for approximately 20%. The majority of published in vitro studies investigating the effects of ketones on cancer cells have focused primarily on BOHB, although some studies suggest different effects of the addition of Acac compared to BOHB [Vallejo et al, J Neurooncol 2020;147:317-326]. Acac was also included in the study to further mimic the in vivo ketogenic situation in which both ketones are present and to investigate possible differences in the effects of BOHB and Acac.

[0060] material and method

[0061] cell line All cell lines were purchased from ATCC (ATCC, LGC standards), except for RCC4, which was from Sigma-Aldrich (Merck). Human primary renal cell carcinoma cells were isolated from nephrectomies performed at Sahlgrenska University Hospital, Gothenburg, Sweden, after informed consent of the patients and with permission from the local ethical committee. Optimal seeding density was determined for each cell line in 96-well plates cultured for 3 days in standard cell culture medium.

[0062] Culture conditions and supplements Cells were maintained in RPMI-1640 medium (31870-025 GIBCO) supplemented with 10% serum, 200 mM L-glutamine and 1% penicillin-streptomycin (PEST) in a humidified chamber at 37°C and 5% CO. For hypoxic conditions (5% O), cells were maintained in a Galaxy 14 S CO incubator (Eppendorf) and N was used to adjust O levels to 5%.

[0063] Media A, B and C were prepared as follows.

[0064] Medium A: RPMI 1640 supplemented with 1% PEST, 200 mM L-glutamine and 10% dialyzed serum (31870-025, GIBCO). Dialyzed serum was used to reduce the amount of small molecules such as amino acids.

[0065] Media B and C were prepared from RPMI1640 modified medium powder without L-glutamine, glucose and amino acids (R9010-01, US Biological Life Sciences). For 1 L medium, 7.4 g of powder was dissolved in 900 ml of sterile water without heating and 2 g of sodium bicarbonate was added. The amino acids listed in Table 5 were added at the same concentrations as in complete RPMI1640 medium (Table 5). After all additions, the medium was sterilized by filtering through a 0.22 um membrane and divided into two bottles. In B medium, to mimic physiological conditions, glutamine, serine, glycine, arginine and glucose levels were set to the median levels measured in human serum, based on data from Mayo Clinic Laboratories (https: / / www.mayocliniclabs.com / test-catalog / Clinical+and+Interpretive / 9265). To model cancer dialysis conditions in medium C, the levels of these nutrients were reduced to 50% of physiological levels. Similar to medium A, 1% PEST and 10% dialyzed serum were added to media B and C. The concentrations of selected nutrients in media A-C are summarized in Table 6. Sodium pyruvate, a common additive in cell culture media, was not present in any of the media used.

[0066] [Table 5]

[0067] [Table 6]

[0068] Amino acids and other additives were purchased from Sigma Aldrich. After adding all nutrients, the pH was measured and the pH values ​​were as follows: complete RPMI 1640 containing 10% non-dialysed FBS, 1% PEST and 200 mM L-glutamine pH 7.78; medium A pH 7.56; medium B pH 7.62 and medium C pH 7.57.

[0069] Stock solutions of DL-β-hydroxybutyric acid sodium salt (H6501, Sigma Aldrich) and lithium acetoacetate (A8509, Sigma Aldrich) were prepared in water, sterile filtered, aliquoted, and stored at −20° C. Lithium chloride (L7026, Sigma Aldrich) was used as a control for the addition of lithium in Li-Acac.

[0070] Viability assay The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used as a readout for cell number according to the manufacturer's instructions. In the experiments shown in Figures 7-8, two plates were seeded and processed. One set of plates was used for lactate measurements (see below) and collection of medium for the CellTiterGlo-assay. The other set of plates was frozen at -80°C at the end of the experiment. The frozen plates were intended for the CyQuant cell proliferation assay (Thermo Fisher), which measures the amount of DNA per well. Analysis of cell quantity by both the CellTiterGlo assay and the CyQuant assay ensures that the effects of culture conditions on viability or proliferation rate are not masked by concomitant changes in ATP levels per cell.

[0071] Collection of medium for lactate measurement In the experiments shown in Figures 7-8, cell culture fluid was collected on day 3, transferred to a new 96-well plate, and frozen at -80 °C. This medium can be used to analyze the amount of lactate excreted as a measure of metabolic status. Several kits for lactate measurement are available, for example, the Lactate-Glo assay (J5021, Promega), which is designed for use in assays in the presence of serum.

[0072] result

[0073] Study 2 was designed to answer the following questions: -Is the growth of selected cancer cell lines affected by cancer dialysis conditions mimicked with Medium C under normoxia or hypoxia?

[0074] Growth on media A, B and C As a first step, media A-C were prepared as described in Materials and Methods, and the ability of cancer cell lines to grow in these media was tested. Growth curves over time in each medium were established for selected cell lines. Cell counts were analyzed after 1, 2 and 3 days of culture in media A-C in normoxic conditions (21% O2). As shown in Figure 5 , reducing selected nutrients to more physiological levels, as in medium B, significantly reduced the proliferation rates of A549 lung cancer and RCC4 renal cancer cell lines compared to medium A.

[0075] Growth of cancer cells in media A to C under normoxic and hypoxic conditions In Figure 6, the amount of cells after 3 days of culture in media A-C in normoxia and hypoxia is shown for A549 lung cancer and RCC4 renal cancer cell lines and primary renal cancer (RCC) cells. Also, the proliferation rate in normoxia was decreased in media B and C compared to media A. The same pattern was found in cells cultured at 5% O2. Also, in RCC4, the low nutrient levels of media C and the addition of BOHB had no significant additional effect compared to media B conditions. For A549, a small but significant decrease in growth rate was observed between media B and C, but only under normoxia.

[0076] Primary renal cancer cells from three patients were included in this study. Similar to established cell lines, these cells showed decreased proliferation rates in media B and C compared to media A.

[0077] Overall, changing oxygen tension from 21% (normoxia) to 5% O2 (hypoxia) had very limited effect on the proliferation rate of these cells.

[0078] We then included Acac in the study and analyzed the viability of cells cultured in the presence of BOHB and Acac alone or in combination in media A-C. Experiments were performed at 21% and 5% O2. Experiments were performed with glioma cell lines A172 and U118MG.

[0079] BOHB, a chiral molecule, exists as two optical isomers, D- and L-BOHB. D-BOHB is normally produced and metabolized in humans. The BOHB salt used in this study contains a 50:50 mixture of D- and L-BOHB. To ensure a high presence of the active D form, the total concentration of BOHB added was increased to 16 mM to obtain a level of 8 mM D-BOHB. To keep the total concentration of active ketones constant, 8 mM Acac was used, and for the combination of both ketones, the levels were adjusted to 4 mM Acac and 8 mM BOHB (containing 4 mM D-BOHB). In vitro available Acac is in the form of a lithium salt. Because lithium itself can affect cancer cell viability [Cohen-Harazi et al, Anticancer Res 2020;40:3831-3837], 8 mM LiCl was used as a control for the 8 mM Acac data points and 4 mM LiCl was used as a control for the 4 mM Acac / 8 mM BOHB data. At the end of the experiment, culture medium from each well was collected and frozen to allow for subsequent determination of lactate levels as a measure of metabolic status. In addition, two experiments were performed, where one set of plates was frozen for subsequent quantification of cell number using a CyQuant proliferation assay, and the amount of cells in the other set was analyzed by the CellTiterGlo viability assay.

[0080] Effect of adding BOHB and Acac alone or in combination As shown in Figures 7-8, initial experiments provided encouraging data regarding the effect of high ketone concentrations in nutrient-reduced conditions on the growth of A172 and U118MG glioma cell lines.

[0081] In medium A, in both normoxic and hypoxic conditions, the addition of 16 mM BOHB alone had no growth inhibitory effect in A172 or U118MG cells, and 8 mM Acac did not further reduce the number of cells compared to the 8 mM LiCl control. However, the addition of 4 mM Acac in combination with 8 mM BOHB significantly reduced the number of A172 cells in medium A under normoxia compared to the 4 mM LiCl control.

[0082] Interpretation of results from medium B was hampered by technical errors in normoxic control samples. However, in hypoxic A172 cells, BOHB significantly reduced cell number to approximately 70% of the amount in medium B without BOHB. A similar reduction was seen in hypoxic U118MG cells. Furthermore, in medium B, 8 mM Acac significantly reduced cell numbers compared to the 8 mM LiCl control in both cell lines, but only at 21% O2.

[0083] In medium C, the addition of 8 mM Acac alone did not significantly reduce the amount of cells compared to the 8 mM LiCl control. However, in both cell lines and at both 21% and 5% O2, the addition of 16 mM BOHB significantly reduced the number of cells to approximately 30% compared to medium C without BOHB. Also, the combination of BOHB and Acac resulted in significantly fewer cells in Medium C compared to the 4 mM LiCl control for both cell lines and at both oxygen levels.

[0084] These results suggest that the addition of high levels of BOHB or Acac alone does not inhibit the growth of glioma cells in a nutrient-rich environment such as medium A. Also, only minor differences were seen when ketones were added in medium B, which has more physiological nutrient levels. The greatest effect was found in medium C, where both 16 mM BOHB alone and the combination of 8 mM BOHB and 4 mM Acac significantly reduced cell numbers compared to their respective controls in both hypoxic and normoxic conditions. This was not seen when 8 mM Acac was added alone.

[0085] However, repetition of these experiments, focusing on media B and C in normoxic conditions, gave inconsistent results. Figures 9-11 show the combined results from experiments 2-6 for the glioma cell lines A172 and U118MG, and the renal carcinoma cell line RCC4. When BOHB or Acac was added separately, a trend towards reduced proliferation was observed in medium C, especially in the A172 cell line.

[0086] The results from the first and second parts of Example 2 showed that cancer cell lines grow slower in nutrient-limited environments. The tested cell lines appeared to be viable in media B and C, although their growth rates were reduced. Optical inspection of the cells on day 3 did not reveal any floating cells, which may be a sign of dead cells.

[0087] Data from the third part of this study show increased sensitivity of glioma cell lines to high levels of BOHB or the combination of BOHB and Acac in nutrient-limited Medium C. However, such sensitivity was not found for the renal carcinoma cell line RCC4.

[0088] Example 3 In this example, rats were given water and a ketogenic diet and then underwent dialysis. The effect of dialysis on blood ketones, lactate, and actual HCO3 was determined.

[0089] method

[0090] animal Experiments were performed in six male Sprague-Dawley rats with an average weight of 316 g (305–318), which were fed fluids and a ketogenic diet (Kliba-Nafag 2201 Ketogenic diet XL75:XP10) 5 days prior to the experiment. Animals were treated in accordance with the US National Institutes of Health guidelines for the care and use of laboratory animals. The Animal Research Ethics Committee of Lund University approved the experiment (Dnr 5.8.18-08386 / 2022). Rats were carefully placed in a covered glass container connected to a continuous supply of 5% isoflurane in air (Isoban, Abbot Stockholm, Sweden). After being fully anesthetized, the rats were gently lifted out of the container. Anesthesia was delivered and maintained with 1.6–1.8% isoflurane in air, delivered with a small mask. After tracheotomy, rats were connected to a volume-controlled ventilator (Ugo Basile; Biological Research Apparatus, Comerio, Italy) with a positive end-expiratory pressure of 4 cmH2O. Body temperature was maintained at 37.1 to 37.3°C using a feedback-controlled heating pad. End-expiratory pCO2 was maintained between 4.8 and 5.5 kPa (Capstar-100, CWE, Ardmore, Pa). The right femoral artery was cannulated for continuous monitoring of heart rate and mean arterial pressure (MAP), and to obtain blood samples (95 μL) for measuring glucose, urea, electrolytes, hemoglobin and hematocrit (I-STAT, Abbott, Abbott Park, IL) and blood ketones (FreeStyle Precision Neo, Abbott, Abbott Park, IL). The right femoral vein was cannulated and connected to a dialyzer using plastic tubing. The right femoral artery was also cannulated and connected to a pressure transducer for continuous monitoring of arterial line pressure and a blood pump (Masterflex Ismatec, Cole-Parmer, IL) connected to the dialyzer via plastic tubing.Prior to connection, the blood circuit was primed with 4% albumin (Albunorm, Octapharma Nordic AB, Sweden) supplemented with heparin 50 IE (Heparin LEO 5000 IE / ml, Leo Pharma AB, Sweden). The dialysis circuit was connected to the pump with an inlet from a glass cylinder containing fresh dialysis fluid (Hemosol B0, Baxter Healthcare, IL) with the following composition: JPEG2024541652000009.jpg85162

[0091] The outlet from the dialyzer was also connected to a peristaltic pump that pumped the spent dialysate into a glass cylinder. Both glass cylinders were placed on a balance to ensure that no fluid was removed from the animal (Figure 12). 51 The right internal jugular vein was cannulated for infusion of maintenance solution containing Cr-EDTA. Hematocrit was determined by centrifugation of fine capillary glass tubes. After the experiment, animals were euthanized by intravenous bolus injection of potassium chloride.

[0092] Experimental protocol A 3-h hemodialysis session was performed using a minicapillary dialyzer device obtained from Baxter Healthcare (Hechingen, Germany) containing a high-flux membrane (Polyflux Revaclear®, HF-Revaclear®). Arterial blood samples were taken before and 30 min after dialysis, and at 60, 90, 120, and 180 min during HD. Dialysate samples were taken before dialysis and at 10, 20, 40, 60, 90, 120, 150, and 180 min. 51 To determine Cr-EDTA activity, the samples were analyzed in a gamma counter (Wizard 1480, LKP Wallac, Turku, Finland).

[0093] statistical methods Data are presented as median (interquartile range) unless otherwise stated. Significant differences were assessed using the asymptotic Friedman omnibus test (COIN package) followed by Wilcoxon-Nemenyi-McDonald-Thompson post-hoc test if significant. Two blood ketone values ​​were above the measurement range (8.0 mmol / L). They were set to 8.0 mmol / L prior to statistical analysis. As they were the highest values ​​in the data set, the value of 8 mmol / L ensures that (i) the median (IQR) is not affected and (ii) the data points get the highest and equal rank. P values ​​less than 5% were considered significant. Calculations were performed using R for mac version 4.1.1.

[0094] result

[0095] Baseline parameters Six Sprague-Dawley rats were fed a ketogenic diet for 5 days, increasing body weight between 10 and 37 g (min-max). Before dialysis, median arterial pH was 7.43 (7.42 to 7.44), actual bicarbonate was 20.4 mmol / L (19.8 to 21.3), standard base excess was -3.5 mmol / L (-4.0 to -2.2), pO2 was 12.4 kPa (12.2 to 12.7), and pCO2 was 4.0 kPa (4.0 to 4.3). Dialysis was performed for 3 hours, after which blood was returned to the animals, followed by a 30-minute rest period before the end of the experiment. Treatment and baseline parameters were as follows: JPEG2024541652000010.jpg178162

[0096] Hemodialysis does not significantly reduce blood ketone levels Blood ketone levels before and after the 180 min dialysis were similar, 3.5 mmol / l (2.2-5.6) and 3.8 mmol / l (2.2-5.1), respectively (P=0.53), with no significant difference in ketone concentrations during dialysis (Figure 13A). The data are presented in the table below: JPEG2024541652000011.jpg107162

[0097] The urea reduction rate (URR) was 38% (25-42) and the glucose reduction rate was 36% (29-43), with concentrations decreasing over the course of treatment (Figures 13C, 13B). The data are presented in the table below: JPEG2024541652000012.jpg176162

[0098] 51 The clearance of Cr-EDTA from blood to dialysate was stable at 1.0 ml / min (0.7-1.1) throughout the dialysis session. The single-pool Kt / V urea was 0.57 (0.52-0.63), calculated as Kt / V = -log(1-URR-0.024). Assuming total body water of 70% of body weight, this implies a clearance from urea plasma to dialysate of 0.67 ml / min (0.62-0.80).

[0099] Effects on acid-base and blood chemistry during dialysis Plasma base excess increased during dialysis (Fig. 13D). Consistent with this, arterial pH increased from 7.42 (7.42-7.44) to 7.51 (7.49-7.52) after 180 min of dialysis (P = 0.005). Arterial pCO2 was not significantly different after 180 min of hemodialysis, 4.0 (4.0-4.3) kPa before dialysis and 4.0 (3.7-4.2) kPa after dialysis (P = 0.81). Blood hemoglobin levels were not significantly different between predialysis and postdialysis, 113 g / L (106-118) and 116 g / L (111-118), respectively (P = 1.00). Plasma potassium increased from 3.8 mmol / l (3.8 to 3.9) before dialysis to 4.5 mmol / l 180 min later (P = 0.03), but there was no significant difference in plasma sodium (Table 3).

[0100] discussion

[0101] This example shows that blood ketone levels in rats fed a ketogenic diet were relatively unaffected by hemodialysis, meaning that low molecular weight ketones (β-hydroxybutyrate, acetoacetate, acetone) are efficiently removed by dialysis. In fact, molecules roughly the same size as the ketoacids (e.g., urea) are effectively removed, 51 Even larger molecules such as Cr-EDTA were effectively removed from the blood.

[0102] Here the dialysate blood flow rate (Qb) was set at 1 ml / min, which is similar to other experimental models of hemodialysis in rats (Fukunaga et al, PLoS One. 2020;15:e0233925). The pre-filter arterial pressure was stable and positive at this blood flow rate, with 40-50 mmHg being typical values. To elucidate how this blood flow rate corresponds to humans undergoing hemodialysis, it can be set in relation to the volume of distribution. For example, for urea, the volume of distribution is approximately equal to total body water (TBW), which in rats is about 70% of body weight. Thus, a 300 g rat has a TBW of 210 ml, meaning that a blood flow rate of 1 ml removes about 0.47% of TBW from urea. For a 70 kg adult with a TBW of 42 L, this corresponds to a blood flow rate of 42 × 0.47% = 200 ml / min. Currently, blood flow rates are usually more than 250 ml / min, especially in patients treated with hemodiafiltration. On the other hand, we used a dialysate flow rate of 5 ml / min, which is much higher than Qb, implying that solute transport is limited by blood flow rather than the dialysis membrane or dialysate flow. Indeed, consistent with this, urea and 51 The estimated plasma clearances of Cr-EDTA (a much larger molecule than urea) from the dialysate were nearly identical, 0.67 ml / min for urea and 0.66 ml / min for 51Cr-EDTA.

[0103] We also found that the ketogenic diet resulted in a mild metabolic acidosis in rats, and the increase in plasma lactate was very mild. Slightly elevated lactate has been previously observed in cattle fed a ketogenic diet (Zhang et al, Research in Vetrenary Science, 2016, 107:246-256). Also, potassium concentration increased slightly to 4.5 mmol / L during dialysis. This could be attributed to the fact that 20 mmol of potassium was manually added to a 5 L bag of unused dialysate, which may result in an actual dialysate concentration that differs from 4 mmol / L due to tolerances in the volume and composition of the potassium solution. Finally, there is mild hyperglycemia before dialysis, which is also consistent with a previous study in mice (Meidenbauer et al, Faseb Journal, 2013, 27) that points out that insulin levels are reduced after a ketogenic diet.

Claims

1. A dialysis solution containing ketone bodies including at least one of acetoacetic acid, β-hydroxybutyric acid or a pharmaceutically acceptable derivative, ester, and salt thereof, for use in peritoneal dialysis therapy for treating cancer.

2. 10. The dialysis solution of claim 1, further comprising bicarbonate ions.

3. 2. The dialysis solution according to claim 1, wherein the concentration of the ketone bodies is 1 to 15 mM.

4. The dialysis solution according to claim 3, wherein the concentration of the ketone bodies is 2 to 12 mM.

5. The dialysis solution according to claim 2, wherein the concentration of the bicarbonate ions is 15 to 50 mM.

6. 6. The dialysis solution according to claim 5, wherein the concentration of the bicarbonate ions is 20 to 40 mM.

7. 2. The dialysis solution according to claim 1, wherein the total hydrolysis equivalent concentration of glucose, pyruvate, and an amino acid selected from the group consisting of serine, cysteine, glycine, alanine, glutamic acid, glutamine, proline, aspartic acid, asparagine, threonine, and derivatives thereof is 3.3 mM at most.

8. The dialysis solution according to claim 7, wherein the dialysis solution does not contain pyruvate, serine, cysteine, glutamic acid, proline, aspartic acid, asparagine, threonine, or derivatives thereof.

9. 9. The dialysis solution of claim 8, wherein the dialysis solution comprises a hydrolysis equivalent concentration of glutamine or a derivative thereof of up to 0.3 mM.

10. 10. The dialysis solution of claim 1, wherein the dialysis solution also comprises a pharmaceutically acceptable amount of a pharmaceutically acceptable cytostatic agent.

11. The dialysis solution according to any one of claims 1 to 10, wherein the dialysis solution also comprises an osmotic agent selected from the group consisting of polyethylene glycol and albumin.

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