WO2004014355A1

WO2004014355A1 - A peritoneal dialysis solution comprising a pyruvate

Info

Publication number: WO2004014355A1
Application number: PCT/IL2003/000663
Authority: WO
Inventors: Shraga Braverman
Original assignee: Talgon Invest Limited
Priority date: 2002-08-09
Filing date: 2003-08-10
Publication date: 2004-02-19
Also published as: IL151169A0; AU2003249563A1

Abstract

The invention provides a peritoneal dialysis solution comprising a pyruvate in a concentration of about 7-9 mMol/L in combination with at least one further antioxidant present in physiologically acceptable concentrations, the pyruvate and further antioxidant being present in concentrations affective to prevent the early acceleration in the cell cycle of the mesothelial monolayer, and the consequent premature senescence derived from a high glucose concentration in the dialysis fluid.

Description

A PERITONEAL DIALYSIS SOLUTION COMPRISING A PYRUVATE

The present invention provides a new formulation of a solution for peritoneal dialysis. More specifically, the present invention provides a peritoneal dialysis solution containing pyruvate salts and a further antioxidant in physiologically acceptable concentrations which solution prevents the alterations of the mesothelial cells life cycle, derived from the oxidative stress that result from exposure to glucose (the osmotic agent).

More particularly, the invention applies to peritoneal dialysis solutions used in the practice of continuous ambulatory peritoneal dialysis (CAPD), automated peritoneal dialysis (APD), intermittent peritoneal dialysis (IPD), continuous cycling peritoneal dialysis (CCPD), continuous flow peritoneal dialysis (CFPD), and tidal peritoneal dialysis (TPD).

Peritoneal dialysis (PD) became an effective and quite commonly used tool for life maintenance of patients affected by end stage renal disease. Use of this technique implies the instillation of a specially designed dialysis solution to remove from blood, through the peritoneal membrane, substances that are at the core of the uremic intoxication.

Despite the fact that peritoneal dialysis was introduced for clinical use about 80 years ago, biocompatibility of PD solutions is still a major issue limiting the application of this therapeutic approach for long periods of time. Poor biocompatibility results in loss of the ultrafiltration capabilities of the membrane required to maintain the fluid balance of the patient unable to eliminate water through the ailing kidneys. Additionally, the permeability of the membrane for the toxic substances is also affected, leading to a situation in which PD cannot provide even the minimally required amount of removal of toxins. Such a development demands interruption or termination of this therapy, and transfer of the patient to other methods of blood purification that, due to technical problems or coexisting diseases, is not always possible. Furthermore, a significant number of patients in whom the peritoneum lost its capabilities of functioning as a reusable dialysis membrane develop peritoneal fibrosis or sclerosis. This complication, also derived from poor biocompatibility of the dialysis solutions, is one additional indication to definitely stop the use of PD as a life maintenance tool for the affected patient. Biopsies taken from humans on CAPD showed evidence indicating that the solutions used created a situation of continuous mesothelial cell injury, coexistent with a simultaneous process of regeneration. The end point, the structure and function of the monolayer as a dialysis membrane depends on a balance between both situations. The question of poor biocompatibility has been explored in the in- vivo and in-situ set-up, using the mouse experiment model of taking -imprints of the mesothelial monolayer from the anterior liver surface, before, during, and after exposure of the tissue to different commercially available as well as experimental solutions.

Early studies provided evidence directing the attention to the fact that long- term exposure (IPD, one exchange a day) of the mesothelium (up to 30 days) to commercially available 4.25% dialysis solution resulted in a marked hypertrophy of the mesothelial cells. These changes were manifested by increased enzymatic activity at the level of the cell membrane, cytoplasma and nuclear enzymes. In addition, the density of the mesothelial cell population appeared reduced; mean cytoplasmic surface area increased at a point that cells became overly hypertrophic, doubling the normally measured area; a substantial proportion of large cells assumed the character of multinucleated (through defective cytokinesis); the mitotic index was dramatically reduced; the index of cell's viability turned into significantly lower levels; whereas evaluation of the prevalence of cells in apoptosis appeared substantially higher compared with values seen in unexposed normal control.

Further investigations of the cavitary aspect of the peritoneal membrane showed evidence indicating that these alterations were unrelated to the low pH (5.2 - 5.5) hyperosmolarity (346 - 485 mOsm/L), or the presence of buffer lactate or bicarbonate in the solution. On the other hand, an extremely relevant conclusion of these investigations was that the core of the problem was clearly connected to the presence of glucose in the dialysis fluid. The higher the concentration of glucose, the more severe the observed changes.

Two additional problems were found. The first was that exposure of the mesothelial monolayer to Mannitol, a known oxygen radicals scavenger, failed to induce any of the alterations derived from use of glucose as the osmotic agent. The other, and not a less relevant emerging concept, was that daily short exposure of cultured mesothelial cells to high glucose concentration resulted in an early peak (at 24 hours) of the mitotic index that became nil, later on, along the whole period of observation. This reduced regenerative capability coincided with signs of cell injury and death.

Thus it has been found that all commercially available osmotic agents, glucose, aminoacids, as well as ₍glucose polymers like lcodextrin induce per se, in non infected patients, substantial changes in the life cycle of the exposed mesothelial population. In the case of glucose, the effect is dose related: in mice exposed to the 1.5% glucose enriched peritoneal dialysis solution, the alterations of the cell cycle engine were present, but at a lower degree than those seen in animals treated with the 4.25% glucose concentration fluid. The aminoacids, as well as the 7.5% lcodextrin solutions also induced substantial changes in the life cycle of the exposed mesothelial monolayer. In all cases, the observed end result _, is a depopulated monolayer made up by hypertrophic cells that reached the point of replicative senescence.

Long-term use of the peritoneal dialysis solutions, as shown with glucose, leads to a situation in which the mechanisms of regeneration and repopulation of the monolayer are hampered, due to the practically continuous presence of the osmotic agent into the abdominal cavity. This derives in repair by connective tissue, which, in turn, results in fibrosis and/or sclerosis of the peritoneal membrane. From a clinical point of view, in those circumstances, failure of ultrafiltration is evident, and the possibility of the peritoneum to function as a reusable dialysis membrane is lost. Furthermore, a significant number of patients affected by peritoneal sclerosis die because of development of thick intestinal adhesions. All these developments are unrelated to the frequency and/or incidence of infectious peritonitis.

It has further been found that the described alterations of the life cycle of the exposed mesothelial cell population derive from different degrees of oxidative stress launched by the aforementioned osmotic agents. Indeed, it has now been demonstrated that glucose, in a dose related manner, can induce generation of hydrogen peroxide from cultured mesothelial cells, and that this reaction can be prevented by the introduction of antioxidants in the formulation of the glucose enriched solution. This development leads to the described changes in the mesothelial cells life cycle and, additionally, stimulate growth of fibroblasts.

At this point, the question of glucose derived oxidative stress, being at the origin of the mesothelial changes, was raised. Further research demonstrated that mesothelial cells in culture exposed to peritoneal dialysis solutions, having high glucose concentration (4.25%), generated hydrogen peroxide which eventually results in oxidative stress, and that this effect can be prevented by the addition of buffer pyruvate, a physiological oxidants' scavenger, to the dialysis solution.

Other line of research showed that in-vivo and in-situ exposure of the monolayer to the 4.25% glucose;'enriched dialysis solution determined a substantial change in the life cycle of the cell population. Namely, after a short lived early acceleration (for up to 3 days), the cell population reached a situation of premature replicative senescence, characterized by large multinucleated cells, low mitotic index, as well as significantly reduced Thymidine incorporation and PCNA expression (proliferative cell nuclear antigen) near zero and, at the same time, a substantially increased expression of B-galactosidase, a marker of cell senescence.

In EP 658,353 there are described pyruvate containing dialysis solutions however said patent relates to concentrations of about 25-40 mMol/L.

As is known the normal blood concentration of pyruvate is about 0.0140-0.11 mMol/L. Furthermore, the rate of pyruvate removal from blood as detected in isolated perfused ratt liver is: 4.46 micromol/L/min/gram of liver wet weight. (Ross, B.D., Hews, H.A. the rate of gluconeogenesis from various precursors in the perfused rat liver. (Biochem. J. 102-942-1967). This implies an eventual clearance of 6.7mM/L/min for 1500 grams of liver, the approximate weight of an adult human liver.

It is also known that blood levels of pyruvate are substantially increased in situations of liver dysfunction and that high concentrations of pyruvate can potentially induce a metabolic deviation, changing the lactate/pyruvate blood concentration ration.

With this state of the art in mind there is now provided according to the present invention a peritonea! dialysis solution comprising a pyruvate in a concentration of about 7-9 mMol/L in combination with at least one further antioxidant present in physiologically acceptable concentrations, said pyruvate and further antioxidant being present in concentrations effective to prevent the early acceleration in the cell cycle of the mesothelial monolayer, and the consequent premature senescence derived from a high glucose concentration in the dialysis fluid. Said further antioxidant can be selected from the group consisting of ascorbic acid and aldose reductase inhibitors, and in preferred embodiments of the present invention said antioxidant is ascorbic acid.

As shown in Figures 15, 16 and 17 described and discussed hereinafter, pyruvate alone, in a concentration of 8mMol/L, failed to prevent the acceleration of the cell cycle induced by the oxidant effect of glucose, while pyruvate in said concentration together with ascorbic acid was effective.

In especially preferred embodiments of the present invention said ascorbic acid is present in concentrations of about 1-95 mg/L.

In a most preferred embodiment of the present invention there is provided a peritoneal dialysis solution as defined above, comprising water-, and having dissolved therein the following components in the respective concentrations indicated:

Cations Anions

Na: 128 - 160 mEq/L Cl: 90 - 115 mEq/L

K: 0 - 4 mEq/L Lactate: 0 - 40 mEq/L

Ca: 1.5 - 4 mEq/L Pyruvate: 7 - 9 mEq/L

Mg: 0 - 1.5 mEq/L Ascorbic acid: 1 - 95 mg/L

Osmotic agent

Dextrose hydrous USP: 1.5 - 4.25gr/100ml.

Osmolar concentration and acidity

Osmolar concentration for the 1.5% glucose solution: approximately 346 mOsm/L. For the 4.25% glucose solution: approximately

485 mOsm/L.

Ph: 5.2 - 5.6.

As will be realized, the pyruvate buffer used in the present invention is especially effective because of its protective effect against hydrogen peroxide (H₂0₂) dependent degradation of DNA, reducing, at the same time, the amount of the H₂θ₂ dependent generation of detectable hydroxyl radicals. This evidence suggests, in turn, that pyruvate appears as playing the role of intracellular antioxidant and, consequently, this protective effect could be of relevance for the mitochondria. Furthermore, the preferred ascorbic acid used in the solutions of the present invention as the further antioxidant, has been found to behave as an effective antioxidant, especially in situations wherein the oxidative injury derives from exposure to glucose in high concentration.

In this context it is worthwhile to note that Normal healthy and adequately nourished people have a total body storage of around 2500mg. of ascorbic acid, maintained by an oral daily intake of around 200mg. Some groups postulate a minimal intake of 138mg/day to keep the total body pool. Then, the plasma concentration remains higher than 4mg/dl (the lower limit below which signs and symptoms of scurvy can develop (Jacob JA, et al. Am. J. Clin. Nutr. 46:818, 1987).

It has been shown that healthy men destroy by oxidation 3-4% of the ascorbic acid storage/daily; and when having an ascorbic acid deficient- diet, they evidence a reduction in glutathion blood levels, as well as a decrease of other antioxidant indices (Henning SM. et al. J. Nutri. 121 :1969, 1991 ).

This reduction of antioxidant defenses becomes much more, significant in patients suffering from different degrees of chronic renal failure, a condition characterized by continuous and severe oxidative stress, even before starting renal replacement therapy by means of blood purification (Inagi R. et al. Blood Purif. 17:95, 1999. Mimic-Oka J. Clin. Nephrol, 51 :233, 1999). Consequently, some investigators proposed a supplementation of around 100mg/day of ascorbic acid to be prescribed to chronic renal patients before starting dialytic therapy (Stein G. et al. Contrib. Nephrol. 65:33, 1988).

Besides those increased requirements of ascorbic acid during chronic uremia, dialysis can markedly aggravate the problem because of substantial losses of the vitamin in the dialysate fluid. Patients on peritoneal dialysis showed a 50-64 % reduction of ascorbic acid plasma levels after completion of the procedure, and losses in the dialysis fluid ranged between 92.5 - 333.6mg in one dialytic session (Bohm V. et al. Int. J. Vitam. Nutr. Research 67:262, 1997. Boeschoten EW. et al. Nephrol Dial. Transplant. 3:187,1988). In this sense, it should be noticed that most patients on peritoneal dialysis have a daily intake of ascorbic acid lower than 75mg (Bohm V. et al. Int. J. Vitam. Nutr. Research 67:262, 1997). Indeed, it has been shown that 40% of patients on peritoneal dialysis have ascorbic acid plasma concentrations lower than that commonly seen in a normal healthy population (Lim SL et al. Adv. Perit. Dial. 17:215,2001).

So far, most investigators proposed that patients on peritoneal dialysis (also those on continuous ambulatory peritoneal dialysis) should be supplemented with ascorbic acid, using a dosage ranging between 80-200mg/daily (Alkhunalzi AM. et al. JASN 7:2320, 1996. Descombes E. et al. Kidney Int. 43:1319,1993. Salusky IB. Adv. Perit. Dial. 6:245, 1990. Morgan SH. et al. Nephrol Dial. Transplant. 3:28, 1988. Shah GM. et al. Am. J. Kidney Dis. 20:42,1992. Boeschoten EW. et al. Nephrol Dial Transplant 3:187, 1988. Lim SL. et al. Adv. Perit. Dial. 17:215,2001 ). Increased serum oxalate levels in this patient population can be predicted with daily dosage of ascorbic acid higher than 500mg/day (Tomson CR. et al. Clin. Chim. Acta 180:255,1989). Lower dosage does not play a role in the secondary oxalosis seen in patients on peritoneal dialysis (Costello JF. et al. J. Am. Soc. Nephrol. 1 : 1289, 1991 ). Besides, the peritoneal clearance of oxalic acid is low and not effective enough for a permanent reduction of its concentration in plasma (Mydlik M. et al. Kidney Int. Suppl. February 78:S304, 2001. Mydlik M. et al. Artif. Organs 22:784,1998).

Additionally, it should be noticed that intravenous administration of 300- 500mg. of ascorbic acid three times a week to patients on maintenance hemodialysis has been found effective to overcome the problem of functional iron deficiency, quite frequently observed in chronic uremics showing low hemoglobin levels, in spite of adequate iron storage (Tarng DC. et al. Nephrol. Dial. Transpl. 13:2867, 1998. Tarng DC. et al. Kidney Int. 55 (Suppl. 69): S107, 1999. Giancaspro V. et al. J. Nephrol. 13:444,2000. Melendez O. Seminars Dial. 13:335, 2000. Petralulo F. et al. Nephrol. Dial. Transplant. 15:1717,2000. Tarng DC. et al. Kidney Int. 55:2477, 1999. Gastaldello K. et al. Nephrol. Dial. Transplant. 10 (Suppl. 6):S44, 1995). The aforementioned authors did not observe significant increase in serum levels of oxalic acid.

The problem of ascorbic acid absorption requires a special comment. Unlike most mammals, man is unable to synthesize vitamin C and it must therefore be acquired from the diet (MacDonald L. et al. Br. J. Nutr. 87:97, 2002). Recent work clearly demonstrated that intestinal absorption of ascorbic acid is achieved by two transporters: SVCT1 and SVCT2, recently cloned from rat and human kidney (MacDonald L. et al. Br. J. Nutr. 87:97, 2002). This same mechanism of active transport was detected in human lymphocytes, whereas the first component: SVCT1 , was defined as concentration and temperature dependent, saturable and generated ascorbic acid accumulation against a concentration gradient (Bergsten P. et al. Arch. Biochem. Biophys. 317:208,1995. Bergsten P. et al. J. Biol. Chem. 265:2584,1990). Additional research identified both components of the ascorbic acid transporter activity in human fibroblasts (Welch RW. et al. Biochem. J. 294:505, 1993. Butler JD. et al. Am. J. Clin. Nutr. 54:1 44S, 1991), intestinal epithelial cells (Fujita I. et al. Res. Commun. Mol. Pathol. Pharmacol. 107:219, 2000), as well as in porcine choroids plexus cells in culture (Hakvoort A. et al. Brain Res. 795:247,1998). Absorption efficiency of ascorbic acid is about 1/5 -1/10 of that of 2- deoxy-D-glucose, an alternative of glucose (Fujita I. et al. Res. Commun. Mol. Pathol. Pharmacol. 107:219,2000). All this information provides evidence that a two- component ascorbic acid transport system may be a generalized mechanism for accumulation of this vitamin in humans, at the cellular level (Welch RW. et al. Biochem J. 294:505, 1993). However, this activity of ascorbic acid transporters is substantially reduced in rats with chronic renal failure in-vivo, indicating some inhibitory influence present- in the serum of uremic animals (Pahl MV. et al. Proc. Soc. Exp. Biol. Med. 191 :332, 1989).

So far, taking into account all these considerations, it may well be assumed that a patient on continuous ambulatory peritoneal dialysis will eventually absorb no more than 30-40% of the ascorbic acid present in the^'dialysate, during a dwell time of 4 hours. If the used concentration is of 45mg/L, then the total amount of intraperitoneally infused ascorbic acid will be 360mg/24%. A 30% to 40% absorption will eventually reach a total amount of 144mg/24h, a value well within the range of the dosage required to maintain normal ascorbic acid storage without the risk of inducing secondary oxalosis.

In summary, it is believed that the combined use of two physiological antioxidant agents in low concentration, preferably pyruvate and ascorbic acid, preserve the mesothelial cell monolayer, preventing the alterations leading to the failure of the peritoneum as a dialyzing membrane. Besides, administration of both substances may well offer protective effects against the general and permanent oxidative stress derived from uremia. Additionally, the administration of ascorbic acid becomes a key element to correct functional iron deficiency and for this reason also its use as the further antioxidant is preferred.

While the invention will now be described in connection with certain preferred embodiments in the following examples and with reference to the attached Figures so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

In the drawings:

Figure 1 is a graphical representation of density of cell population using a control and using a solution according to the present invention;

Figure 2 is a graphical representation of mean cytoplasmic surface area using a control and using a solution according to the present invention;

Figure 3 is a graphical representation of mean nuclear surface area using a control and using a solution according to the present invention;

Figure 4 is a graphical representation of prevalence of multinucleation using a control and using a solution according to the present invention;

Figure 5 is a graphical representation of prevalence of mitosis using a control and using a solution according to the present invention;

Figure 6 is a graphical representation of prevalence of non viable cells using a control and using a solution according to the present invention;

Figure 7 is a graphical representation of prevalence of apoptosis using a control and using a solution according to the present invention;

Figure 8 is a graphical representation of population cell density at 30 days using different dialysis solutions and using a solution according to the present invention; Figure 9 is a graphical representation of mean cytoplasmic surface area at 30 days using different dialysis solutions and using a solution according to the present invention;

Figure 10 is a graphical representation of prevalence of large cells at 30 days using different dialysis solutions and using a solution according to the present invention;

Figure 11 is a graphical representation of multinucleation using different dialysis solutions and using a solution according to the present invention;

Figure 12 is a graphical representation of mitosis: intact vs 30 day exposure using different dialysis solutions and using a solution according to the present invention;

Figure 13 is a graphical representation of prevalence of non viable cells using different dialysis solutions and using a solution according to the present invention;

Figure 14 is a graphical representation of prevalence of apoptosis using different dialysis solutions and using a solution according to the present invention;

Figure 15 is a graphical representation of density of the mesothelial cells using a control, two different test solutions, and using a solution according to the present invention;

Figure 16 is a graphical representation of prevalence of mitosis at two hours using a control, two different test solutions, and using a solution according to the present invention; and

Figure 17 is a graphical representation of TB stained cells using a control, two different test solutions, and using a solution according to the present invention. The experiments recorded hereinafter, using different PD solutions for comparison, were carried out in albino mice weighing 20-25g. Animals were handled according to the NIH guidelines for care of laboratory animals, and housed in plastic cages with a floor area of 513cm². The number of animals per cage was calculated according to the formula A = n x 2 x BW, where A = minimal required area; n = number of animals per cage; and BW = body weight in grams. Accordingly, 8-10 mice were kept in each cage (minimal required area: 460 cm²), maintained under a 12 hours light/dark cycle, and fed with normal mouse Purina chow and water ad libitum. 1. Evaluated parameters and experimental groups.

Morphometric information on cell's population density, mean surface area of mesothelial cells, prevalence of large cells, mean cytoplasmic surface area, prevalence of multinucleated cells, mitosis as well as mesothelial cells' viability was obtained from the following groups of experimental animals: a. Intact unexposed mice. . b. Mice injected with 4.25% glucose lactated solution for peritoneal dialysis. c. Mice injected with 4.25% glucose bicarbonated solution for peritoneal dialysis. d. Mice injected with L-lactate solution without glucose or any other osmotic agent. e. Mice injected with 4.25% Mannitol lactated solution for peritoneal dialysis. f. Mice injected with a commercially available 1.1 % Aminoaci'ds solution for peritoneal dialysis. g. Mice injected with a 4.25% glucose peritoneal dialysis solution prepared with Hank's BSS fluid and sterilized by bacteriological filtration. h. Mice injected with a 7 5% lcodextrin commercially available solution for peritoneal dialysis, i. Mice injected with the new solution containing pyruvate buffer in a concentration of 8mMo!/L and ascorbic acid in a concentration of 45 mg/dl.

2. The Experimental Protocol

Each experimental group included at least 30 mice. Intraperitoneal injections of the corresponding peritoneal dialysis solution were performed once a day (5ml for each injection), during a period of time of up to 30 days. Of course, the intact unexposed control group of animals was exempted from injections. One additional control group of animals was included to check the needle effect. Since observations made on this last group were not significantly different from those obtained from the group of unexposed mice, results are not presented. Samples of the mesothelial monolayer (imprints) were taken 2 hours after the first injection (acute effect), as well as after 15 and 30 days of exposure to the experimental solutions (long term effect). 3. Preparation of the mesothelial cell imprints for morphology

Imprints of the mesothelial monolayer were taken immediately after the animals had been sacrificed by neck dislocation. After laparotomy, slides coated with 1 % Agar were applied for 15-20 seconds to the anterior liver surface, peeling off the monolayer. Two imprints were taken from each animal, making 20 slides for each experimental group. Fixation was immediately done with 70% ethanol for 2 minutes and, after washing, samples were stained with Hematoxylin Eosin and Pyronin B, and examined by light microscopy.

4. Morphometric techniques

All data concerning morphometric information was obtained using an image . analysis system (Sigma Scan Pro), connected to a light microscope. The quality of the exfoliated monolayers was initially evaluated by scanning each slide at low magnification. The main goal was to detect and choose observation areas free from artefactual lack of cells, resulting from poor initial attachment and/or subsequent loss during the several steps of processing. Then, imprints were observed at higher magnification and projected on the computer screen.

5. Imprints for evaluation of mesothelial cells viability

Thirty additional mice for each experimental solution were included in this part of the study. Forty to 60 seconds before being sacrificed, mice received an intraperitoneal injection of a 0.4% Trypan Blue solution in PBS (15ml/100g body weight). Fixation was done by means of 70% ethanol. Mesothelial cells viability was evaluated by Trypan Blue exclusion, according to observations made in 2 imprints obtained from every mouse of each experimental group, and at each time interval (2 hours - 15 and 30 days). Prevalence of Trypan Blue stained cells was determined using the image analysis system already mentioned. Results are presented as the percentage of stained cells present in the whole observed cell population.

6. Prevalence of apoptosis.

This parameter was evaluated in the mesothelial cell imprints stained with Hematoxylin-Eosin and Pyronin B. Apoptotic cells were morphologically identified by light microscopy. Results are presented as the percentage of apoptotic cells seen in the whole observed cell population. 7. Statistical methods.

Data are presented as mean ± standard deviation. Means of intact, unexposed controls and the experimental groups were initially compared by oneway analysis of variance. The level of signification was fixed at P<0.05. As a second step, the appropriate sample size was calculated on the basis of Power = 0.95, calculated for α = β error. Longitudinal comparison of each experimental group against intact, unexposed controls, showing a number of cases lower than the minimal required sample size, were not considered significantly different and, consequently, became excluded from further statistical comparison. Experimental groups displaying a number of cases within the limits based on power = 0.95 were compared with the intact group of animals, using the non-parametric Dunnet test for multiple comparisons against a single control. Statistical analysis of differences between two proportions (nominal scale) was done using the Two Tailed Fisher's Exact Test. Example 1

As shown in Fig. 1 , the new solution for peritoneal dialysis, containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, induced a modest reduction in the density of the mesothelial cell population, reaching anyway statistical significance after 15 and 30 days of exposure, (xx = PO.01 ). Example 2

Exposure of the mesothelial monolayer to the new solution induced a limited increase of the cytoplasmic surface area, even though, in no instance, values reached the limit between normal size and large, hypertrophic cells (518.8 μm²). So, large cells were not observed at all. (Fig. 2. xxx=P<0.001 ). Example 3

Cells exposed to the new solution for peritoneal dialysis showed a slight, but significant increase of nuclear area, which reached a statistical significance of P<0.01 after 15 and 30 days of follow-up. (Fig. 3. x = P<0.05. xx = PO.01 ). Example 4

The monolayer of mice treated with the new peritoneal dialysis solution exposed to view a marginal 2% increase in the prevalence of multinucleated cells, mostly binucleated, after 15 and 30 days of exposure. (Fig. 4. x. =

PO.05).

Example 5

As shown in Fig. 5, the prevalence of mitosis did not significantly change during the whole period of follow-up, compared with that observed in intact unexposed controls. This finding supports the contention that the new solution for peritoneal dialysis, containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, neutralized the early acceleration on the life cycle of the mesothelial cell population exposed to 4.25% glucose. Example 6

As shown in Fig. 6, exposure of the mesothelial monolayer to the new peritoneal dialysis solution failed to induce a significant change in the prevalence of non-viable cells. Indeed, values observed at each experimental interval (2 hours, 15 and 30 days) were not statistically different from that detected in intact, unexposed control mice. This example is a clear indication that the damage of the cells' membrane derived from the exposure to high concentration of glucose was absolutely prevented by the new formulation of the solution, containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl. Example 7

Mice exposed to the new solution at each time interval showed a . prevalence of apoptosis of around 2% and, consequently, not significantly different from that seen in unexposed, intact control animals (1.8 ± 0.9%). This information, presented in Fig. 7, completes those exposed in previous examples, indicating that under the exposure of the new peritoneal dialysis solution, there was no detectable change in the life cycle of the mesothelial cell population. Neither there was nor early acceleration, nor premature senescence leading to programmed cell death in apoptosis. Example 8

In Fig. 8 we compare the density of the mesothelial cell population observed in unexposed intact control mice (C) with a series of experimental groups treated with the different dialysis solutions including, of course, the new solution for peritoneal dialysis containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45mg/dl: 4.25% glucose with buffer lactate (HGL), 4.25% glucose with buffer bicarbonate (HGB), L-D lactate without any osmotic agent (LDL), 4.25% mannitol solution with buffer lactate (ML), 1.1 % aminoacids solution with buffer lactate (AA), 4.25% glucose prepared in Hank's BSS fluid (HGH), and 7.5% lcodextrin fluid with buffer lactate (lc). As it can be seen, besides the mannitol solution (a radical oxygen scavenger), all other fluids induced a significant reduction of the population cells' density, at a level ranging between PO.01 and P .001 (xx = PO.01. xxx = PO.001 ). In animals exposed to the new solution for peritoneal dialysis containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, the reduction was less marked (x = P .05), and not significantly different from that observed in an additional group of mice injected for 30 consecutive days with 20ml/100g of body weight of Hartman solution (78 ± 5 cells/33000μm² for the new solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, vs. 83±7 cells in mice exposed to the Hartman solution). (Fig. 8).

So far, this modest reduction of the cell density population, observed after 30 days of exposure to the new solution, most probably reflects the effect of the mechanical lavage, as well as a change in the microenvironment leading to a dilution of growth factors, as well as nutritional elements. This is perhaps the moment to remind that peritoneal dialysis implies the use of a biological membrane created by Nature, to fill a series of physiological functions absolutely unrelated to our purpose of making from it a reusable membrane for renal replacement therapy. Example 9

Analysis of data presented in Fig. 9 reveals that LDL was the only experimental solution that failed to significantly increase the mean cell surface area, compared with the group of unexposed, intact control animals. As stated before, glucose was absolutely absent from this formulation. Both 4.25% solutions, possessing antioxidant properties (the 4.25% Mannitol fluid and the solution for peritoneal dialysis containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl), showed a modest increase in the cell surface area, which, anyway, was below the upper normal limit of 518.8μm². This information suggests that large hypertrophic mesothelial cells, present in the monolayer of mice treated with the other experimental fluids, should be absent in animals exposed to the new peritoneal dialysis solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl. The statistical difference was at the level .of PO.01. Example 10

This experiment confirmed the supposition postulated in Example 9. Indeed, as shown in fig. 10, the prevalence of large, hypertrophic cells in mice exposed to the new solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl was nil, and not significantly different from values seen in control unexposed mice, neither in those treated with the 4.25% Mannitol fluid or the LD lactate glucose free solution. Consequently, the new solution containing pyruvate buffer in a concentration of 8r Mol/L and ascorbic acid in a concentration of 45 mg/dl, in spite of being enriched with glucose in high concentration (4.25%), prevented the development of a population basically made by large, hypertrophic and senescent cells. The differences with the other formulations containing glucose in high concentration like HGL (High Glucose Lactate), HGB (High Glucose Bicarbonate) and HGHC (High Glucose in Hank's BSS) were at the PO.001 level (xxx = PO.001 ). Example 11

The phenomenon of multinucleation indicating defective cytokinesis, a major alteration in the cell cycle engine, was evident in mice exposed to high glucose with bicarbonate buffer, as well as in the 4.25% glucose fluid prepared on the basis of Hank's BSS. Also, it was significantly higher in animals exposed to the 1.1 % aminoacids fluid and to the 7.5% lcodextrin solution (Fig. 11 ). Exposure to the high glucose lactated fluid failed to induce a significant increase in the prevalence of multinucleation, possibly due to the high percentage of non-viable cells (Fig. 13). Noteworthy, in animals treated with the new solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, the prevalence of multinucleated cells (basically binucleated) was not significantly higher than that seen in intact, unexposed control mice (Fig. 11 ). Example 12

As shown in Fig. 12, the mitotic index was not affected in mice treated with the new peritoneal dialysis solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, whereas use of the 3 other glucose-enriched solutions resulted in a prevalence of mitosis near zero.

It is known that a low intensity oxidative stress can induce acceleration of the life cycle of the exposed cell population. One of the early manifestations is the increase of the mitotic index, as observed with the Mannitol solution, which, even though being a radical scavenger, failed to render the full protection the new peritoneal dialysis solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl provided (Fig. 12). This, in spite of the fact that the differences were not statistically significant, even though the biological implications cannot be overlooked. Example 13

As can be appreciated in Fig. 13, cell viability evaluated by Trypan Blue exclusion was not affected by exposure of the monolayer to the new peritoneal dialysis solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, when compared with intact, . unexposed control mice. This observation is in open contradiction when compared with the 2 most commonly used commercially available dialytic formulations: HGL (4.25% glucose with Lactate buffer) and I (7.5% lcodextrin). The level of significance was P .01 (xx = P .01 ). Example 14

Prevalence of cells dying in apoptosis, seen in imprints taken from mice exposed to the new peritoneal dialysis solution containing pyruvate buffer in a concentration of 8mMol/L and ascorbic acid in a concentration of 45 mg/dl, was not significantly different from values observed in intact, unexposed control animals. On the other hand, values were significantly lower than those detected in animals treated with the 7.5% lcodextrin solution, (xx = PO.01 ) (Fig. 14). Example 15

This experiment, as well as those described in Figs.16 and 17, was performed to evaluate the effect of Pyruvate alone, in a concentration of 8mMol/L, with Lactate 32mMol/L as buffers, with and without Ascorbic Acid in a concentration of 45mg/dl. As stated in Page 11^- line 15, the detrimental effects of glucose can be seen after a short exposure of two hours to the dialytic solution. The described early acceleration of the cell cycle results in a process of early senescence, depopulation, decreased cell viability and increased prevalence of cells dying in apoptosis.

Fig. 15 shows results of one experiment comparing control- intact- unexposed mice with three groups of animals exposed to three different experimental solutions: 4.25% glucose with Lactate buffer in a concentration of 40mMol/L; 4,25% glucose with Lactate buffer in a concentration of 32mMol/L and Pyruvate in a concentration of 8mMol/L; and 4.25% glucose with Lactate buffer in a concentration of 32mMol/L, Pyruvate buffer in a concentration of 8mMol/L and Ascorbic Acid in a concentration of 45mg/dl. As can be seen in Fig.15, density of the mesothelial cell population, evaluated according to the methodology described in preferred embodiments 3 - 4 - 5, seen in the four groups of animals were not significantly different. Example 16

This experiment brings to view the prevalence of mitosis after a two hours exposure in the same experimental groups mentioned in Example 15. As it can be seen, 4.25% glucose with Lactate buffer in a concentration of 40mMol/L induced a significant early acceleration of the life cycle of the exposed mesothelial cell population (PO.001 ). A similar effect at the same level of significance (PO.001) was detected in mice exposed to 4.25% glucose with Lactate buffer in a concentration of 32mMol/L and Pyruvate in a concentration of 8mMol/L. Quite the contrary, the early acceleration of the life cycle of the mesothelial monolayer exposed to 4.25% glucose, Lactate buffer in a concentration of 32mMol/L, Pyruvate in a concentration of 8mMol/L and Ascorbic Acid in a concentration of 45mg/dl was absolutely prevented. Prevalence of mitosis observed in this experimental group was not significantly different from that seen in unexposed-intact control mice. Example 17

This experiment was performed in mice whose mesothelial monolayer was exposed for two hours to the same dialysis solutions mentioned in Examples 15 and 16. We investigated here cells' viability according to Trypan-Blue exclusion. The methodology followed was described in preferred embodiment No. 6.

As shown in fig. 17, mesothelial cells of mice exposed to 4.25% glucose with 40mMol/L Lactate buffer as well as 4.25% glucose with 32mMol/L Lactate buffer and 8mMo!/L Pyruvate buffer showed a prevalence of Trypan-Blue stained cells (non viable) significantly higher (P .001) than that seen in intact-unexposed animals, as well as higher than that detected in mice treated with the solution containing 4.25% glucose with 32mMol/L Lactate buffer, 8mMol/L Pyruvate and Ascorbic Acid in a concentration of 45mg/dl. Additionally, differences between observations made in this latter experimental group of mice and intact-unexposed control animals were. not statistically significant.

As shown in Figures 15, 16 and 17, use of Pyruvate alone, in a concentration of 8mMol/L, failed to prevent the early acceleration of the life cycle of the mesothelial cell population. On the other hand, the combination of Pyruvate, in a concentration of 8mMol/L, with Ascorbic Acid in a concentration of. 45mg/dl, prevented the above mentioned change of the mesothelial life cycle keeping density, prevalence of mitosis and cells' viability at a level not significantly different from that observed in normal-unexposed animals.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A peritoneal dialysis solution comprising a pyruvate in a concentration of about 7-9 mMol/L in combination with at least one further antioxidant present in physiologically acceptable concentrations, said pyruvate and further antioxidant being present in concentrations affective to prevent the early acceleration in the cell cycle of the mesothelial monolayer, and the consequent premature senescence derived from a high glucose concentration in the dialysis fluid.

2. A peritoneal dialysis solution according to Claim 1 wherein said further antioxidant is selected from the group consisting of ascorbic acid and aldose reductase inhibitors.

3. A peritoneal dialysis solution according to Claim 1 wherein said further antioxidant is ascorbic acid.

4. A peritoneal dialysis solution according to Claim 3 wherein said ascorbic acid is present in concentrations of about 1 -95 mg/L.

5. A peritoneal dialysis solution according to Claim 1 , including glucose as an osmotic agent, in concentrations ranging between 1.5% and 4.25%.

6. A peritoneal dialysis solution according to Claim 1 , comprising water, having dissolved therein the following components in the respective concentrations indicated: