WO2001002004A1

WO2001002004A1 - Peritoneal dialysis solution containing antioxidant for treating renal failure

Info

Publication number: WO2001002004A1
Application number: PCT/KR2000/000654
Authority: WO
Inventors: Hi Bahl Lee; Hunjoo Ha; Sungil Kim
Original assignee: Lee Hibahl; Hunjoo Ha; Sungil Kim
Priority date: 1999-07-02
Filing date: 2000-06-21
Publication date: 2001-01-11
Also published as: KR20010008659A; KR100390630B1

Abstract

The present invention relates to peritoneal dialysis solutions containing antioxidant(s) for patients with end-stage renal failure undergoing peritoneal dialysis. More specifically, the present invention relates to peritoneal dialysis solutions containing electrolyte (Na?+, Mg2+, Ca2+ and C1-¿), buffer (lactate and/or bicarbonate), osmotic agent(s) (glucose, polyglucose, amino acid, glycerol, polypeptide, or combinations thereof) and antioxidant(s) (catalase, taurine, ascorbic acid, α-tocopherol, N-acetylcysteine, glutathione, α-lipoic acid, superoxide dismutase, or combinations thereof) that inhibits reactive oxygen species. By inhibiting reactive oxygen species that may be generated by the stimulation of high concentration of glucose contained in peritoneal dialysis solutions, the peritoneal dialysis solution of the present invention, unlike the currently used peritoneal dialysis solutions, can prevent oxidative stress and subsequent peritoneal injury.

Description

PERITONEAL DIALYSIS SOLUTION CONTAINING ANTIOXIDANT FOR TREATING RENAL FAILURE

Technical Field of the invention

The present invention relates to peritoneal dialysis solutions containing

antioxidants for treating renal failures. More specifically, the present invention

relates to peritoneal dialysis solutions for patients with end-stage renal failure undergoing peritoneal dialysis, containing electrolytes including Na⁺, Mg²⁺, Ca²⁺

and CI^"; a buffer; an osmotic agent; and at least one antioxidant that inhibits the

generation of the reactive oxygen species

Background of the Invention

Kidneys which exist symmetrically with respect to the spine behind

peritoneum are important organs that remove metabolic wastes and unnecessary

excess water, that control the blood concentrations of calcium and phosphorus, that enhance the absorption of calcium from intestines by activating vitamin D

produced in the body, that control blood pressure by regulating sodium excretion and by renin-angiotensin, and that produces hemoglobin by secreting

erythropoietin.

Renal failure refers to a state where renal function is decreased or completely lost, and can be categorized into acute renal failure and chronic renal failure. Acute renal failure refers to a state where renal function is temporarily lost, and typically results from renal ischemia, sepsis, or drug toxicity. Generally, kidneys of patients suffering from the acute renal failure recover to normal state. When the renal damage continues, however, due to diabetes, chronic glomerulonephritis, high blood pressure, congenital polycystic renal disease, renal failure progresses to chronic renal failure.

Chronic renal failure refers to a state where renal function is lost permanently. For some serious cases, it can ultimately result in death.

As one method for the treatment of patients suffering from acute or chronic renal failure, the dialytic therapy has been in application for a long time. The dialysis therapy is divided into hemodialysis and peritoneal dialysis.

Hemodialysis which is widely used is effected by drawing the patient's blood out of body, introducing the blood into an artificial kidney equipped with artificial dialysis membranes, allowing metabolic wastes (for instance, urea and creatinine) to diffuse through the artificial membrane and pass into a dialysis solution, and removing excess water through ultrafiltration utilizing a negative pressure. The hemodialysis provides satisfactory results in treating the renal failure. However, The hemodialysis has inherent disadvantages because it is an extracorporeal treatment that requires special machinery, and it also has a disadvantage that it requires patients' regular visits to hospital (usually, three times a week or more than 12 hours a week).

Peritoneal dialysis is effected by directly infusing dialysis solution into the patient's abdominal peritoneal cavity in which a peritoneal catheter is pre- implanted, allowing the dialysis solution to dwell for 4-6 hours such that metabolic wastes (for instance, urea and creatinine) diffuse from capillary blood into the peritoneal cavity and passed into the dialysis solution, and removing excess water by virtue of the difference in the osmotic pressure produced between the

infused hypertonic dialysis solution and the body fluid.

In the past, peritoneal dialysis has been selectively adopted in which

hemodialysis is not suitable. Peritoneal dialysis, however, is increasingly used

since the continuous ambulatory peritoneal dialysis (CAPD) has been developed.

These new peritoneal dialysis has advantages that it can be carried out by the

patient oneself at 3 to 4 times a day, the cost involved is low, and the time

required for the therapy can be changed depending on the patient's daily life.

Patients receiving peritoneal dialysis have equal or better chance of survival

when compared to patients receiving hemodialysis during the first 3-5 years of

dialysis.

Conventional dialysis solution used for peritoneal dialysis is an acidic,

hypertonic solution containing: electrolytes represented by Na⁺, Ca²⁺, Mg²⁺ or CI^",

buffer typified by lactate; and osmotic agents including glucose and polyglucose.

Recently, to avoid the problems caused by acidic solution, neutral dialysis

solution containing bicarbonate instead of lactate has been developed.

However, there are many reports that diabetiform changes in the

peritoneum may result from high concentration of glucose used in the dilaysis solution to obtain osmotic gradient across peritoneum and that a peritoneal

membrane damage may result from oxidative stress which are caused by high concentration of glucose and a long-term administration of peritoneal dialysis solution. In the current invention, the term "oxidative stress" refers to a tissue or

cell damage induced by reactive oxygen species (examples: superoxide anion,

hydrogen peroxide, hydroxyl radical). Accordingly, patients administered with peritoneal dialysis solution containing high concentration of glucose for a long time are subjected to a

peritoneal membrane damage leading to a failure to remove water and metabolic

waste products which requires more peritoneal dialysis solution containing higher

concentration of glucose. By the repeated vicious cycles, the peritoneal

membrane damage becomes worse, and finally peritoneal dialysis should be stopped in the end.

Summary of the Invention

Therefore, an objective of the present invention is to provide a new

peritoneal dialysis solution to solve the problems caused by the conventional

peritoneal dialysis solutions. The objective of the present invention can be

achieved by providing peritoneal dialysis solutions containing antioxidants, more

specifically by providing the peritoneal dialysis solution containing electrolytes

including Na⁺, Mg²⁺, Ca²⁺ and CI^", buffer, osmotic pressure regulating agent, and

at least one antioxidant that inhibits reactive oxygen species.

Detailed Description of the Present Invention

Inventors of the present invention have been working on the effects of

high glucose on the peritoneal mesothelial cell biology and found that reactive oxygen species mediate peritoneal tissue injury and that reactive oxygen species can be satisfactorily inhibited by a peritoneal dialysis solution containing antioxidants, without separately administering drugs to the patient suffering from

renal failure. The peritoneal dialysis solution according to the present invention comprises 1) electrolytes, 2) buffer, 3) osmotic pressure regulating agent and 4) at least one antioxidant.

The electrolytes include Na⁺, Mg²⁺, Ca²⁺ and CI^", and can be supplied by sodium chloride, magnesium chloride and/or calcium chloride.

The buffer that controls the pH of the dialysis solution to an appropriate range is selected from the group consisting of bicarbonate and lactate. Bicarbonate is preferred buffer.

The osmotic agent used to remove excess water from the blood into the dialysis solution includes glucose, polyglucose, and glycerol, but are not limited thereto. Amino acids and polypeptides can also be used as an osmotic agent in the present invention.

The antioxidant used in the present invention for the inhibition of the generation of reactive oxygen species is preferably selected from the group

consisting of catalase, taurine, ascorbic acid, α-tocopherol, N-acetylcysteine,

glutathione, α-lipoic acid, superoxide dismutase, or combinations thereof.

Ruiz-Munoz LM et al have reported that high concentration of glucose increases the generation of hydrogen peroxide in the mesangial cells, but this has been inhibited by the combination of catalase, an enzyme that catalyze the decomposition of hydrogen peroxide into water and oxygen, with enalaprilat used for the treatment of diabetic renal disease (Nephrol Dial Transplant 12:456-464, 1997).

Ha H et al have reported that long-term administration of taurine to diabetic rats results in a decrease of proteinuria, an indicator of diabetic nephropathy, and a meaningful decrease in the gene expression of transforming

growth factor-β that is a fibrosis enhancing growth factor and fibronectin that is a

cell metrix protein (Free Biol Med 26:944-950, 1999).

King GL et al have reported for the first time that diabetic retinopathy or

nephropathy can be reduced by administering α-tocopherol to the diabetes-

mellitus patients (Diabetes Care, 1999). Also, Craven PA et al have shown that

a long-term administration of ascorbic acid or α-tocopherol to diabetic rats

caused a significant reduction of the indicators of diabetic nephropathy (J Am Soc Nephrol 8:1405-1411 , 1997). Studer RK et al have also reported that administration of an antioxidant such as N-acetylcystein to diabetic rats significantly inhibited activation of protein kinase C that mediates tissue damage and reduced the generation of

transforming growth factor-β (Metabolism 46:918-925, 1997).

Marocutti A et al have reported that the growth of fibroblast separated from the skin of the patient suffering from diabetic nephropathy has been inhibited, but it returned to normal state by addition of glutathion or superoxide dismutase (J Am Soc Nephro 9:1060-1066, 1998).

Hofmann MA et al have shown that NF-kappaB that alters the transcription of many genes as one of the results of oxidative stress in the tissue is related to diabetic nephropathy, and the activity of NF-kappaB can be inhibited

by administration of α-lipoic acid (Diabetologia 42:222-232, 1999).

Nishikawa T et al have reported that an increase in the concentration of the reactive oxygen species mediated by high concentration of glucose originates from the increase in the glucose metabolism in mitochondria. They have also showed that the activation of protein kinase C, known as the mechanism of tissue damage due to high glucose level, the procudtion of AGE (Advanced glycosylation end product) and the activation of aldose reductase can be significantly reduced by inhibiting the superoxide production by superoxide dismutase (Nature 404:787-790, 2000).

The amount of antioxidants contained in the dialytic solution depends on the kind of antioxidant used, since the inhibition ability against reactive oxygen species is different for each antioxidant.

The preferable amount for each antioxidant used is as follows: based on 100 ml of dialysis solution, catalase 10000 - 50000 unit; taurine 0.001 - 0.1 g;

ascorbic acid 0.02 - 0.2 g; α-tocopherol 0.004 - 0.04 g; N-acetylcystein 0.008 -

0.32 g; glutathione 0.016 - 0.031 g; α-lipoic acid 0.021 - 0.042 g; or superoxide

dismutase 10 - 100 unit.

Generally, the peritoneal dialysis solution of the present invention is administered 4 times a day and 2 L for a single dose. The solution is administered to the patients suffering from renal failure according to the commonly used method. More particularly, the solution is administered to the peritoneum through the pre-implanted catheter in the peritoneum. About 4-6 hours are generally required for removing metabolic wastes including urea and creatinine as well as excess water.

The invention will be further illustrated by the following examples, but the scope of the present invention is not limited to the examples given. Example 1

Peritoneal dialysis solutions were prepared by adding water up to 100ml to a mixture of glucose 1.5 g, sodium chloride 358 mg, sodium lactate 446 mg,

calcium chloride - 2H₂0 25.7 mg, magnesium chloride - 6H₂0 5.08 mg and

antioxidant described in the Table 1. Table 1

Example 2

In the same manner described in Example 1 , peritoneal dialysis solutions were prepared except that the different amount of glucose was added. The amount of glucose used in the peritoneal dialysis solution is shown in Table 2 below.

Table 2

Example 3

Changes in the Concentration of the Reactive Oxygen Species according to the Changes in the Glucose Concentration and Effect of Antioxidants 3-a) Preparation of peritoneal mesothelial cell solution

To the M199 culture medium containing 10 % bovine fetal serum (GIBCO, USA), the human peritoneal mesothelial cells were added at the concentration 5

x 10⁴ cell/2 ml/well. The cells were attached onto a slide glass pre-treated with

poly-L-lysine and cultured at 37 °C. When the human peritoneal mesothelial

cells were ca. 80 % confluent, the medium was exchanged with a serum-free medium and cultured again (2^nd culture) at the same temperature for 24 hours.

3-b) Preparation of Samples

Second culture medium was replaced with the third culture medium as below, and cells were cultured for an hour.

1. culture medium containing 30 mM glucose;

2. culture medium containing 100 mM glucose;

3. culture medium containing 30 mM glucose and 500 unit/ml catalase; and,

4. culture medium containing normal 5.6 mM glucose, but not containing

antioxidant (will be refered to as "control group" hereinafter);

After each culture product was washed with Ca²⁺ and Mg²⁺-containing

phosphate buffer solution, a reagent (obtained by dissolving 2.69 μg of 5-

chloromethyl-2,7-dichloro dihydro fluorescein in 5 μl dimethyl sulfoxide) and 1 ml phosphate buffer solution were added and left standing for 15 min at room temperature. Using Ca²⁺ and Mg²⁺-containing phosphate buffer solution, the excess reagent that was not taken up by the cells were washed out such that 4 samples were obtained.

3-c) Evaluation of Fluorescence Level

Using a laser scanning confocol microscope (Leica TSC NT, Germany), the fluorescence level for 4 samples obtained from 3-b) was calculated and the results were summarized in Table 3.

Table 3

(The relative fluorescence refers to the ratio of the sample's fluorescence to the control group's fluorescence, In other words, it means relative values calculated by setting the fluorescence of the control group to 1) The above Table 3 shows that the fluorescence increases as the glucose concentration increases, but the fluorescence of the sample containing catalase, an antioxidant, is virtually identical to that of the control group. That is, results shows that the generation of the reactive oxygen species increases proportionally to the amount of glucose added, but this increase can be inhibited by the addition of antioxidant. Example 4

Generation of the Reactive Oxygen Species by the Commercially Available Peritoneal Dialysis Solutions

4-a) Preparation of peritoneal mesothelial cells Peritoneal mesothelial cells were prepared in the same manner described in Example 3-a).

4-b) Preparation of the samples

Sample: 10 ml of unused commercially available peritoneal dialysis solution containing 4.25 % glucose and 10 ml of each drained dialysis solutions from the patient immediately after injection (time 0) and after 15, 30, 60, 120 and 240 minutes

After washing the peritoneal mesothelial cells treated with serum-free medium for 24 hours, 1 ml of the samples were added, and cells were cultured

for 1 hour in the cell incubator. A reagent obtained by dissolving 2.69 μg of 5-

chloromethyl-2,7-dichloro dihydro fluorescein in 5 μl dimethyl sulfoxide and 1 ml

phosphate buffer solution were added and standed for 15 min at room temperature. Using Ca²⁺ and Mg²⁺-containing phosphate buffer solution, the excess reagent that was not taken up by the cells were washed out such that samples were obtained.

4-c) Evaluation of Fluorescence Level

Using a laser scanning confocol microscope (Leica TSC NT, Germany), the fluorescence level for 4 samples obtained from 3-b) was calculated and the results were summarized in Table 4.

Table 4

Table 4 shows that not only the commercially available peritoneal dialysis solution itself but also the drained dialysis solutions obtained from the dialysis patient during the dialysis process stimulate the generation of the reactive oxygen species from the peritoneal mesothelial cells.

The above Example 3 shows that high concentration of glucose increases the generation of the reactive oxygen species, whereas catalase, an antioxidant, reduces the generation of the reactive oxygen species. It is well

known to the skilled person that taurine, ascorbic acid, α-tocopherol, N-

acetylcysteine, glutathione, α-lipoic acid, superoxide dismutase, or their mixures

as well as catalase can inhibit the generation of the reactive oxygen species. Therefore, it is expected that the peritoneal dialysis solution additionally containing at least one antioxitant selected from the group consisting of catalase,

taurine, ascorbic acid, α-tocopherol, N-acetylcysteine, glutathione, α-lipoic acid,

superoxide dismutase, or combinations thereof, unlike the currently used peritoneal dialysis solutions, can also prohibit the damage of the peritoneal mesothelial cells and the loss of the function of the peritoneal membranes.

Claims

1. A peritoneal dialysis solution for the treatment of renal failure, comprising

electrolytes including Na⁺, Mg²⁺, Ca²⁺ and CI^", buffer, osmotic agent and at

least one antioxidant that inhibits the generation of the reactive oxygen

species.

2. The peritoneal dialysis solution according to claim 1 wherein the antioxidant

is selected from the group comprising catalase, taurine, ascorbic acid, α-

tocopherol, N-acetylcysteine, glutathione, α-lipoic acid, superoxide

dismutase, or combinations thereof.

3. The peritoneal dialysis solution according to claim 2 wherein the antioxidant

is catalase.

4. The peritoneal dialysis solution according to claim 2, wherein the amount of

antioxidant use is, based on 100 ml of the peritoneal dialysis solution,

catalase 10000 - 50000 unit, taurine 0.001 - 0.1 g, ascorbic acid 0.02 - 0.2 g,

α-tocopherol 0.004 - 0.04 g, N-acetylcystein 0.008 - 0.32 g, glutathione

0.016 - 0.031 g, α-lipoic aicd 0.021 - 0.042 g or superoxide dismutase 10 -

100 unit.

5. The peritoneal dialysis solution according to claim 1 , wherein the buffer is

bicarbonate or lactate.

6. The peritoneal dialysis solution according to claim 1 , wherein the osmotic pressure regulating agent is selected from the group consisting of glucose, polyglucose, glycerol, amino acid, polypeptide and combinations thereof.

7. The peritoneal dialysis solution according to claim 1 , wherein the electrolytes are supplied by sodium chloride, magnesium chloride and/or calcium chloride.

8. The peritoneal dialysis solution according to claim 1 , wherein the renal failure includes acute renal failure and chronic renal failure.