WO1998001141A1

WO1998001141A1 - Alternative osmotic agents and related solutions for peritoneal dialysis

Info

Publication number: WO1998001141A1
Application number: PCT/IB1997/000747
Authority: WO
Inventors: Antonella Bisio; Annamaria Naggi; Enrico Petrella; Alberto Siccardi; Giangiacomo Torri
Original assignee: Bieffe Medital S.P.A.
Priority date: 1996-07-09
Filing date: 1997-06-20
Publication date: 1998-01-15
Also published as: IT1283137B1; AU2975297A; ITMI961419A1; ITMI961419A0

Abstract

The osmotic agents alternative to glucose for solutions for peritoneal dialysis are selected from the group comprising: chondroitin sulfates (ChS) as such, or their mixtures, products obtained through the desulfation of the same, derivates of these obtained through depolymerization either chemical or enzymatic, etc., hyaluronic acid and its products of depolymerization, products of chemical derivation of other glycosaminoglycans, as such, or depolymerized.

Description

Title: ALTERNATIVE OSMOTIC AGENTS AND RELATED SOLUTIONS FOR PERITONEAL DIALYSIS.

The present invention concerns new osmotic agents alternative to glucose, and related solutions for peritoneal dialysis.

Peritoneal dialysis is a method of treating end-stage renal failure (both acute and chronic) that had already been envisaged at the end of the last century but came into clinical practice only in the 1950s. Since the 1970s a particular form of this technology, CAPD (Continuous Ambulatory Peritoneal Dialysis), has had very good clinical resilts and is today widely used. It is estimated that currently over 11% of uremic patients in the terminal phase are treated with such methodology. The principle on which CAPD is based is that of dialysis, i.e. the diffusive passage of several solutes from a solution where their concentration is higher (in this case the blood of the patient) to another solution where their concentration is lower, or absent (in this case the dialysis fluid) , there being interposed between the two solutions a semipermeable membrane (the peritoneum of the patient) through which these solutes can pass. The severe compromising, or failure, of the excretory and detoxifying function of the kidneys hinders the elimination with the urine of some toxic substances that form in the body during normal metabolic processes. Furthermore when the diuresis of these patients is extremely low, or even absent, the patient's body also accumulates water, that must definitely be eliminated so as not to provoke a life threatening cardiac insufficiency. To favor the elimination of such excess water the fluid used for peritoneal dialysis is made hyperosmotic with respect to the blood so that water passes from the blood to the dialysis solution. The osmotic force depends on the osmolality of a solution that represents a measure of the total number of osmotically active molecules in the solution itself. It refers to the molal concentration, i.e. to the number of moles per Kg of solvent In clinical practice the concentration of a solute is measured as a percentage value, or as its weight per liter of solution In treating renal insufficiency, peritoneal dialysis (CAPD) is today a valid alternative to extracorporeal dialysis (hemodialysis), that however still remains the most widespread Despite its undoubted economic advantages, (e g there is no need for sophisticated and expensive equipment, and treatment can be carried out by the patients themselves at home) and its capacity to allow the social and economic recovery of the patients (CAPD is compatibile with normal working activity) several drawbacks have limited its wider diffusion Among these are frequent infections and the loss of peritoneal permeability with time Glucose is generally considered responsible for the latter

The presence of glucose in the dialysis fluid (in quantities usually varying between 2 25 and 4 25%) causes several negative consequences, These are

1 ) an abnormally low pH of the dialysis fluid, with possible irritating effects on the peritoneum,

2) an excessive metabolic load, from 150 to 300 g/day, that tends to give rise to hypertriglyceπdemia and/or obesity

3) a marked fluctuation of the blood sugar levels during the day

4) difficulty in controlling giycemic values, especially in insulin-dependent diabetics (whose number is tending to increase)

5) because of hyperglycemia, a tendency of the glucose metabolism to take an alternative route (polyols) with tissue hyperosmolality, that at the level of the peritoneal membrane can be very detrimental

For these reasons several osmotic agents have been proposed for use as alternatives to glucose

As CAPD calls for the introduction of 8 liters of dialysis fluid per day (almost 3000 liters a year) consideration must be given to the principal criteria that such solutions must fulfil These are criteria 1 ) a capacity to remove about two liters of body fluid per day 2) a capacity to remove toxic nitrogenous catabo tes

3) not introducing into the cavity substances that can potentially damage or irritate the peritoneal membrane or produce mutagenic or oncogenic effects in the long term or with repeated use

4) non-accumulation in body fluids with possible toxic effects

5) not be antigenic

6) able to be sterilized and not pyrogenic

The osmotic agents that have been proposed as alternatives to glucose have several drawbacks Fructose¹ and

have the disadvantages of being metabolized by the polyol route and poorly tolerated by diabetics and can provoke neuropathy Mannitol ³ and xy tol ⁴ frequently provoke lactic acidosis and hyperuπcemia and are potentially carcinogenic Solutions containing gelatine could be dangerous due to possible anaphylactic reactions and their high viscosity ⁵ presents technical problems Dextran 6 has been demonstrated to be inefficient and, furthermore, presents risks of possible anaphylactic reactions The use of ammo acids⁷ (apart from the high cost of their solutions) is limited due to their tendency to worsen the patient's actdotic state and to increase urea synthesis Furthermore it is not possible to add glucose to these solutions as steam sterilization of these two families of compounds generates (Maillard reaction) carametization Several glucose polymers (dextπns), obtained by the hydrolysis of corn starch, have shown desirable properties from the point of view of water ultrafiitration 8 However, patients have been observed to accumulate several polymer degradation products such as maltose and maltotπose, whose long term effects have not been determined, such accumulation certainly cannot be defined as physiolgical

The reabsorption rate of the osmotic agent from the peritoneal cavity is determined by its molecular weight

Substances of low molecular weight, such as glucose, glycerol and the ammo acids, are rapidly reabsorbed Large molecules, for instance albumin, cannot easily cross the peritoneal membrane and their reabsorption (that occurs through the lymphatic route) is minimal. Thus an osmotic agent of hign molecular weight remains in the peritoneal fluid for a long time and can fully exert its action of attracting water Nevertheless it has been observed that the initial ultrafiltration rate obtained with substances of high molecular weight is lower than that obtained with substances of lower molecular weight

'Raja RM.Kxamer MS.Manch.anda R et al Peritoneal dialvsis with fructose, dialysate-prcvention of hyperglycemia and hyperosmolality Ann Int Med 1973.79 51 1-517

²Yutuc W. Ward G. Silipelar G. et al Substitution of Sorbitol for Dextrose in peritoneal irrigation fluid Trans Am Soc Artif Organs 1967.13 168-171

Wang YM. Van E s J Nutritional significance of fructose and sugar alcohols Ann Rev Nutr

1981.1 437

Bazzato G. Coli U. Landini S et al Xylitol and low doses of insulin new perspectives for diabetic ureπuc patients in CAPD Perit Dial Bull 1982.2 161-164

^Winchester J Alternative osmotic agents to dextrose for peritoneal dialysis In LaGreca G et al

(eds) Peritoneal dialysis Wichtig Editore Milano 1985. 135-142

G|essing J The use of dextran as a dialvsmg fluid in pentoneal dialvsis Acta Med Scand

1969.185 237-239

Gjessing J The use of dextran as a dial sing fluid in pentoneal dialvsis Acta Med Scand

1969.185 237-239

Mistry CD.Gokal R. Mallick NP Glucose polimer as an osmotic agent In Maher JF et al (eds)

Frontiers in Peritoneal Dialysis. Field. Rich and Assoc Inc NY 1986. 241 -248

SUMMARY OF THE INVENTION

The present invention concerns new osmotic agents alternative to glucose, and related solutions for peritoneal dialysis

According to the first aspect of the invention alternative osmotic agents are glycosaminoglycans devoid of anticoagulant and pro-hemorrhagic activity

Compounds with such characteristics include both natural chondroitin sulfates and hyaluronic acid and other products that can be obtained by chemical or enzymatic modification of these and other glycsoaminoglycans, or their precursors

According to another aspect of the present invention the preferred osmotic agents are selected from the group comprising

• chondroitin sulfates (ChS) as such or their mixtures, products obtained through the desulfation of same, their derivatives obtained through depolymerization either chemical or enzymatic, • hyaluronic acid (HA) and its depolymerization products,

_• products derived from chemical modification (e g desulfation, reduction, oxidation etc ) whether of the said glycosaminoglycans or of other glycosaminoglycans as such or depolymeπzed

According to another aspect of the present invention it was surprisingly found that agents with molecular weight from 1000 to 30000 Dalton, preferably around 25000, give optimal ultrafiltration, even if according to the prior art only agents of very low molecular weight should have given high ultrafiltration values

The choice of these macromolecules as osmotic agents was prompted by their physiological origin and by the fact that, differently from other glycosaminoglycans such as hepaπn and heparan sulfate, they show no pro- hemorrhagic activity Furthermore these are polyelectrolytes and this is advantageous as the osmotic properties depend on the molal concentration of the osmotic agent, and pharmacologically significant osmotic activity is unlikely to be achieved with completely neutral macromolecules for both solubility problems and the rheological characteristics of the final solutions

ChS can be obtained from animal tissues through extraction processes, or by the chemical modification of precursors, of microbial origin, with a chondroitin type skeleton (A Naggi in "Non anticoagulant actions of glycosaminoglycans" ed J Haremberg, B Casu, Plenum Press, New York,

1996, 59-64)

For the application of the invention commercial ChS (SIGMA, Laboratoπ Deπvati Organic! (LDO) and purified HA (Cπnos) are used Commercial ChS are generally mixtures of chondroitin 4 sulfate (Ch4S or Ch-S-A) and chondroitin 6 sulfate ( Ch6S or Ch-S-C) or hybrid structures containing in the same chain residues of both Nacetil galactosamine 6 sulfate and Nacetil galactosamine 4 sulfate (L A Fransson, Mammalian Glycosaminoglycans, in G O Aspinal, ed , 'The Polysacchaπdes" vol 3, Academic Press, New York, 1982, pp337~415) To the best of our knowledge there has never been observed, in extracted compounds, the exclusive presence of a single component The said mixtures can contain dermatan sulfate, known also as chondroitin sulfate B (DeS or ChS-B), as both a component and as an impurity The structural formulae of the prevailing disacchaπde units of the three ChS are shown in figure 1

The structural characterization of the glycosaminoglycans was carried out efficiently with techniques of nuclear magnetic resonance spectroscopy (NMR) (A Perlin, B Casu, Spectroscopic Methods In G O Aspinal, ed , 'The Polysacchandes" vol 11 , Academic Press, New York, 1982, pp133-193) obtaining also information on the presence of different components and/or impurities, on the distribution of the sulfate groups, and on the end groups The homogeneity of the preparations was normally determined by the combined use of NMR and electrophoretic techniques (G Torn, "Electrophoretic and Nuclear Magnetic Resonance characterisation of non heparin glycosaminoglycans", Seminars in Thrombosis and Hemostasis, vol 17, suppl. 1 , 1991 )

The molecular weights of the ChS are generally dispersed, being between 5000 and 50000 Dalton The apparent value of the mean molecular weight can vary as a function of the evaluation method used (gel permeation, NMR, viscosity etc.)

Chondroitin sulfates (ChS) are present in the body in the form of proteoglycans In such a form they are components of the extracellular matrix, to whose elastic properties they contribute through the inter- and intrachain repulsion of the negative charges of their sulfate and carboxylic groups This charge disposition also controls the diffusion of water and small cations through the matrix

With respect to the macromolecular osmotic agents proposed biopoiymers of animal origin, such as albumin and ChS, have the advantage of being recognized and catabolized by the body The use of ChS as the inhibitor of free radical formation in peritoneal dialysis solutions and/or as a regulator of the permeability of peritoneal membrane is cited in the international patent application W093/14797, that refers however to compositions for use during and after peritonitis and that use glucose or glycerol as the osmotic agent, and that contain as the principal additive a mixture of ammo acids, at least one of which is in the dipeptide form Note that the concentration of ChS used as the inhibitor of radicals varies from 0 1 (example 3) to 1 % and is too low to generate an osmotic effect The same claimed solutions can contain a degradation product of hyaluronic acid for the regeneration of peritoneal mesothelium without the formation of fibrous tissue The concentration of added ChS is very low and the indications of this patent application do not in any way at all imply the use of ChS as a possible osmotic agent, in fact as glucose is the indicated osmotic agent the indications are absolutely misleading

Samples of non anticoagulating glycosaminoglycans, particularly ChS, have been tested with regard to their dialytic properties in vitro, and in vivo in peritoneal dialysis experiments in rats

IN VITRO TESTS (dialysis with membranes)

The samples described in examples 1 , 4, 5, and 6 were subjected to tests of diafiltration (see Example 7) At determined times (0,1 , 2, 3, 4, 5, 6, 24 h) the increase in volume of the dialytic solutions was evaluated by weight, and expressed as percentage increase with respect to the volume of the initial solution (Δw%) Furthermore an evaluation was made of the osmola ty before and after the dialysis tests The results reported in table 1 have been compared with those relative to glucose, hyaluronic acid (HA) and two samples of commercial maltodextπn (Cerestar-01910, 01934) The data reported in table 1 show that in the working conditions indicated, at concentration values lower than or equal to 1 25% w/v, glycosaminoglycans are inefficient as osmotic agents, while for concentration values equal to or higher than 2 5%, significant and sometimes noteable increases in volume of the dialytic solution can be observed Surprisingly such increases do not seem to be influenced significantly by the factor of molecular weight For confirmation see examples 1b (ChS 24,000 Dalton) and 3a (ChS<1 ,000) where both solutions at 5% w/v concentration show an analogous increase in volume after six hours, despite the starting values of osmolahty being significantly different In several cases after 24 h better results were observed by employing high molecular weight polymers

Table 1

IN VIVO TESTS

In conditions simulating clinical use in vivo tests (see Example 9) were carried out to verify the behavior of solutions containing ChS as the osmotic agent, compared with ultrafiltration and dialyses of low molecular weight solutes (urea, creat ine) With the aim of reproducing the characteristics of dialysis fluids used in clinical practice several electrolytes (Na, Cl, Ca, Mg and lactate) were added to the chondroitin sulfates

The following graphs (G1 , 2, 3, 4, 5) allow comparison of the different solutions prepared, in as far as concerns the ultrafiltration entity, the osmolahty and the sodium and ChS content (expressed in absolute value) in the dialysis fluid

Analysis of these data indicates that the reabsorption of the chondroitin sulfates by the peritoneum occurs, surprisingly, quite slowly as shown by graph 5 This permits a prolonged osmotic effect of the solution, differing from that which occurs for glucose and other conventional, low molecular weight, osmotic agents It is of particular interest to observe that the ultrafiltration is particularly significant after the third hour, by which time the osmolahty of the solution reaches equilibrium values Equally interesting is the observation that at low concentration (1 25%) ChS is not able to potentiate the ultrafiltration of the peritoneal dialysis solution, not even in the in vivo experiment In effect, starting from the first hour of dwell time there is reabsorption (or an inverse movement of fluids) that at the sixth hour becomes more significant Concentration values of ChS equal to 7% appear to exceed the limit of tolerabi ty if extrapolated to a clinical condition, given the high degree of ultrafiltration achieved In vivo, analogous behavior can be noted for solutions of ChS of different molecular weight that do not correlate with the value of the osmolahty measured initially The addition of co-solutes of low molecular weight like glucose and glycerol, in concentrations that alone would not generate significant ultrafiltration, allow further improvement in the performance of the ChS solutions, as shown in graph G6

Different aspects and advantages of the invention appear more clearly from the following (illustrative but not limiting) examples and relative figures and graphs In the drawings, the figures show respectively

Figure 1 Three ( 3) formulae of the basic compounds

Figure 2 ¹³C-NMR spectrum of G1845 (Chondroitin sulfate A)

Figure 3 ³C-NMR spectrum of G1381 (Chondroitin sulfate A)

Figure 4 ¹³C-NMR spectrum of G1394c (Commercial chondroitin sulfate of low molecular weight)

Figure 5 ¹³C-NMR spectrum of G1845b (Chondroitin sulfate partially depolymeπzed with chondroitinases ABC)

Figure 6 ^{1 3}C-NMR spectrum of G2001 (Chondroitin sulfate partially depolymeπzed with hyaluronidases)

Figure 7 ¹³C-NMR spectrum of G 1863 (Desulfated chondroitin sulfate)

Figure 8 ³C-NMR spectrum of G1093 (Hyaluronic acid)

Figure 9 ^{1 3}C-NMR spectrum of G2198 (2-O-Desulfated hepann)

G1 Ultrafiltration

G2 Percentage of ultrafiltration

G3 Sodium in dialysis fluid (absolute value)

G4 Osmolahty in dialysis fluid

G5 ChS recovery in dialysis fluid

G6 Ultrafiltration in presence of co-solutes

G1

Ultrafiltration

50

40ι

10

0

0 3 4 h

♦ G1394 D2(sol5%) -,, G1845 C(sol5%) » G1394D3(sol7%) _*-G1381(3%) *G1845 (1,25%)

G2

Percentage of ultrafiltration

200

150

100

50

0

1

G1394D2(sol5%)

G1845C(sol5%)

G1394D3(sol7%) G3

Sodium in dialysis fluid (absolute value)

6 I-

• G1394 D2(sol5%) - G1845 C(sol5%)

* G1394 D3 (sol7%) → G1381 (3%)

G4

Osmolahty in dialysis fluid

500 _

300

-» G1394 D2(sol5%) ^ G1845 C(sol5%) _. G1394 D3 (S0l7%) G1381 (3%) G5

ChS recovery in dialysis fluid

mg

500

400^-

— fc-

300 ^¥

2001 ιoo :

o

0

• ChS3%

-». ChS3%+glycerol

G6

Ultrafiltration in presence of co-solutes

30

10-

⁵F 0

0 1 2 3 4 h - G1381(3%)

» G1381( %+08%glycerol) ^ G1381(3%+08%glucose) EXAMPLES

Example 1 - Chondroitin sulfates (G1845)

a) The sample used was commercial ChS of type A (Laboratoπ Deπvati Organici), desalted through dialysis, and characterized with regard to the degree of sulfation and its distribution by means of conductimetric titration (B Casu, U Gennaro Carbohydrate Research 39, 168,1975) and with ¹³C-NMR spectroscopy for the mean molecular weight and its distribution by gel permeation at high pressure (GPC-HPLC) (J Harenberg and J X De Vπes J Biol Chem , 265 (1990) 7292-7300) Figure 2 shows the ¹³C-NMR spectrum of the desalted chondroitin A (G1845) The ratio between the signals of the carbon 2 of the ammo sugar, at 52 ppm, corresponds to the ratio between the sulfate structure in position 4 and 6 of N-acetyl galactosamine and is about 40/60 (A Naggi Drugs Exptl Chn Res XVII (1 ) 21-32 (1991 )) The profile obtained through GPC-HPLC shows a PM equal to 24000 Dalton and a degree of polydispersion of 1 4 The conductimetric titration provided molar ratio data between the sulfate and carboxy c groups equal to 1 1

b) The sample used was commercial ChS of type A (SIGMA) characterized as above Figure 3 shows the ¹³C-NMR spectrum of chondroitin A (G1381 ) The ratio between the signals of the carbon 2 of the a mo sugar, at 52 ppm, corresponds to the ratio between the sulfate structure in positions 4 and 6 of N-acetyl galactosamine and is about 42/58 The profile obtained through GPC-HPLC shows a PM equal to 24000 Dalton and a polydispersity of 1 4 The conductimetric titration provides molar ratio data between the sulfate and carboxyhc groups equal to 1 1 , the moisture content, determined by drying to constant weight, is equal to 12% w/w Example 2 - Chondroitin sulfates of low molecuar weight

a) - Partial acid hydrolysis (G1394/C and G1473)

A sample of chondroitin of low molecular weight was obtained by acid hydrolysis of the commercial ChS sample The thus obtained sample (G1394/C, G1473) was characterized as in example 1 Figure 4 shows the ¹³C-NMR spectrum the pattern of the main signals shows no variations, signals being evident at about 93 and 96 5 ppm due to the reducing end units GPC-HPLC analyses reveal the mean molecular weight to be 13000 Dalton The molar ratio between the sulfate and carboxyhc groups, obtained by conductimetric measurements, is equal to 1 1

b)- Ethanohc fractιonatιon (G2159a, G2159b, G2159c, G1394D1 ,

G1394D2, G1394D3) 40g each of two preparations of chondroitin of low molecular weight (G1394/C and G1394/d) were dissolved in 400 mL of water and precipitated with 600 mL of ethanol (solution at 60%) at 4°C The milky-white solutions were centrifuged at 5,000 rpm for 20', obtaining precipitates G2159A and G1394D1 , respectively Ethanol was added to the supernatants until reaching a concentration equal to 70%, centπfugation then gave precipitates G2159B and G1394D3 and the respective supernatants G2159C and G1394D2 GPC-HPLC analyses gave the mean molecular weight of each fraction and the polydispersity to be the following

Example 3 - Oligosacchandes from chondroitin sulfates

a) - Enzymatic hydrolysis with Chondroitinases ABC (G1845b, G1845c, G1845d)

Three batches each of 2g of ChS of high molecular weight G1845 were incubated at 37° C with 5 units of Chondroitinases ABC After 4 h another 5 units were added and after 20 h another 5 units Digestion was continued for another 24 h after which the enzyme was denatured by heating at 100° C The solution was filtered through membrane of 022 μm and the product recovered by evaporation at reduced pressure

GPC-HPLC analyses revealed the mean molecular weight of the samples to be less than 1000 Dalton

Figure 5 shows the ¹³C-NMR spectrum of the product G1845b, it is possible to observe signals due to the anomeπc carbons of the reducing end units, and further downfield, those due to carbons 4 and 5 of the unsaturated non- reducing end units

b) - Enzymatic hydrolysis with hyaluronidases (G2001 )

1 g of ChS G1845 was incubated with 30 mg (10200U) of testicular hyaluronidases at pH5 in acetate buffer at 37°C for 48 h The enzyme was denatured by heating at 100^CC The solution was filtered through a membrane of 022 μm the product (G2001 ) was recovered by evaporation at reduced pressure Figure 6 shows the ¹³C-NMR spectrum of the obtained product The signals due to the anomeπc carbons of the reducing end units can also be seen Example 4 - Desulfated chondroitin sulfates (G1863)

Preparations of 80 g of desulfated ChS were made by mixing 10 batches obtained starting from 10 g of each ChS The solvolytic desulfation was carried out according to the procedure (K Nagasawa, Y Inoue, and T Kamata Carbohydrate Research 58 1997 47-55) using the pyπdine salt of chondroitin sulfate dissolved in a DMSO solution containing 10% water, kept under stirring at 80°C for 24 h The solution was then dialysed against water and the product recovered by evaporation at reduced pressure Figure 7 shows the spectrum of the desulfated sample (G 1863) where the absence of overlapping of the signals A2, A1 and G1 is evident, as expected in the case of fully desulfated samples

Example 5 - Hyaluronic acid

A sample of commercial hyaluronic acid (G 1093) was characterized by ¹³C- NMR spectroscopy (fig 8)

Example 6 - 2-O-Desulfated hepann (G 2198)

1g of G479 (sodium hepann of high molecular weight from the intestinal mucosa of the pig) was dissolved in 100mL of NaOH 0 1 N The solution was frozen and lyophohzed, the sample was redisolved in the minimum quantity of distilled H2O and neutralized with 4% HCI The solution was dialyzed using a membrane with cut-off 6000-8000 for 48 h in distilled H2O and desalted by gel filtration on Sephadex G25 The obtained product (G2198) shows a sulfate carboxyl ratio equal to 1 2. The ¹³C-NMR spectrum (fig. 9) shows the signal at 104 6 ppm typical of a desulfated hepann at position 2 of the iduronic acid

Example 7 - In vitro evaluation of the dialytic capability of the samples

Small dialysis bags with Spectra Pore membrane with a cut-off of 500 Dalton (diameter 15 mm, 15 cm high) were used They were filled with 3 mL of solutions at different concentrations (1 25, 2 5, 5 0, 7 0% w/v of the samples) The bags were immersed in 200 mL of distilled water at 37° C with stirring of the extradialysis solution At given times (0, 1 , 2, 3, 4, 5, 6, 24 h) the increase in volume inside the dialysis bag was evaluated by weight and expressed as percentage increase compared to the starting volume (Δw%), furthermore the osmolahty was evaluated with a Roeb ng microosmometer before and after the dialysis tests The results are shown in Table 1 and compared with those related to glucose, hyaluronic acid (HA) and two samples of commercial maltodextπn (Cerestar-01910, 01934) with the following composition percentages.

01910 01934

glucose maltose maltotπose oligo-sacchaπdes at high PM

Example 8 - Preparation of solutions for peritoneal dialysis

a) The ChS samples of high and low molecular weight and the ohgosaccharides, characterized in examples 1a, 1b, 2b and 3a, were dissolved in electrolyte solutions in concentrations respectively equal to 1 25% (G1845), 3% (G1381), 5% (G 1845C and G 1394 D2) and at 7% (G 1394 D3) The percentage expressed as weight per volume (w/v) was calculated on the product itself, not taking into account the moisture present in the sample The composition of the used electrolyte solution was the following mEq/L mEq/L mEq/L mEq/L

b) The sample G1381 was dissolved at a concentration equal to 3% in two electrolytic solutions of the following concentrations

All the solutions were filtered through 0 22 μm membrane, then sealed and sterilized for 30 mm at 121 ° C

Example 9 - In vivo evaluation of the dialytic capability of the solut.ons

Male Sprague-Dawley rats each weighing between 250 and 350 grams were used in the experiments The animals were fed a standard diet until the evening before the experiment They also had free access to drinking water The rats were anesthetized by an injection into the neck of sodium pentobarbital (Nembutal), dosage 60 mg/kg, and kept under the anesthetic for the duration of the experiment

The animals were laid in the supine position on a heated pad, 37° C, and then given a tracheotomy The carotid was exposed and a tube inserted for blood withdrawal In addition the jugular vein was exposed and a suitable catheter inserted, through this there was the continuous infusion into the animal of the physiological solution containing Nembutal in the proportion of 0 3 mU10 mL, the rate of infusion was 0 0425 mL/min The arterial pressure of the animal was monitored throughout the experiment At this point a catheter was introduced into the peritoneum, allowing it to penetrate 1 cm to the left of the median line of the abdomen Through this catheter was dripped into the cavity 15 mL of the dialytic solution under investigation (see example 8) At zero time (T 0) samples of the dialysis fluid and blood were collected for chemical analysis and the measurement of osmolahty

After 1 , 3 and 6 hours of dwell time, starting from the running in of the dialysis fluid, a blood sample was taken for hematological examination, measuring Na, K, urea, creatinine (enzymatic method), glucose, total proteins and osmolahty and ChS residues (colorimetπc treatise Bitter Muir Anal Biochem 4, 1962 (330-334)) The animals were then sacrificed by bleeding The abdomen was opened carefully with an incision along the median line All the peritoneal fluid was then aspirated using a 10 mL syringe Measurements were made of both the volume and the weight of the fluid removed. Also measured in the fluid was the osmolahty, the concentration of Na, K, creatinine (enzymatic method), urea, total proteins and total ChS (colonmetnc method T. Bitter H M. Muir Anal. Biochem 4, 1962 (330-334)

The following tables summarize the obtained results, that refer to each and every one of the chondroitin sulfate solutions tested

G 1845 (example la)

G 1381 (example 1b)

The more important characteristics of the alternative osmotic agents and the relative solutions for peritoneal dialysis according to the invention are listed in the claim written below.

Claims

1 Use of glycosaminoglycans devoid of anticoagulant and hemorrhagic activity, as osmotic agents alternative to glucose in peritoneal dialysis

2 Alternative osmotic agents according to claim 1 , characterized in that they are selected from the group comprising

• chondroitin sulfates (ChS) as such, or their mixtures, products obtained through the desulfation of the same, derivatives of these obtained through depolymerization either chemical or enzymatic etc ,

• hyaluronic acid and its products of depolymerization,

• products of chemical derivation of other glycosaminoglycans, as such, or depolymeπzed

3 Alternative osmotic agents according to claim 2 characterized as being in the form of a single salt or mixed salts, preferably alkaline and/or alkaline- earth salts or better still Li, Na, K, Ca and Mg

4 Alternative osmotic agents according to claim 2, characterized by the NMR spectra reported in figures 2 to 8

5 Alternative osmotic agents characterized by molecular weights lower than 50,000 Dalton, preferably between 5,000 and 30,000 but ideally around 25,000

6 Aqueous solutions for peritoneal dialysis containing, apart from additives known as such, osmotic agents according to claims 1 to 4, in concentrations greater than 1 25% and lower than 10%, preferably between 2% and 7%

7 Solutions for peritoneal dialysis according to claim 5, characterized by the presence of co-solutes of low molecular weight such as glucose, glycerol etc

8 Solutions for peritoneal dialysis with ultrafiltration as shown in graphs 1 to 6

9 Dialytic solutions that are substantially according to what has been exemplified in the in vitro and in vivo tests

10 Methods for the preparation of the alternative osmotic agents of claims 1 to 5, substantially according to what is described in examples 1 to 6

11 Methods for the preparation and utilization of the solutions of claims 6 to 9, substantially according to what was described in the in vitro and in vivo tests, and with ultrafiltration performances as represented in graphs 1 to 6