The present invention relates to a peritoneal dialysis fluid (in the following also referred to as „PDF“).

Peritoneal dialysis fluids remove solutes and water from the uremic patient. The mechanism of PDFs is known and does not need to be elaborated in any more detail here.

PDF solutions comprise an osmotic agent. For the purposes of the present invention, the osmotic agent is glucose. PDFs containing glucose as the osmotic agent will in the following be referred to as „glucose-based“ PDFs. The amount of glucose in such glucose-based PDFs is typically from about 0.5 to 4 g/L.

However, Glucose is known for being prone to degradation and forming glucose degradation products (GDP) ((1), EP 2 962 683 Al).

Therefore, it is known to store the glucose containing osmotic solution at rather acidic pH values (1).

Known embodiments of PDFs such as the “Dianeal® PDG4” product range manufactured by Messrs. Baxter (https://www.baxterpi.com/pi-pdf/Dianeal PI. pdf, retrieved 23.06.2022) comprise one single glucose solution at a pH-value of around pH 5.0-5.5. However the extent of GDPs in such products is high.

Other embodiments therefore, employ an even lower pH-value of the osmotic solution, such as 3 to 5. However, peritoneal administration of a solution having a low pH value like this would not be possible or at least painful.

Thus, these further embodiments contain a second solution, which is an alkaline buffered solution, which is mixed with the osmotic solution before administration so as to form a final solution which is ready-to-use (“RTU”) having about a neutral pH value such as from 6.9 to 7.5.

The two solutions are manufactured at a predetermined ratio of their respective amount. They are stored separately but are mixed together upon administration. As a convenient means for storage, multi-chamber bags, especially double-chamber bags, containing the solutions in separated compartments which, however, can be connected with each other for mixing upon administration (e.g. by peelable seals), are known.

As a buffer, both lactate buffers and bicarbonate buffers and mixtures of the aforementioned are employed.

Various commercial solutions of the state of the prior art provide different approaches with regard to

- the pH-values of the two solutions

- the respective amounts of the two solutions as well as

- the amount of buffer in the alkaline solution:

In the product range “Physioneal® 40 Glucose Clear-Flex” as manufactured by Messrs. Baxter, the glucose containing solution and the alkaline buffered solution are mixed in a ratio of 3 : 1. The alkaline buffered solution contains lactate buffer in an amount such that upon mixing of the solutions a total amount of 15 mmol/L results, and, furthermore, a bicarbonate buffer in an amount such that upon mixing of the solutions a total amount of 25 mmol/L results (https://www.baxter.ca/sitesZ2/files/ebysai 143 I /files/2018-

12/Phy si on eal Cl earfl ex EN . pdf, retrieved 24 June 2022). The pH-values of the solutions are not disclosed but are assumed to be 2.1 (glucose containing solution) and >9 (alkaline buffer), respectively.

In the product range “Physioneal® 40 Glucose Viaflex” as also manufactured by Messrs. Baxter, the glucose containing solution and the alkaline buffered solution are mixed in a ratio of 725: 1275. The alkaline buffered solution contains lactate buffer in an amount such that upon mixing of the solutions a total amount of 15 mmol/L results and, furthermore, a bicarbonate buffer in an amount such that upon mixing of the solutions a total amount of 25 mmol/L results. (https://www.baxter.ca/sites/g/files/ebysai 1431 /files/2018-

11/Physioneal Vi afl ex EN.pdf, retrieved 24 June 2022). The pH-values of the solutions are not disclosed but are assumed to be 4.2 (glucose containing solution) and 7.6 (alkaline buffer), respectively.

The product range “Balance®” as currently manufactured by Messrs. Fresenius also contains two chambers. The glucose containing solution and the alkaline buffered solution are mixed in a ratio of 1 : 1. The alkaline buffered solution contains lactate buffer in an amount such that upon mixing of the solutions a total amount of 35 mmol/L results. The pH-values of the solutions are not disclosed but were measured on the basis of a sample product to be about 3.0 (glucose containing solution) and about 8.6 (alkaline buffer), respectively.

According to the product information relating to this product, sodium hydrogen carbonate is mentioned as an excipient.

In a brochure “Safety and biocompatibility in perfect balance” published by Messrs.

Fresenius in 2006 (2) a pH-value of the acidic solution of appr. 3.1 and a pH-value of the buffered solution of appr. 8.0 are reported. The mixing ratio of the two solutions is not disclosed.

Thus, from these prior art solutions it is known that in case of using a glucose solution with a low pH value such as 3 or even only 2 (which is favourable in terms of GDP formation), the pH value of the alkaline solution needs to be high (even 9 or more).

EP 2 962 683 Al, for the purposes of reducing GDP formation, proposes a fluid to be prepared by mixing of two or more solutions, wherein the sodium content is low and wherein no sodium ions are contained in the glucose containing solution. No pH-values are disclosed.

EP 1 465 688 B2, in the context of hemodialysis, employs a bicarbonate-based solution and an electrolyte (glucose) solution, and discloses several pH ranges for both solutions under moderate or extreme pH conditions.

Further state of the art discussing double chamber products for peritoneal dialysis is disclosed in EP 1 038 552, EP 1 131 077, EP 1 744 768, EP 2 647 397 and WO 2018/201443.

Several clinical and experimental observations have shown that PDF is cytotoxic, associated with a risk of technical failure of up to 30% with long term peritoneal dialysis (PD) treatment (2).

WO 2008/106702A1 provides a glucose-based peritoneal fluid containing a protective agent in the form of L-glutamine and dipeptides which are capable of releasing L-glutamine, said dipeptide being L-glutaminyl-L-glycine, L-glycinyl-L-glutamine, L-glutaminyl-L-alanine or L-alanyl-L-glutamine, or a mixture of two or more of said dipeptides, wherein the concentration of said dipeptide in the dialysis fluid is 2 mM to 25 mM.

According to this patent application, it was found that these dipeptides, especially L-alanyl- L-glutamine (in the following abbreviated as “Ala/Gln”), contribute to the prevention of Technical Failure.

Several publications have further investigated the effect of Ala/Gln in a glucose-based PDF ((3) to (27)).

In the following, the term L-alanyl-L-glutamine or “Ala/Gln” stands both for Ala/Gln as such as well as for all other protective agents within the scope of the present invention.

The problem underlying the present invention is to provide a glucose-based PDF containing said protective agent, especially Ala/Gln, and at the same time providing optimal results in terms of GDP formation.

This problem is solved by the subject matter of claim 1. Preferred embodiments are disclosed in the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

In the context of formulating a ready -to-use glucose-based peritoneal dialysis fluid containing said protective agent, problems are encountered.

On the one hand, it was found that mixing of Ala/Gln and the glucose in one single solution would lead to reactions between the dipeptide and the glucose. Furthermore, at low pH values Ala/Gln itselfs tends to decompose.

Thus, a single-solution embodiment would not be storable over a longer period of time, which however is necessary for a PDF.

On the other hand, dipeptides such as Ala/Gln are known for not being stable under alkaline conditions ((28), (29)) and to form degradation products. This is even more the case given the need that a PDF must undergo a heat sterilisation of the solution at about 120°C. However, as can be seen from the solutions discussed above, if a low pH-value for the glucose-containing solution is envisaged for reasons of minimizing GDP formation, the necessary pH value of the alkaline buffered solution needs to be high.

Surprisingly, it was found, however, that a PDF containing the two solutions as defined in claim 1 solves the problem underlying the invention.

The first solution provided by the present invention is a solution containing the osmotic agent glucose in a strongly acidic environment.

The second solution provided by the present invention is an alkaline buffered solution containing said protective agent, however at only a weakly alkaline pH-value.

Surprisingly, it was found that even if a low pH-value such as 2.2 to 3.5 of the glucose containing solution is employed (thus minimizing GDP formation), only a very low alkalinity of the alkaline buffered solution containing the Ala/Gln, such as 7.4 to 8.2, was sufficient to establish a neutral fluid upon mixing the first and the second solution. At the same time, the degradation of Ala/Gln upon storage can also be minimized due to the low alkalinity of the solution.

It needs to be noted that Ala/Gln itself would not be expected to have any effect on pH.

In a preferred embodiment of the present invention, the pH-value of said first solution is from 2.2 to 2.9, more preferred from 2.2 to 2.8, most preferred from 2.5 to 2.8.

In a further preferred embodiment in that the pH-value of said second solution is from 7.4 to 7.9, more preferred from 7.4 to 7.8, most preferred from 7.5 to 7.8.

The amount of lactate in said second solution is from 30 to 45 mmol/L, preferably from 34 to 41 mmol/L, most preferred about 35 mmol/L. All these values refer to the amount in the resulting (“ready-to-use” - RTU) fluid upon mixing said first and second solution. For example, if the mixing ratio of the first solution and the second solution is 1 : 1, with a target amount of 34 to 41 mmol/L in the RTU fluid, the amount of lactate in the second solution is from 68 to 82, preferably about 70 mmol/L.

This would resemble an amount of lactate such as known from the currently marketed “Balance ®” product range as currently marketed by Messrs. Fresenius. However, while in that product the glucose containing solution is at a pH-value of of 3.0 (i.e. less acidic than at least in the preferred embodiments of the present invention), the pH-value of the alkaline solution is about 8.6 (i.e. more alkaline than in the present invention), the respective amounts of both solutions being the same.

The lactate is preferably provided in the form of sodium lactate.

In the present invention, the ratio of the amount of said first solution to the amount of said second solution may be from 3 : 1 to 1 :3, and is preferably from 2: 1 to 1 :2, even more preferred 1.5: 1 to 1 : 1.5.

In an especially preferred embodiment, said ratio is about 1 : 1.

The term “amount” with regard to the first and the second solutions refers to the respective volumes of the solutions.

It is again surprising that when mixing quite equal to even absolutely equal amounts of the two solutions with each other, a neutral and, thus, physiologically acceptable PDF ready-to- be-used („RTU“) results.

A further embodiment of the present invention is characterized in that both said first solution and said second solution contain one or more sodium compounds in a physiologically acceptable amount (calculated, again, on the resulting amount in the fluid aupon mixing of the two solutions). In other prior art proposals, the sodium compounds are only present in one of the two solutions.

The total amount of sodium ions in both solutions may range from 130 mmol to 140 mmol/L.

The first solution of the PDF of the present invention preferably further contains a physiologically active agent selected from the group consisting of physiologically acceptable compounds containing sodium ions, calcium ions, magnesium ions and mixtures thereof.

The need for physiologically active agents comprising the afore-mentioned ions in a PDF is known to the skilled person and thus does not need to be discussed in more detail. Typical physiologically active agents that may be employed in the fluid can be selected from sodium chloride, calcium chloride and magnesium chloride.

For the purposes of adjusting the pH-value of the first and second solution to the desired value, physiologically acceptable acids (for example HC1) and acceptable bases (for example NaOH) can be employed, respectively.

The amount of the protective agent, especially the amount of Ala/Gln preferably is from 2 mM to 25 mM, more preferably 5 mM to 10 mM, most preferably about 8 mM, again calculated as amount in the fluid upon mixing said first and second solution.

Preferably the amount of said protective agent after at least one year of storage, preferably after two years of storage, is 95% or more of its original amount. It has been found in longtime studies that the amount of degradation of Ala/Gln in the second solution indeed is very low.

Furthermore, it has also been found that the amount of glucose degradation, i.e. formation of GDP in said first solution during storage is low and is basically in the same range as that of commercially available products.

In an especially preferred embodiment said protective agent comprises, or even more preferably consists of L-alanyl-L-glutamine.

The two solutions of the PDF of the present invention may be manufactured by mixing the ingredients mentioned above in water, respectively, and heat sterilizing the resulting solutions.

Surprisingly, it has been found that the addition of only very small amounts of a bicarbonate compound to the second solution has apparently a further effect of stabilizing the protective agents against degradation, both during heat sterilization and during storage.

Furthermore, without wishing to be bound to theory, the addition of this amount of bicarbonate compound is believed to assist in achieving the target neutral pH-value upon mixing the two solutions.

Thus, in a further aspect the present invention provides a process for manufacturing the second solution of the PDF of the present invention, comprising the step of mixing together the ingredients of the second solution and, in addition, a bicarbonate buffer in an amount of 1 mmol/L to 6 mmol/L, calculated as amount in the fluid upon mixing said first and second solution, and sterilizing the mixed solution.

The amount of said bicarbonate buffer preferably is from 2 mmol/L to 4 mmol/L, even more preferably about 3 mmol/L.

This amount, again, is too small as to expect such a big influence of the pH-value upon mixing the two solutions with each other. Furthermore, this amount is even so small that the bicarbonate may no longer be detected in the second solution or in the fluid after mixing after a longer period of storage, such as 1 or 2 years. This is because of the equilibrum between the bicarbonate anion and CO2, the latter possibly being able to penetrate through the film containing the solution at least partly.

Yet, it can be shown that the target neutral pH-value upon mixing the first and the second solution can also be obtained after longer storage time.

The bicarbonate compound preferably is sodium hydrogen carbonate.

The first and second solutions of the present invention may conveniently be filled into a bag comprising several chambers separated from each other by e.g. peelable seals, especially into a double chamber bag as previously described.

After filling into the bag, the solutions are sterilized, optionally further packaged with an overpoach, and stored.

EXAMPLES

Example 1 - pH-shift of alanyl-glutamine-dipeptide (AGP) containing solutions after sterilization:

The pH value of AGD containing solutions was adjusted in 0.5 pH steps. These samples were used for analyzing the pH behaviour of the solutions during sterilization and to find the most appropriate combination of AGD containing alkaline solutions to reach the required pH range in the ready-to-use-solution (RTU solution). The final target composition of the AGD containing alkaline solutions in the RTU solution after mixing and the actual composition of the alkaline solution before mixing (based on a mixing ratio of acidic and alkaline solution of 1 : 1) were as follows:

For the preparation of 1 L solution the following quantities of raw materials were weighed by means of a calibrated balance:

To prepare the AGD containing alkaline solutions, approximately 250 mL of deionized water were placed in a beaker. All ingredients were added with stirring and the solution was transferred to a plactic canister. While stirring deionized water was added and the pH value was adjusted with 1 M NaOH solution until the required value was met. Deionized water was filled up to the final volume of 1 L and pH was controlled. The required volume of solution was transferred to the bag through the filling tube. Sterilisation (steam-air mixture method) was performed with a standard programme for 5000 ml bags with sterilizable overwrapping under the conditions as shown in Table 3:

Table 3: Sterilisation programme for 5000 ml bags. Solutions at six different pH-values were prepared (pH 6.0 to 8.5 in 0.5 steps) and pH values were determined before and after sterilisation for the determination of the pH shift after sterilisation.

All pH investigations were performed according to the current valid monograph 2.2.3 “Potentiometric Determination of pH” of the European Pharmacopoeia using a qualified pH electrode, calibrated for the corresponding pH range (alkaline, neutral or acidic) with certified NIST traceable buffer solutions pH 1.0, 4.0, 7.0, 9.0, and 10.0.

Table 4: Results of the pH-value evaluation of alkaline solutions with AGD before and after sterilisation.

The results in Table 4 show a significant influence of the sterilisation process on the pH value of the alkaline solution of about 1 to 1.5 units within an acidic range of pH 6 to 7.5. The difference was smaller (approx. 0.3 units) at higher pH values of 8.00 and 8.50. These results indicate a superior stabilization of the pH value of the solutions by AGD at alkaline pH-values.

Example 2 - Stabilization of AGD by addition of small amounts of a bicarbonate compound (sodium hydrogen carbonate):

In order to determine the influence of sodium hydrogen carbonate on the pH shift during sterilisation, solutions with a constant bicarbonate content and varying pH values were prepared. The final concentration in the RTU solution was as follows:

Table 5: Composition of alkaline solution comprising sodium hydrogen carbonate in the RTU solution.

For the preparation of 1 L alkaline solution (again based on a 1 : 1 mixing ratio) the following quantities of raw materials were weighed by means of a calibrated balance:

Table 6: Quantities of raw materials for preparing the alkaline solution comprising sodium lydrogen carbonate.

Approximately 500 mL of deionized water were placed in a beaker. All ingredients were added with stirring and the solution was transferred to a plactic canister. While stirring, deionized water was added and the pH value was adjusted with 1 M NaOH solution until the required value was met. Deionized water was filled up to the final volume of 1 L and pH was controlled. The required volume of solution was transferred to the bag through the filling tube. Sterilisation was performed with a standard programme for 3000 ml bags with sterilized overwrapping.

Table 7: Sterilisation programme for 3000 mL bags.

The pH values were determined before and after sterilisation for the determination of the pH shift after sterilisation. Table 8: Results of the pH-value evaluation of alkaline solutions with AGD and NaHCCh before and after sterilisation.

The results in Table 8 exhibit no significant influence of the sterilization process on the pH- value of alkaline solutions in the presence of 3 mmol/L NaHCCE. In comparison to Example 1, the presence of small amounts of bicarbonate obviously has an influence on the pH value during the sterilisation process. It is assumed, that the hydrogen carbonate has kind of a buffer effect on the solution.

Example 3 - Mixing of acidic solution and alkaline solution:

For the pH mixing experiments acidic glucose containing and basic AGD containing solutions were prepared. In the first step, the pH value of the solutions were adjusted in 0.5 pH steps. These samples were used for analyzing the pH behaviour of the solutions during sterilization and to analyze the result of the combination of acidic and basic solutions with regard to the pH range in the mixed solution. As the target pH-value for the mixed, i.e. ready-to-use solution (RTU solution), a range of 6.9 to 7.5 was set.

Table 9: Composition of acidic solution in the RTU solution.

Table 10: Quantities of raw materials for preparing the acidic solution.

The pH-value of the acidic solution was adjusted by adding 1 M HC1 until the desired pH- value was reached.

The alkaline solutions were prepared as described in Example 2.

The ready -to-use solutions were prepared by mixing of samples (acidic and basic) with a ratio of 1 : 1 followed by an immediate analysis of the pH value.

Table 11 : Results of mixing the solutions, with the pH-value of the acidic solution indicated in the first column of the table, and the pH-value of the alkaline solution being indicated in the first row of the table.

The results in Table 11 show that the target pH range of 6.9 to 7.5 could not be reached after mixing acidic solution and alkaline solution

Example 4 - Mixing of acidic solution and alkaline solution:

In a further trial, the pH values were set as as follows:

- Acidic solutions pH 2.0 to 3.4 (0.2 steps)

- Alkaline solutions pH 7.6 to 9.0 (0.2 steps)

Additionally, 3 mmol/L NaHCCh (referred as to the ready -to-use-solution) was added to the alkaline solution before the adjustment of the pH value. The acidic solutions were prepared as described in Example 3 in a pH range from 2.0 to 3.4 in 0.2 steps. Basic solutions were prepared according to Example 2, and pH-values were adjusted in a range of 7.2 to 9.0 in 0.2 steps. Sterilistion of both solutions was performed with the standard programme for 3000 mL bags as shown in Table 7.

The ready-to-use solutions were prepared by mixing acidic and alkaline solutions with a ratio of 1 : 1 followed by an immediate analysis of the pH value.

The pH values marked in grey correspond to the pH range of 6.9 to 7.5, which has been defined as target range for the final product. Based on the results of the mixing tests, there are several possibilities regarding the adjustment of the pH values of acidic and alkaline solutions in order to achieve the desired target range of the pH value of the RTU solution of 6.9 - 7.5.

Example 5 - Examples for acidic and alkaline solutions and mixed ready-to-use solution:

Three examples for acidic and alkaline solutions, according to the invention, with different contents of glucose, respectively, are presented in the following. For all examples, the mixing ratio of acidic and alkaline solution is set at 1 : 1 (volume based).

Table 13: Composition of acidic solution and alkaline solution for a ready -to-use solution

RTU solution) comprising 3,86 % glucose.

Table 14: Composition of acidic solution and alkaline solution for a ready-to-use solution

RTU solution) comprising 2,27 % glucose.

Table 15 : Composition of acidic solution and alkaline solution for a ready -to-use solution (RTU solution) comprising 1.36 % glucose.

Table 16: Composition or RTU solution upon mixing of acidic and alkaline solution in a ratio of 1 : 1

The pH value of the mixed, i.e. RTU solution in each case was in the targeted pH-range of 6.9 to 7.5.

Example 6 - Long-term stability of AGP:

The long-term stability tests of AGD in an alkaline solution according to the present invention have been performed. Two sample solutions with pH values of 7.4 and 7.8 respectively, were prepared containing the same amounts, respectively, of AGD (16 mmol/L), NaHCCh (6 mmol/L) and sodium lactate (70 mmol/L) . The pH of each sample was adjusted with 1 M NaOH. Aliquots of each sample were prepared under controlled conditions and stored under the defined conditions at 25°C/ 60% rH.

Sterilization was perfomed under following conditions:

Table 17: Sterilisation programe for stability tests.

The stability of AGD has been investigated by AS analysis. Analysis was performed by qualitative amino acid analysis with IEX and post column derivatization with Ninhydrine.

Both investigated samples showed the same trend in terms of only a very slight decrease of the AGD (less than 5% of original amount) after storage at 25°C/60% rH during a storage time of 52 weeks.

The least decrease in AGD was found in the sample with a pH-value of 7.4 (98% of original amount), with the decrease in AGD in the sample with a pH-value of 7.8 being >95% of the original amount.

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(2) Safety and biocompatibility in perfect balance. Fresenius Medical Care Deutschland GmbH. 2006.

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