ES2958165A1 - Peritoneal dialysis solutions containing a natural flavonoid as an osmotic agent - Google Patents

Abstract

Peritoneal dialysis solutions containing a natural flavonoid as an osmotic agent. The present invention relates to peritoneal dialysis solutions comprising troxerutin or vitamin P4 as an osmotic agent. The invention also relates to the process for preparing such solutions and to the novel use of troxerutin.

Description

DESCRIPTION

PERITONEAL DIALYSIS SOLUTIONS CONTAINING A FLAVONOID

NATURAL AS AN OSMOTIC AGENT

The invention relates to peritoneal dialysis solutions containing troxerutin or vitamin P4 as an osmotic agent. The invention also relates to the new use of troxerutin in the preparation of said solutions.

Therefore, the present invention belongs to the field of biotechnological medicine, in particular, that of dialysis solutions used for the treatment of kidney disease (nephropathy).

STATE OF THE TECHNIQUE

Currently, approximately 850 million people suffer from different types of kidney disorders, while one in ten adults worldwide has chronic kidney disease (CKD) (Li PK,et al. Kidney Int.2020 Feb;97(2 :226-32). By 2040, CKD is predicted to become the 5th most common cause of death worldwide (Foreman KJ,et al.Lancet. 2018;392(10159):2052-90).

In 2020, approximately 4.5 million patients with chronic kidney disease were treated worldwide. Of these patients, approximately 3.7 million received dialysis treatments, 11% of them with peritoneal dialysis (between 5 and 70% of dialysis programs depending on the country) (Fresenius Annual Report 2020).

In Spain, far from stopping its advance, CKD continues to grow, and in 2019 its prevalence reached 1,367 people per million inhabitants, with more than 64,000 people undergoing kidney replacement treatment (dialysis or transplant), according to 2019 data. from the Spanish Registry of Renal Diseases (REER).

Peritoneal dialysis (PD) is a form of renal replacement therapy that uses the abdominal cavity as a reservoir to exchange water and solutes across the peritoneal membrane (PM). The PM is covered by a semipermeable monolayer barrier of mesothelial cells (MC).

Glucose is the most commonly used osmotic agent for PD solutions. It is well known that glucose produces local and systemic complications leading to PM failure and cardiovascular complications due to metabolic disorders. Glucose degradation products (GDPs) are partly responsible for this deterioration of PM and are essential for the formation of advanced glycation end-products (AGEs). AGEs are generally incorporated into tissues, inducing hyperproduction of cytokines and growth factors (VEGF and TGF-®), initiating and perpetuating PM injury through uncontrolled extracellular matrix (ECM) synthesis (fibrosis). ), angiogenesis and the mesothelial-mesenchymal transition (MMT) of CM. These structural changes are characteristic of type I failure of MP.

Although there are PD fluids not based on glucose as an osmotic agent (icodextrin and amino acids), none of them can be used for all PD exchanges throughout the day. One reason to limit its use is its molecular weight. While glucose shows a molecular weight of approximately 180 Da, that of icodextrin is greater than 7000 Da.

Some of the documents that describe osmotic agents other than glucose are the following: WO 2016/066672, US4339433, US4761237, US6770148, US4649050.

In view of the above, there remains a need to provide new compounds that have low or no toxicity, water solubility, suitable molecular weight, high stability and suitable molality values for use as alternative osmotic agents to glucose.

In this context, the present invention discloses a compound for use as an alternative osmotic agent to glucose that meets all of the above characteristics and that can solve a health problem that affects a significant number of kidney patients receiving PD treatment.

DESCRIPTION OF THE INVENTION

The inventors have demonstrated that troxerutin (also called vitamin P4) is a compound with optimal properties for use as an osmotic agent in peritoneal dialysis solutions.

Troxerutin is a natural flavonoid with a molecular formula C<33>H<42>O<19>and a molecular weight of 743 g/mol that is present in tea, coffee, cereal grains, and a variety of fruits and vegetables. It shows anti-inflammatory and anti-apoptotic properties. It acts as an antioxidant and is used as a vasoprotective drug marketed in humans, in addition to having other interesting reported effects. (M. Zamanian, S. Shirooie,et al. Current Neuropharmacology,2021, 19, 97-110). Its CAS No. is 7085-55-4 and according to the European Pharmacopoeia (10.0 Vol. 111), it is defined as a mixture of O-hydroxyethylated derivatives of rutoside containing a minimum of 80 percent 2-[3, 4-bis(2-hydroxyethoxy)phenyl]-3-[[6-O-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-5-hydroxy-7-(2-hydroxyethoxy)-4H- 1-benzopyran-4-one(tris(hydroxyethyl)rutin).

A first aspect of the invention relates to a peritoneal dialysis solution comprising troxerutin as an osmotic agent.

The term "peritoneal dialysis solution" is known in the art and is intended to mean a solution comprising an electrolyte, a buffer and an osmotic agent, wherein the electrolyte comprises ions, such as sodium, potassium, calcium and magnesium; The buffer comprises components, such as acetate, lactate and bicarbonate. Examples of medical solutions for use as peritoneal dialysis solutions can be found in Wieslanderet al.,1991, Kidney Int 40:77-79.

The term "osmotic agent", also known in the art, is intended to mean a substance in a liquid that causes an osmotic force to draw fluid from the patient into the dialysis fluid if it is present at a sufficiently high concentration. Therefore, the fluid is transported over the peritoneal membrane into the PD fluid.

In a preferred embodiment, the peritoneal dialysis solution contains between 1% and 6% w/v troxerutin, more preferably between 1% and 3% w/v. %w/v= 100 * [mass of troxerutin (g)/volume of solution (ml)]

The procedure for preparing the peritoneal dialysis solution comprising troxerutin as an osmotic agent, comprises mixing all the components of the peritoneal dialysis solution and sterilizing the resulting mixture, preferably by heating at a temperature between 110 and 130 ° C for 0.5 to 2 h, more preferably at 121 °C and/or for 1 hour. It can be prepared in a single-chamber bag, without the need to divide the product into different chambers.

Another aspect of the present invention relates to the use of troxerutin for use as an osmotic agent in a peritoneal dialysis solution, the solution being preferred as described in the first aspect of the present invention. Troxerutin is then used to prepare a peritoneal dialysis solution.

In summary, in the examples provided in the present invention it has been shown that troxerutin (vitamin P4) has many advantages with respect to a conventional osmotic agent known in the state of the art. Specifically, troxerutin has an ideal molecular weight, high water solubility, and an osmotic capacity 3-4 times greater than that of glucose and is a safe molecule in an animal model.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as normally understood by one skilled in the art to which the present invention pertains. Methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or steps. Additional objects, advantages and features of the invention will be apparent to those skilled in the art upon examination of the description, or may be learned by practicing the invention. The following examples and drawings are provided by way of illustration and are not intended to limit the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Representation of the structural formula of troxerutin (major component).

FIG. 2: Graph showing the ultrafiltration capacities of different concentrations of troxerutin (referred to as F4 in the examples) and glucose.

FIG. 3: mRNA analysis of TMM markers in cultured human omental mesothelial cells (OHMCs) treated with 1% F4 or 4.25% glucose, as well as with TGF-beta (positive control) or without treatment (negative control).

FIG. 4: mRNA analysis of TMM markers in Met5a cells exposed to high concentrations of F4.

FIG. 5: Optical microscopy images of cultured Met5a cells exposed to high concentrations of F4 for 72 h and measurement of cell viability (%).

FIG. 6: Drained cells in peritoneal effluents from mice exposed to 4.25% glucose, 2.3% glucose, or 3% and 1.5% F4.

FIG. 7: Thickness of the peritoneal membrane of mice and weight of the animals throughout the treatment.

FIG. 8: Evaluation of hypertrophic markers in cardiac tissues

FIG. 9: Systolic function of mice treated with different PD liquids.

EXAMPLES

The following are some tests carried out by the inventors with troxerutin (cited in the examples as F4) to demonstrate its effectiveness with respect to the claimed use. Troxerutin was purchased from Biosynth Carbosynth®.

Example 1: Ultrafiltration Capacity (UF) of F4

The ultrafiltration capacity (UF) of F4 was analyzed in vitro at different concentrations. These analyzes were carried out at the facilities of the Fresenius Medical Care company. UF experiments were performed using dialysis membranes (ZelluTrans/Roth V series) with a molecular weight cutoff (MWCO) of 25 kDa purchased from Carl Roth. An 11 cm long dialysis tube was used in all experiments. The filling volume of the dialysis tubes at the beginning of the experiments was 10 ml, closed by two clamps, each set with a plumb line and a swimmer. The filled bags were placed in a beaker with 900 ± 0.1 g of lactate matrix warmed to 37 °C and to determine the change in mass during the 30 minutes over a 24 hour period the weight of each bag was measured. . The mass increase for each residence time interval was calculated. The experiments were carried out 3 times, the mean value and standard deviation were calculated. Additionally, osmolality was measured by freezing point depression (or cryoscopic descent) (Osmomat 030, Gonotec GmbH). The determination of the concentration of the osmotic agent was carried out by measuring the refractive index (DR 6300-T Refractometer, KRÜSS Optronic). For each substance a calibration curve was prepared and the test solution was measured after a 24-h ultrafiltration test.

The results showed that F4 at concentrations of 1% w/v has a similar UF pattern to that of 4.25% glucose, the highest glucose concentration used in clinical practice (Fig. 2). Given these results, it was decided to use F4 at 1% w/v to perform additional in vitro analyzes to check toxicity.

Example 2: Effects of F4 on mesothelial cells

The effect of F4 on mesothelial cells was analyzed in vitro in cultured human omental mesothelial cells (OHCM), containing the liquids prepared by Fresenius Medical Care, glucose or F4, and using the buffer for PD liquids and sterilized as commercial PD liquids (in the In the case of 4.25% w/v glucose, this process generates degradation products, PDG, which are known to promote peritoneal damage). It was observed that F4 at 1% w/v induces less TMM than glucose at 4.25% w/v, since there is less induction of mesenchymal genes (VEGF-A, Fibronectin), and less repression of epithelial genes (Calretinin and E-cadherin) (Fig. 3). It was also observed that 4.25% w/v glucose induced a higher rate of mesothelial cell death compared to 1% w/v F4.

Example 3: Cytotoxicity assays

Additionally, the Met5a cell line was used to analyze TMM at higher concentrations of F4 (1%, 2%, and 3% w/v). F4 was diluted in Lactate buffer containing 1.25 mmol/L calcium, 134 mmol/L sodium, 0.5 mmol/L magnesium, 100.5 mmol/L chloride, 25 mmol/L lactate, with a pH = 7 ,0. To compare with the results of F4, 4.25% w/v (Stay Safe solution, Fresenius Medical Care) and 2.3% w/v (Balance solution, Fresenius Medical Care) glucose were used. All solutions were diluted in 20% FBS M199 culture medium with a ratio of 50:50. CTRL and CTRL+TGF-beta contained M199 medium+20% FBS: 50:50 Lactate Buffer. TGF-beta was used to induce MMT. F4 did not induce cell death or TMM at any of the concentrations tested (Fig. 4 and Fig. 5).

To completely rule out cytotoxicity, even higher concentrations of F4 (5%, 7%, 9% w/v) were added to Met5a cultures for 72 h and the cells retained a cobblestone morphology and good viability (Fig. 5). .

Example 4: Osmotic Agent Testing in a Mouse Model of Peritoneal Exposure to PD Liquids

Following the in vitro results, the suitability of this compound as an osmotic agent was tested in a mouse model of peritoneal exposure to PD fluids. First, mice with an implanted catheter underwent PD for 50 days, receiving glucose at concentrations of 4.25% w/v, 3% F4, or 1.5% w/v F4. All mice successfully completed treatment with F4 and presented a lower number of granulocytic cells in the peritoneal washings, while a greater presence of anti-inflammatory macrophages was detected in these groups within the myeloid population, indicating a protective inflammatory pattern (Fig. .6).

Also to mimic kidney failure in humans, mice underwent 5/6 nephrectomy. To do this, the right kidney was removed and a catheter was inserted into the peritoneal cavity, and after a week of recovery, the poles of the left kidney were removed by cauterization. After 2 weeks of recovery, mice received DP Stay Safe fluid (4.25% w/v glucose, with a high concentration of PDG) and F4-based LDP at a concentration of 1% w/v (the concentration which showed the same UF pattern as 4.25% w/v glucose as previously tested in vitro) for 2 months. Control mice received saline and two additional control groups were performed, using mice without surgery or LDP exposure, mice exposed only to surgery. It was observed that F4 induced less fibrosis of the peritoneal membrane than Stay-Safe with 4.25% w/v glucose (Fig. 7).

During the experiment, the weight of the mice was monitored, cardiovascular damage was evaluated by ultrasound, and cells drained in peritoneal lavage were analyzed by flow cytometry.

F4 induced less peritoneal membrane fibrosis as well as less cellular infiltration into the peritoneal membrane (Fig. 7). These results suggest that F4 may have less local impact on peritoneal membrane functionality. The weight of the animals increased equally in all groups.

Additionally, we were interested in the evaluation of heart failure in the PD-treated mice. Initially, heart samples were weighed and the ratio of heart weight to body weight, which is an established measure of cardiac hypertrophy, was measured. The ratio of heart weight to body weight was significantly elevated in the SS treatment group and the effect was attenuated in the F4-based treatment group (Fig. 8). To confirm the hypertrophic state, we looked at cardiac stress markers. The RNA expression of atrial and brain natriuretic peptides (ANP and PNC) in cardiac tissues was not affected by the F4-based solution compared to the SS treatment group (Fig. 8).

Furthermore, VEGF-A (vascular endothelial growth factor A) whose function is to promote angiogenesis in myocardial tissue, MCP-1 (monocyte chemoattractant protein 1) and the fibrotic marker fibronectin were improved in mice treated with solution F4 (Fig. 8). These results suggest that F4 may have less impact on the heart by improving cardiac function.

To measure the systolic function of mice treated with different PD fluids we used the echocardiography technique. Measurements were taken in both systole and diastole. From these values, the values of fractional shortening (FA), LV end-systolic and end-diastolic volumes (LVESV and LVEDV), ejection fraction (EF) and stroke volume (SV) were calculated. Echocardiography data revealed that the levels of VS, AF and FE, which measure heart muscle contractility, were reduced in the SS-treated group after 20 and 60 days of PD (Fig. 9). This indicates that the efficiency of the heart to eject blood is affected by the glucose-based PD that was rescued by the F4 solution.

Claims (5)

1. A peritoneal dialysis solution comprising troxerutin as an osmotic agent. 2. A peritoneal dialysis solution, according to claim 1, wherein the concentration of troxerutin varies between 1 and 6% w/v. 3. A peritoneal dialysis solution, according to claim 2, wherein the concentration of troxerutin varies between 1 and 3% w/v. 4. Use of troxerutin as an osmotic agent in a peritoneal dialysis solution. 5. Use according to claim 4, wherein troxerutin is in a concentration between 1 and 6% w/v in the solution.