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Title:
REDUCTION OF OXIDATIVE STRESS DERIVED FROM THE PROCESS OF HEMODIALYSIS BY COVALENT CONJUGATION OF MOLECULES WITH HIGH ANTIOXIDANT POWER ON THE FIBER CONSTITUTING THE DIALYTIC MEMBRANE: POST-PRODUCTION PROCESS OF THE CARTRIDGE
Document Type and Number:
WIPO Patent Application WO/2019/097492
Kind Code:
A1
Abstract:
The present invention relates to a process for the preparation of dialysis cartridges functionalized with antioxidant molecules and to functionalized dialysis cartridges which can be obtained with such process.

Inventors:
PUOCI FRANCESCO (IT)
PEZZI VINCENZO (IT)
PARISI ILARIA ORTENSIA (IT)
AMONE FABIO (IT)
MALIVINDI ROCCO (IT)
SCRIVANO LUCA (IT)
MELICCHIO ALESSANDRO (IT)
PERRI ANNA (IT)
LOFARO DANILO (IT)
Application Number:
PCT/IB2018/059097
Publication Date:
May 23, 2019
Filing Date:
November 19, 2018
Export Citation:
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Assignee:
UNIV DELLA CALABRIA (IT)
International Classes:
B01D71/68; A61M1/16
Foreign References:
US20170209836A12017-07-27
US3669954A1972-06-13
Other References:
YAMADERA ET AL: "Vitamin E-Coated Dialyzer Inhibits Oxidative Stress.", 25 October 2017 (2017-10-25), XP055483182, Retrieved from the Internet [retrieved on 20180612]
GODDARD ET AL: "Polymer surface modification for the attachment of bioactive compounds", PROGRESS IN POLYMER SCIENCE, PERGAMON PRESS, OXFORD, GB, vol. 32, no. 7, 1 July 2007 (2007-07-01), pages 698 - 725, XP022133245, ISSN: 0079-6700, DOI: 10.1016/J.PROGPOLYMSCI.2007.04.002
Attorney, Agent or Firm:
DI GIOVINE, Paolo (IT)
Download PDF:
Claims:
CLAIMS

1. A process for the preparation of a dialysis cartridge functionalized with antioxidant compounds comprising the following steps: i) washing a dialysis cartridge with a solution containing a redox pair; ii) washing said dialysis cartridge with a solution containing one or more antioxidant compounds.

2. The process according to claim 1 wherein said dialysis cartridge comprises a polymer selected from the group consisting of polypropylene (PP), polyvinylpyrrolidone (PVP), polybenzoimidazole (PBI), polyvinylalcohol (PVA), polyethersulfone (PES), cellulose acetate, polyacrylonitrile (PAN), polysulfone (PSu), polymethacrylic acids (PMAA), polymethylmethacrylates (PMMA), polyacrylamides (PAM), polyurethanes (PU), polystyrenes (PS) or derivatives thereof.

3. The process according to claim 1 or 2, wherein said antioxidant compound is selected from the group consisting of: gallic acid, ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives thereof, anthocyanins, cinnamic acid and derivatives thereof.

4. The process according to anyone of claims 1 to 3, wherein the solvent of said solution in said first and/or second washing is selected from the group consisting of: water, ethyl alcohol, isopropyl alcohol, dimethylsulfoxide and mixtures thereof.

5. The process according to anyone of claims 1 to 4, wherein said first and/or second washing is carried out via the dialysis circuit.

6. The process according to anyone of claims 1 to 5, comprising a further step of washing with heparin. 7. The process according to anyone of claims 1 to 6, wherein the reducing agent of said redox pair is selected from bisulfite or ascorbic acid.

8. The process according to anyone of claims 1 to 7, wherein the oxidizing agent of said redox pair is selected from hydrogen peroxide, acetyl peroxide, ammonium persulfate, sodium perborate, potassium persulphate or sodium persulfate.

9. The process according to anyone of claims 1 to 6, wherein the reducing agent of said redox pair is ascorbic acid and the oxidizing agent of said redox pair is hydrogen peroxide. 10. The process according to anyone of claims 1 to 9, wherein said redox pair is in a concentration between 2 and 15% by weight with respect to the washing solution.

11. The process according to anyone of claims 1 to 10, wherein said redox pair is in a concentration ranging from 0.1 % to 5% by weight with respect to the washing solution.

12. The process according to anyone of claims 1 to 11 , wherein said dialysis cartridge comprises a polymer with a molecular weight between 20,000 and 10,000,000 Da.

13. The process according to anyone of claims 1 to 12, wherein the pH of the first and/or second washing solution is between 4.0 and 8.0.

14. The process according to anyone of claims 1 to 13, wherein the temperature thereat the process is performed is between 15 and 45 °C.

15. The process according to anyone of claims 1 to 14, wherein the process is carried out at atmospheric pressure. 16. A dialysis cartridge functionalized with antioxidant compounds obtainable by the process according to anyone of claims 1 to 15.

17. The dialysis cartridge functionalized with antioxidant compounds comprising a polymer covalently bonded to one or more antioxidant compounds.

18. The functionalized dialysis cartridge according to claim 17 wherein said polymer is selected from the group consisting of polypropylene (PP), polyvinylpyrrolidone (PVP), polybenzoimidazole (PBI), polyvinylalcohol (PVA), polyethersulfone (PES), cellulose acetate, polyacrylonitrile (PAN), polysulfone (PSu), polymethacrylic acids (PMAA), polymethylmethacrylates (PMMA), polyacrylamides (PAM), polyurethanes (PU), polystyrenes (PS) or derivatives thereof.

19. The functionalized dialysis cartridge according to claim 17 or 18 wherein said antioxidant compound is selected from gallic acid, ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives thereof, anthocyanins, cinnamic acid and derivatives thereof.

20. The functionalized dialysis cartridge according to anyone of claims 17 to 19 wherein said polymer has a molecular weight between 20,000 and 10,000,000 Da.

Description:
Reduction of oxidative stress derived from the process of haemodialysis by covalent conjugation of molecules with high antioxidant power on the fibre constituting the dialytic membrane: post-production process of the cartridge DESCRIPTION

Technical field of the invention

The present invention relates to a process for the preparation of dialysis cartridges functionalized with antioxidant molecules and to functionalized dialysis cartridges which can be obtained with such process. State of art

Dialysis is the therapy replacing the kidney functionality and still nowadays it is the only therapeutic technique capable of guaranteeing to the patient having renal failure a good health status. This technique allows to depurate the blood from the toxic substances which accumulate in circulation when the kidney is no more capable of carrying out such function. By exploiting the scattering and convection physical principles the blood is put in contact with the dialysis liquid (solution with known composition) through a semi-impermeable membrane. By scattering, the toxic substances pass from blood to the dialysis liquid which the kidney is no more capable of eliminating, in the same way ions can pass from dialysis liquid to the blood, such as sodium, calcium, magnesium, potassium, to maintain their values balanced in the blood. By exploiting the convection phenomenon a passage of fluids (ultrafiltration) can be created which allows the removal of the liquids accumulated in the body, without which the patient would be subjected to dangerous phenomena of heart circulatory failure. The ultrafiltration takes place in several ways depending upon the used dialytic technique.

At least two techniques are known to perform dialysis: an intracorporeal one, called peritoneal dialysis, which exploits the peritoneum capability of acting as natural dialyzing membrane, the other extracorporeal one, called haemodialysis, which in turn divides into traditional, haemodiafiltration and haemofiltration. Some dialytic membranes, used in haemodialysis, are capable of activating the oxidative metabolism of the neutrophils and macrophages, by leading to an hyperproduction of free radicals of oxygen which, together with a defect of the antioxidant systems, could be responsible for several complications typical of dialyzed patient. For these reasons, different efforts were concentrated on several strategies to reduce the oxidative stress in dialyzed patients.

Up to now, dialysis cartridges consisting of polymeric material, as well as several antioxidant compounds, such polyphenols, are singularly used in the clinical practice but examples of polymer-antioxidant conjugated cartridges do not exist.

Several strategies have been described for the reduction of oxidative stress of patients subjected to dialysis treatments for treating renal failure, they are used both during dialysis and in the post-treatment phase. Some provide the use of antioxidant agents which remove oxygen free radicals (ROS) which have formed, others are based upon strategies which reduce the production thereof upstream. For example, EP 2373638 A1 describes dialysis membranes consisting of biocompatible polymers and containing at least a derivative of xanthone in the polymeric matrix. WO 2001002004 A1 describes the use of a solution for peritoneal dialysis containing different antioxidants. US 6627659 B1 , on the contrary, describes the intravenous infusion of N-acetylcysteine, after the dialytic treatment. WO 2007147590 A3 instead provides the use of solutions of N- acetylcysteine and, optionally, of other antioxidants to be added to the dialysis liquid. WO 2010115291 A1 provides the treatment of patients subjected to haemodialysis with creatine-based solutions for its antioxidant and anti-apoptosis effects, whereas its function of osmotic agent is described to prevent damages from high integration of glucose in peritoneal dialysis. WO 2014180959 A1 , instead, states that the use of trace elements (such as selenium, cobalt, rubidium, molybdenum and zinc), which restore those which have been lost during the dialysis process, could have antioxidant activity since such trace elements act as co-factors in the operation of several enzymes, notwithstanding their chemical properties inducing oxidation reactions. US 20090045121 A1 limits the reduction of the oxidative stress to a strategy for the removal of the glucose decomposition products which, partially, are the cause thereof. Currently, however, none of the up-to-now used techniques is capable of preventing effectively the damages caused by the oxidative stress in the dialyzed patients. Moreover, the procedures described in the state of art are expensive and very difficult, as already shown none of the products which can be implemented with these procedures in fact is used in the clinical practice. The object of the present invention is to provide dialysis cartridges functionalized with antioxidant molecules and processes for their preparation allowing to reduce the oxidative stress in patients subjected to haemodialysis and to overcome the disadvantages of the products of known art.

Summary of the invention The dialysis treatment results to be correlated to an increase in the production of radical species and, consequently, of the levels of oxidative stress. The main factors linked to such increase are two, the process in itself and the patient uremic state. During dialysis, in fact, the only contact of blood with the materials the equipment is constituted of and the dialysis membranes cause activation of neutrophils, production of ROS and reduction of the antioxidant potential. In particular, the water-soluble molecules spread in dialysate and thus are eliminated by determining a depletion of reduced thiol plasma proteins, such as albumin, and a decrease in the plasma levels of glutathione and of activity of glutathione peroxidase. Even the uremic state takes part in the oxidative stress since the uremic toxins can cause modifications in the three-dimensional structure of proteins, with consequent loss or change in function. Such modifications, together with the oxidation of LDL, determine an increase in the production rate of ROS by neutrophils.

According to the present invention the antioxidant molecules bind covalently to the polymer chains constituting the dialysis membrane. Such process can be applied to several polymers and different antioxidant molecules. The latter carry out an action for removing free radicals from the fluids subjected to dialysis, through their intrinsic chemical properties, given by the electronic delocalization capability. This reduces the levels of oxygen free radicals (ROS) and nitrogen free radicals (RNS) which cause the damage of the cell structures of dialyzed patients. As a consequence of this, in such patients the side effects connected to a high oxidative stress are reduced. The process according to the present invention allows to implement bioactive dialysis cartridges by means of the covalent bond of antioxidant molecules to the polymer chains constituting the conventional dialysis membranes. The process according to the present invention offers different advantages: · use of traditional dialysis cartridges already currently in use in the hospital practice;

• implementation by means of instruments already present in all dialysis centres;

• low implementation costs; · easily modulable procedure and which allows to act under not drastic conditions.

The present invention is based upon the use of the dialysis cartridges, such as for example those which are used nowadays in the hospital practice and upon their functionalization with antioxidant molecules such as for example gallic or ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives, anthocyanins, cinnamic acid and derivatives thereof, through simple procedures which can be performed in hospital or ambulatory field, prior to the dialysis treatment, without jeopardizing the correct operation of the cartridge. The so-obtained cartridges are capable of reducing the oxidative stress in the patients subjected to haemodialysis.

The present invention then relates to the "plasma grafting-like" (PGL) functionalization of different dialysis cartridges with different antioxidant molecules, that is in the process of covalent anchoring of antioxidant molecules to the polymeric membrane of the cartridge so as to make the membrane itself bioactive and with antioxidant power.

The technical problem, placed and solved by the present invention, is then to provide a process for the preparation of dialysis cartridges functionalized with antioxidant molecules according to claim 1 and dialysis cartridges functionalized with antioxidant molecules according to claim 15 having several advantages with respect to the procedures and to the products of the state of art. Preferred features of the present invention are set forth in the depending claims.

Other advantages, features and use modes of the present invention will result evident from the following detailed description of some embodiments, shown by way of example and not for limitative purposes. Brief description of the figures

The figures of the enclosed drawings will be referred to, wherein:

- Figure 1 is a schematisation of the covalent insertion of an antioxidant molecule on a polymeric chain by means of grafting induced by free radicals. A= antioxidant molecule. - Figure 2 is a UV spectrum of catechin alone and of catechin conjugated to a sample of haemodialysis cartridge in polysulfone according to the process of the present invention. The spectrum shown in figure is a confirmation of the conjugation of the antioxidant Catechin to the polysulfone. UV spectra of the conjugate show the presence of absorption peaks in the aromatic region, which are correlated to the presence of catechin. Moreover, the absorption is shifted to higher wavelengths as consequence of the conjugation extension due to the formation of the covalent bonds between the reactive groups of polysulfone and antioxidant. (Bathochromic shift). UV spectra (recorded with spectrophotometer UV-Vis Jasco V-530 UV) are of catechin (10 mM) and of the respective polysulfone conjugate in acetonitrile. In order to remove the interference of the native polymer, the conjugate spectrum was recorded by using virgin polysulfone at the same base concentration.

Detailed description of the invention

The present invention relates to a process for the preparation of dialysis cartridges functionalized with antioxidant molecules comprising the following steps: i) a first washing of a dialysis cartridge with a solution containing a redox pair; ii) a second washing of said dialysis cartridge with a solution containing one or more antioxidant compounds. In the present description under“dialysis cartridge” a cartridge suitable to perform a haemodialysis is meant, then a cartridge for haemodialysis is meant.

The above-described process could advantageously be performed during the process for preparing the dialysis circuit and before the circuit washing with heparin, then by using the same dialysis circuit which will be then used to dialyze the patient.

The polymeric component of the cartridge dialysis which will be functionalized with the antioxidant compounds could be of polysulfone, or other polymer for dialysis membrane such as for example polypropylene, polyvinylpyrrolidone, polybenzoimidazole, polyvinylalcohol, polyethersulfone, cellulose acetate, polyacrylonitrile, polymethacrylic acids, polymethylmethacrylates, polyacrylamides, polyurethanes, polystyrenes and derivatives thereof. According to an embodiment the polymer will have a molecular weight comprised between 20,000 and 10,000,000 Da. In the present invention, the redox pair will consist of an oxidant agent, which will be selected in the group constituted by hydrogen peroxide, acetyl peroxide, ammonium persulfate, sodium perborate, potassium persulphate or sodium persulfate, and by a reducing agent which could be a bisulphite or ascorbic acid. According to an embodiment of the process, the redox pair will be used preferably in which the reducing agent is ascorbic acid, whereas the oxidizing agent of said redox pair is hydrogen peroxide.

The optimum concentrations of the redox pair which will be used in the first washing are comprised between 2% and 15% by weight (that is 0.02 and 0.15 Kg/L). Whereas in the second washing a solution will be used including the antioxidant compounds at a concentration between 0.1 % and 5% by weight (that is 0.001 and 0.05 Kg/L).

In the second passage antioxidant compounds could be used such as for example gallic acid, ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives thereof, anthocyanins, cinnamic acid and derivatives thereof, or mixtures thereof. In the first and/or second washing of the process an aqueous solution could be used such as for example water, ethyl alcohol, isopropyl alcohol, dimethylsulfoxide and mixtures thereof.

Preferably in the first and/or second washing solutions with pH values between 4.0 and 8.0 will be used. The temperature used in the process preferably will be between 15 and 45°C at atmospheric pressure.

According to a preferred embodiment the following use process conditions of the solution containing 2% redox pair, an antioxidant concentration equal to 0.1 %, a temperature between 25 and 40°C and a pH between 5.5 and 7.5 both for the first and the second washing will be used.

The invention further relates to the functionalized dialysis cartridges which can be obtained by means of anyone of the herein described embodiments and uses thereof, the process according to the present invention in fact allows to obtain the dialysis cartridges wherein the polymeric component is covalently bound to one or more antioxidant compounds.

According to an embodiment the used dialysis cartridges are constituted by polymers such as polypropylene (PP), polyvinylpyrrolidone (PVP), polybenzoimidazole (PBI), polyvinylalcohol (PVA), polyethersulfone (PES), cellulose acetate, polyacrylonitrile (PAN), polysulfone (PSu), polymethacrylic acids (PMAA), polymethylmethacrylates (PMMA), polyacrylamides (PAM), polyurethanes (PU), polystyrenes (PS) and derivatives thereof with one or more of the following antioxidant compounds such as for example gallic acid, ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives thereof, anthocyanins, cinnamic acid and derivatives thereof, or mixtures thereof. * * *

They can be subjected to the radical grafting reaction in presence of several antioxidant molecules (gallic acid, ellagic acid, quercetin, resveratrol, catechin, tocopherol, hydroxytyrosol and derivatives thereof, anthocyanins, cinnamic acid and derivatives thereof). All obtained derivatives are effective in reducing the concentration of oxygen and nitrogen free radicals. The examples shown hereinafter further illustrate the invention in a not limiting manner.

EXAMPLES

Example 1.

A 2% aqueous solution containing the redox pair (consisting of hydrogen peroxide and ascorbic acid) is made to pass, by means of peristaltic pump for 20 minutes in recirculation, in a cartridge with dialysis membrane in PMMA. Then, one proceeds with making to pass, for further 20 minutes in recirculation, an aqueous solution of 0.1 % gallic acid. At last, one proceeds with the purification from not reacted chemical species through washing in recirculation with physiological saline.

Example 2.

A 4% aqueous solution containing the redox pair (consisting of hydrogen peroxide and ascorbic acid) is made to pass, by means of peristaltic pump for 15 minutes in recirculation, in a cartridge with dialysis membrane in PSu. Then, one proceeds with making to pass, for further 15 minutes in recirculation, an aqueous solution of 0.1 % ferulic acid. At last, one proceeds with the purification from not reacted chemical species through washing in recirculation with physiological saline.

Example 3.

A 4% aqueous solution containing the redox pair (consisting of hydrogen peroxide and ascorbic acid) is made to pass, by means of peristaltic pump for 20 minutes in recirculation, in a cartridge with dialysis membrane in cellulose acetate. Then, one proceeds with making to pass, for further 20 minutes in recirculation, an aqueous solution of 0.2% resveratrol. At last, one proceeds with the purification from not reacted chemical species through washing in recirculation with physiological saline.

Example 4.

A 5% aqueous solution containing the redox pair (consisting of hydrogen peroxide and ascorbic acid) is made to pass, by means of peristaltic pump for 10 minutes in recirculation, in a cartridge with dialysis membrane di PES. Then, one proceeds with making to pass, for further 20 minutes in recirculation, an aqueous solution of 0.2% catechin. At last, one proceeds with the purification from not reacted chemical species through washing in recirculation with physiological saline.

The present invention has been sofar described with reference to some preferred embodiments. It is to be meant that other embodiments belonging to the same inventive core may exist, as defined by the protective scope of the herebelow reported claims.