Liposomal suspension and system and method of peritoneal administration of liposomal suspension

Abstract

There is provided a liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the suspension comprising citric acid anhydrous and lipids in a ratio of (3.54 g / L) / (6.49 g / L) along with a kit for preparing the liposomal suspension, uses of the liposomal suspension and kits for treating an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof, in a subject in need thereof. Also provided are a system and method for intraperitoneally administering the liposomal suspension to a subject in need thereof.

Description

[0001] LIPOSOMAL SUSPENSION AND SYSTEM AND METHOD OF PERITONEAL ADMINISTRATION OF LIPOSOMAL SUSPENSION FIELD OF THE DISCLOSURE

[0002] The present disclosure is concerned with the treatment of accumulation of ammonia and of other disease-related metabolites.

[0003] BACKGROUND OF THE DISCLOSURE

[0004] Accumulation of ammonia and other disease-related metabolites (LPS, hepatic and uremic toxins) is associated with reduced brain, liver, and kidney function in patients with liver impairments such as decompensated liver cirrhosis and acute-on-chronic liver failure (ACLF).

[0005] Extra-corporeal dialysis is the standard recommended treatment for managing inherited and acquired hyperammonemia that is unresponsive to conventional medications, such as ammonia scavengers, lactulose, and rifaximin. Nevertheless, its availability in community hospitals is limited, and it presents significant challenges. This method is not suitable for patients experiencing hemodynamic instability and may lead to severe complications, including thrombosis, bleeding, hypotension, and renal failure. These issues can significantly delay the start of treatment and worsen the prognosis for patients with severe hyperammonemia, where the mortality rate may reach up to 85%. Peritoneal dialysis (PD), being less invasive and simpler to administer, could serve as a valuable alternative but is infrequently used for acute hyperammonemia due to its limited capacity for ammonia clearance. The advancement of liposome-supported PD represents a significant development, enhancing the peritoneal removal of toxins related to liver and kidney dysfunctions, such as ammonia.

[0006] There is a need for a clinically tested liposome formulation and dosage, as well as an effective method for its peritoneal administration in subjects requiring it.

[0007] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0008] SUMMARY OF THE DISCLOSURE

[0009] The present disclosure presents in a first aspect a liposomal suspension comprising the transmembrane pH-gradient liposomes of the present disclosure in an aqueous solution, the suspension comprising citric acid anhydrous and lipids in a specific ratio. In a further aspect, it presents a specific useful dosage regimen for administrating the liposomal suspension to subjects. In another aspect, it presents a use of this liposomal suspension, and dosage regimen in the treatment of an ammonia-associated disease or disorder, ammonia methylated analog-associated disease or disorder, endotoxemia, or endotoxemia-associated disease or disorder, or a symptom thereof. In a further aspect, it presents a system for intraperitoneal administration in a subject of the liposomal suspension.

[0010] More specifically, in accordance with the present disclosure, there are provided the following items: Item 1. Liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the suspension comprising citric acid anhydrous and lipids in a ratio of (3.54 g / L) / (6.49 g / L).

[0011] Item 2. The liposomal suspension of item 1, for administration to a subject (a) in a daily dose of citric acid anhydrous / lipids of about 71 / 130 mg / kg to about 142 / 260 mg / kg based on the subject’s (dry) body weight, preferably in a daily dose of citric acid anhydrous / lipids of (a-i) about 71 / 130 mg / kg to about 92.9 / 173 mg / kg for a subject having a weight between 40 kg and < 53kg; (a-ii) about 96 / 175 mg / kg to about 142 / 260 mg / kg for a subject having a weight 53 Kg and < 79 kg; and (a-iii) about 106 / 195 mg / kg to about 142 / 260 mg / kg for a subject having a weight 79 Kg and < 140 kg; (b) wherein administration comprises one daily dose or at least two daily doses, or at least three daily doses or four daily doses; or (c) any combination of (a) and (b).

[0012] Item 2’. Liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the suspension comprising citric acid anhydrous and lipids for administration to a subject (a) in a daily dose of citric acid anhydrous / lipids of about 71 / 130 mg / kg to about 142 / 260 mg / kg based on the subject’s (dry) body weight, preferably in a daily dose of citric acid anhydrous / lipids of (a-i) about 71 / 130 mg / kg to about 92.9 / 173 mg / kg for a subject having a weight between 40 kg and < 53kg; (a-ii) about 96 / 175 mg / kg to about 142 / 260 mg / kg for a subject having a weight 53 Kg and < 79 kg; and (a-iii) about 106 / 195 mg / kg to about 142 / 260 mg / kg for a subject having a weight 79 Kg and < 140 kg; (b) wherein administration comprises one daily dose or at least two daily doses, or at least three daily doses or four daily doses; or (c) any combination of (a) and (b). Item 2”. Liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the suspension comprising citric acid anhydrous and lipids in a daily dose of about 1050 g to about 4200 g, or preferably about 1050 g, about 2100 g, about 3150 g or 4200 g.

[0013] Item 3. The liposomal suspension of item 2, 2’ or 2”, wherein (a’) the administration comprises one, two or three daily doses; (b’) the doses are administered on consecutive days; or (o’) a combination of (a’) and (b’). Item 4. The liposomal suspension of any one of claims 1 to 3, 2’ and 2”, wherein (A) the lipids comprise (i) at least one phospholipid as main constituent, wherein the at least one phospholipid preferably comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%; (ii) cholesterol, preferably in a range of 10 to 40 mol%; (iii) 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%; or (iv) any combination of at least two of (i) to (iii), preferably the combination comprise (i) to (iii), and most preferably the lipids contain dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG) at 85.5:14:0.5 mol%; (B) the aqueous solution contains, in addition to water, (i) xylitol, (ii) sodium, (iii) chloride, (iv) magnesium; (v) potassium, (vi) calcium; (vii) citric acid anhydrous, or (viii) any combination of at least two of (i) to (vii), preferably the combination comprises, two of, three of, four of, five of, six of, 7 of or all of (i) to (vii); (C) the liposomes have an average diameter between about 8 pm and 12 pm (e.g., average diameter between about 10 pm and 15 pm); or (D) any combination of at least two of (A) to (C). Item 5. Kit for preparing a liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the kit comprising (a) a citric acid anhydrous solution comprising citric acid anhydrous and water; (b) a liposome aqueous suspension comprising lipids and water; and (c) a neutralizing aqueous solution comprising water and (i) at least one sugar alcohol; (ii) at least one salt; or (iii) a combination of (i) and (ii); wherein the citric acid anhydrous and lipids are in a ratio of (3.54 g / L) / (6.49 g / L) and wherein each of (a), (b) and (c) are in separate enclosures.

[0014] Item 5’. Kit for preparing a liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the kit comprising (a) a citric acid anhydrous solution comprising citric acid anhydrous and water; (b) a liposome aqueous suspension comprising lipids and water; and (c) a neutralizing aqueous solution comprising water and (i) at least one sugar alcohol; (ii) at least one salt; or (iii) a combination of (i) and (ii); wherein each of (a), (b) and (c) are in separate enclosures; and optionally (i) the citric acid anhydrous solution has a volume of 32.2 mL or a multiple thereof; or a weight of about 34 g or a multiple thereof; (ii) the liposome aqueous suspension has a volume of about 68.1 mL or a multiple thereof or weight of about 68.1 g or a multiple thereof; (iii) the neutralizing aqueous solution has a volume of about 950 mL or a multiple thereof or weight of about 960 g or a multiple thereof; or (iv) a combination of at least two of (i) to (ii), preferably at least three thereof. Item 6. The kit of item 5 or 5’, wherein (a) the citric acid anhydrous solution further comprises at least one of sodium chloride, sodium hydroxide, and magnesium chloride hexahydrate, preferably at least two thereof and most preferably all three thereof; (b) the lipids comprise (i) at least one phospholipid as main constituent, wherein the at least one phospholipid preferably comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%; (ii) cholesterol, preferably in a range of 10 to 40 mol%; (iii) 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%; or (iv) any combination of at least two of (i) to (iii), preferably the combination comprise (i) to (iii), and most preferably the lipids contain dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG) at 85.5:14:0.5 mol%; (c) the neutralizing aqueous solution comprises (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride; or (vii) any combination of at least two of (i) to (v), preferably the combination comprises two of, or three of, or four of or all five of (i) to (v); (d) (i) the volume of the citric acid anhydrous solution is between about 20 mL and about 40 mL, preferably about 32.2 mL; (ii) the volume of the liposome aqueous suspension is between about 55 mL and about 75 mL, preferably about 68.1 mL; (iii) the volume of the neutralizing aqueous solution is between about 900 mL and about 1000 mL, preferably about 950 mL; (iv) the combined volume of the citric acid anhydrous solution, the liposome aqueous suspension and the neutralizing aqueous solution is between about 975 mL and about 1115 mL, preferably about 1050 mL; or (v) a combination of at least two of (i) to (iv); (e) (i) the enclosure of the citric acid anhydrous solution is a bottle; (ii) the enclosure of the liposome aqueous suspension is a bottle; (iii) the enclosure of the neutralizing aqueous solution is a bag, such as an infusion bag; or (iv) a combination of at least two of (i) to (iii); or (f) a combination of at least two of (a) to (e).

[0015] Item 7. The liposomal suspension of any one of items 1 to 4, 2’ and 2” or the kit of any one of item 5, 5’ and 6, which is for use in the treatment of an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof, in a subject in need thereof, preferably a human, wherein the treatment preferably includes peritoneal administration of the liposomal suspension to the subject in need thereof.

[0016] Item 8. The liposomal suspension or kit for its use of item 7, wherein the ammonia- or ammonia methylated analog-associated disease or disorder is cirrhosis with ascites or acute-on-chronic liver failure with ascites.

[0017] Item 9. System for intraperitoneal administration in a subject of the liposomal suspension defined in any one of items 1 to 5, 2’, 2”, and 5’, comprising:

[0018] an infusion pump (10) for pumping the liposomal suspension contained in an infusion bag (12) via a fluid line (16);

[0019] a warming device (14) connectable to the fluid line (16) for warming the liposomal suspension before administering the liposomal suspension to the subject; and

[0020] a catheter (18) fluidly connectable to the infusion bag (12) for insertion into a peritoneal space of the subject.

[0021] Item 10. The system of item 9, comprising one, two, three or four infusion pumps (10) each respectively being in fluid communication with one, two, three or four infusion bags (12), each infusion bag preferably containing about 1050 mL of the liposomal suspension the infusion pumps (10) being selectively activated depending on the dry body weight of the subject, wherein a flow rate of each pump is preferably set at 1000 mL / hour, and wherein at dwell time of the liposomal suspension in the peritoneal space of the subject preferably lasts for about 3 hours before being drained.

[0022] Item 11. The system of item 9 or 10, further comprising a drainage bag (20) for removing the liposomal suspension from the peritoneal space of the subject.

[0023] Item 12. The system of item 11, further comprising:

[0024] a washing solution infusion bag (24) containing a washing solution of peritoneal dialysis solution that is optionally warmed before being injected in the peritoneal space of the subject and preferably contains about 1 L of the washing solution; and

[0025] a 3-way-stop cock (22) having a first inlet fluidly connectable to the catheter (18), a second inlet fluidly connectable to the washing solution infusion bag (24), and a third inlet connectable to a syringe (26) for injecting the washing solution in the peritoneal space of the subject.

[0026] Item 13. The system of item 12, further comprising another drainage bag (20) for removing the washing solution in the peritoneal space of the subject.

[0027] Item 14. Method for intraperitoneal administration in a subject of the liposomal suspension defined in any one of items 1 to 4, 2’, 2”, and 5’, comprising:

[0028] pumping the liposomal suspension contained in an infusion bag (12) by means of an infusion pump (10) via a fluid line (16);

[0029] warming the liposomal suspension by means of a warming device (14) connectable to the fluid line (16) before administering the liposomal suspension to the subject; and

[0030] infusing the liposomal suspension from the infusion bag (12) into a peritoneal space of the subject via a catheter (18) inserted therein, the catheter being fluidly connectable to the infusion bag (12).

[0031] Item 15. The method of item 14, further comprising:

[0032] pumping the liposomal suspension contained in one, two, three or four infusion bags (12) by means of respective one, two, three or four infusion pump (10),

[0033] selectively activating the infusion pumps (10) depending on the dry body weight of the subject, wherein a flow rate of the pumps is preferably set at 1000mL / hour, wherein a dwell time of the liposomal suspension in the peritoneal space of the subject preferably lasts for about 3 hours before being drained.

[0034] Item 16. The method of item 14 or 15, further comprising draining the liposomal suspension from the peritoneal space of the subject by means of a drainage bag (20).

[0035] Item 17. The method of item 16, further comprising:

[0036] injecting a washing solution from a washing solution infusion bag (24) in the peritoneal space of the subject by means of a 3-way-stop cock (22) having a first inlet fluidly connectable to the catheter (18), a second inlet fluidly connectable to the washing solution infusion bag (24), and a third inlet connectable to a syringe (26), the washing solution infusion bag (24) being optionally warmed before injecting the washing solution, the washing solution infusion bag (24) preferably containing about 1 L of the washing solution.

[0037] Item 18. The method of item 17, further comprising (a) removing the washing solution from the peritoneal space of the subject by means of another drainage bag (20), and optionally (b) draining ascites from the subject prior to infusing the liposomal suspension from the infusion bag (12) into the peritoneal space of the subject. Definitions

[0038] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0039] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0040] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0041] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0042] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

[0043] No language in the specification should be construed as indicating any non-daimed element as essential to the practice of the disclosure.

[0044] Herein, the term "about" has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0046] Liposomes

[0047] Liposomes according to the present disclosure comprise a lipid bilayer membrane.

[0048] Liposomes composition

[0049] Liposome compositions (e.g., suspension) according to the present disclosure comprise a lipid bilayer membrane enclosing a hydration medium such as an acidic buffer (acidic solution). The term “liposomal suspension” as used herein refers to a liposome composition comprising liposomes of the present disclosure (e.g., transmembrane pH-gradient liposomes of the present disclosure enclosing an acidic and hyperosmotic buffer (e.g., citric acid anhydrous)) suspended in an aqueous solution (e.g., neutralizing aqueous solution) of the present disclosure, containing free an acidic and hyperosmotic buffer (e.g., citric acid anhydrous).

[0050] Lipid bilayer membrane

[0051] In preferred embodiments, the liposome lipid bilayer membrane comprises at least one natural or synthetic phospholipid. Preferred phospholipids are long saturated phospholipids, e.g., those having alkyl chains of more than 12, preferably more than 14, more preferably more than 16, and most preferably more than 18 carbon atoms.

[0052] In specific embodiments, the natural or synthetic phospholipid comprises at least one of 1 ,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC); 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC); 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC); 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC); 1 ,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC); 1,2-Dimyristoyl-sn-Glycero-3-Phosphoelhanolamine (DMPE); 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoelhanolamine (DPPE); 1,2-Distearoyl-sn-Glycero-3-Phosphoelhanolamine (DSPE); 1,2-Dioleoyl-sn-Glycero-3-Phosphoelhanolamine (DOPE); 1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (MPPC); 1-Palmitoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine (PMPC); 1-Stearoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (SPPC); 1-Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine (PSPC); 1 ,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG); 1 ,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG); 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG); 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG); 1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DM PA); 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (DPPA); 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (DPPS); natural L-a-phosphatidylcholine (from chicken egg, EPC, or from soy, SPC). In specific embodiments, the natural or synthetic phospholipid is DPPC. In specific embodiments, the main constituent of the liposome lipid bilayer is the at least one natural or synthetic phospholipid. In specific embodiments, the at least one natural or synthetic phospholipid forms at least 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, or 85 mol% of the liposome bilayer membrane. In specific embodiments, the natural or synthetic phospholipid forms about 85.5 mol% of the liposome bilayer membrane.

[0053] In other embodiments, the liposome lipid bilayer membrane further comprises an ammonia retention-enhancing compound. In specific embodiments, the ammonia retention-enhancing compound comprises a sterol derivative. In other specific embodiments, the sterol derivative is cholesterol. In specific embodiments, the at least one ammonia retention-enhancing compound forms at least 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol% of the liposome bilayer membrane. In specific embodiments, the at least one ammonia retention-enhancing compound forms at least 10 mol% of the liposome bilayer membrane. In specific embodiments, the at least one ammonia retention-enhancing compound forms about 14% of the liposome bilayer membrane.

[0054] In other embodiments, the liposome lipid bilayer membrane further comprises at least one steric stabilizer, such as at least one PEGylated compound, preferably at least one PEGylated lipid, more preferably DSPE-PEG. In specific embodiments, the at least one steric stabilizer forms at least 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, or 10 mol% of the liposome bilayer membrane. In specific embodiments, the at least one steric stabilizer forms about 0.5% of the liposome bilayer membrane.

[0055] In other embodiments, the liposome lipid bilayer membrane comprises 10 to 100 mol%, more preferably 25 to 75 mol%, more preferably 40 to 70 mol%, most preferably 50 to 60 mol% of at least one sphingolipid, preferably sphingomyelin.

[0056] In other embodiments, the liposome lipid bilayer membrane comprises 30 to 100, more preferably 40 to 95, most preferably 45 to 60 mol% of at least one surfactant. In specific embodiments, the at least one surfactant comprises hydrophobic alkyl ether (e.g., Brij), alkyl ester, polysorbate, sorbitan ester, and / or alkyl amide. In other embodiments, the average diameter size of the liposomes is larger than 900 nm, larger than 1000 nm, larger than 2000 nm, larger than 3000 nm; larger than 4000 nm; larger than 5000 nm, larger than 6000 nm; larger than 7000 nm; between 3000 nm and 15 pm, between 4000 nm and 15 pm, between 5000 nm and 15 pm, between 6000 nm and 15 pm, between 7000 nm and 15 pm, between 8000 nm and 15 pm, between 3000 nm and 14 pm, between 4000 nm and 14 pm, between 5000 nm and 14 pm, between 6000 nm and 14 pm, between 7000 nm and 14 pm, between 8000 nm and 14 pm, between 3000 nm and 13 pm, between 4000 nm and 13 pm, between 5000 nm and 13 pm, between 6000 nm and 13 pm, between 7000 nm and 13 pm, between 8000 nm and 13 pm, to avoid too rapid drainage from the peritoneal space. In specific embodiments, the average diameter size of the liposomes is between about 8 pm and about 12 pm (e.g., average diameter between about 10 pm and 15 pm).

[0057] Hydration medium

[0058] Liposomes of the present disclosure contain a hydration medium in the inner compartment of the liposomes. In specific embodiments, the aqueous medium is an acidic buffer.

[0059] Acidic buffer / acidic solution

[0060] The acidic buffer in the inner compartment of the liposomes preferably has a high buffering capacity at low pH for a high retention of basic compounds (e.g., ammonia). The acid is not toxic to animals and does not (or only weakly) permeate out of the liposome membrane.

[0061] Without being so limited, the acid enclosed in the liposomes core is (i) a hydroxy acid such as citric acid, isocitric acid, malic acid, tartaric acid, or lactic acid; (ii) a small chain fatty acid such as acetic acid; (iii) a sugar acid such as uronic acid; (iv) a dicarboxylic acid such as malonic acid; (v) a tricarboxylic acid such as propane-1, 2,3- tricarboxylic acid or aconitic acid; (vi) a tetracarboxylic acid such as 1 ,2,3,4-butanetetracarboxylic acid; (vii) a pentacarboxylic acid such as 1,2,3,4,5-pentanepentacarboxylic acid; (viii) a polymeric poly(carboxylic acid) such as poly(acrylic acid) or poly(methacrylic acid); (ix) a polyaminocarboxylic acid such as ethylenediaminetetraacetic acid; or (x) a combination of at least two thereof. In specific embodiments, the acid is a hydroxy acid such as citric acid (e.g., citric acid anhydrous).

[0062] In specific embodiments, the concentration of acid used in a method such as the osmotic shock method, may be varied between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of between about 100 mM and 900 mM or between about 100 mM and 900 mM, or between about 300 mM and 800 mM, or between about 400 mM and 750 mM, or between about 500 mM and 750 mM, or between about 500 mM and 650 mM or about 600 mM is optimally used; at an osmolality between 500 and 1500 mOsmol / kg, or between 600 and 1400 mOsmol / kg, or between 700 and 1400 mOsmol / kg, between 800 and 1400 mOsmol / kg, or between 800 and 1350 mOsmol / kg, or between 900 and 1350 mOsmol / kg, or between 950 and 1300 mOsmol / kg, or between 950 and 1250 mOsmol / kg, or between 1000 and 1200 mOsmol / kg is optimally used. In another specific embodiment, the concentration of citric acid (e.g., anhydrous) used in the method may be varied between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of between about 500 mM and 600 mM is used with an osmolality of between 1000 and 1200 mOsmol / kg is used in the osmotic shock method. In a preferred embodiment, transmembrane pH-gradient liposomes produced by methods described herein have an inner concentration of citric acid anhydrous of about 200 nM, and an inner osmolarity that is physiological i.e., around 350 mOsm / kg.

[0063] The acid within the core (inner compartment of liposomes) is present in a concentration that produces a pH between 1 and 6 in the core of the liposomes, and in a specific embodiment, a pH between 1.5 and 3, and in a more specific embodiment, a pH of about 2.

[0064] In a specific embodiment, the liposomes contain in their internal compartment / core between 200 nM citric acid (anhydrous), and this core has a pH of about 2.

[0065] In alternative embodiments, liposomes for use in the present disclosure are as described in EP 2 882 421 to Leroux et al.

[0066] Composition

[0067] In accordance with another aspect of the present disclosure, there is provided a composition (in the form of a suspension or otherwise) comprising the liposomes of the present disclosure, and at least one pharmaceutically acceptable excipient or carrier. The compositions of the disclosure can contain a pharmaceutically acceptable carrier / excipient including, without limitation, aqueous or non-aqueous solutions. Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., sugar solutions, saline), neutralizing species (basic or acidic, such as weak bases or weak acids) but also chemical agents used to adjust the osmolarity and / or provide a physiological function. Without being limited excipients encompassed by the present disclosure include glycerol, tris((hydroxymethyl)aminomethane) (TRIS), agents to counteract potential anticoagulant effects of certain weak acids (e.g., citric acid) such as calcium salts (e.g., calcium chloride); other salts such as sodium salts (e.g., sodium chloride), magnesium salts, lactate salts, potassium salts (e.g., potassium chloride); hydroxides (e.g., sodium hydroxide); sugars or polysaccharides (icodextrin, glucose, sorbitol, fructose); amino acids; sugar alcohols (e.g., xylitol, glycerol) or other known carriers / excipients appropriate for the intraperitoneal route. In specific embodiments, the liposomal composition (e.g., suspension) comprise (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride or (vii) any combination of at least two of (i) to (v), preferably the combination comprises all of (i) to (v).

[0068] Method of preparing liposomes

[0069] Osmotic shock method

[0070] Lipid blend

[0071] In specific embodiments, a lipid blend can be prepared by mixing the lipid bilayer components in a solvent such as an alcohol or a mixture of water and of an organic solvent (e.g., alcohol such as ethanol or t-butanol), until complete dissolution to form a homogenous lipid mix. The mix can be conducted at room temperature (i.e., around 20-25 °C) or while heating (e.g., at a temperature of up to 60 °C, preferably up to 45 °C) and optionally slowly mixing.

[0072] The mix can optionally be filtered (e.g., 0.2 pm filter). The organic solvent is then removed e.g., by lyophilization, spray drying (e.g., using liquid nitrogen as drying gas), rotary evaporation or otherwise.

[0073] The resulting dried lipid blend can then be hydrated in the aqueous medium as further described below to form a hydrated lipid blend (also called liposome aqueous suspension).

[0074] Aqueous medium

[0075] In a preferred embodiment, the lipid bilayer components can be directly mixed in an aqueous medium having an osmolarity of not more than 400 mOsm / l (direct lipid hydration method).

[0076] In an embodiment, the aqueous medium has a pH value of around 7, e.g., in the range of 6.0 to 7.5, of 6.1 to 7.4, of 6.2 to 7.3, of 6.3 to 7.2, of 6.4 to 7.1 , of 6.5 to 7.3, of 6.6 to 7.3, of 6.7 to 7.3, of 6.8 to 7.3, of 6.9 to 7.1 , of 6.95 to 7.01, or of about 7.0. In an embodiment, the aqueous medium is chosen from the group consisting of water (e.g., distilled water, deionized water, ultra-pure water or any other kind of purified water), a mixture of water as defined above and organic solvent (e.g., alcohol), aqueous solutions of organic salts, aqueous solutions of inorganic salts, aqueous solutions of organic substances, and combinations thereof. In an embodiment, the aqueous medium is chosen from the group consisting of aqueous solutions of organic salts having a pH value of around 7, aqueous solutions of inorganic salts having a pH value of around 7, aqueous solutions of organic substances having a pH value of around 7, water and combinations thereof.

[0077] When using organic or inorganic salts or other organic compounds, these salts or compounds are present in the aqueous medium, in an embodiment, in a low concentration so as to keep a difference in osmolarity between the aqueous medium and the hyperosmotic buffer provoking the osmotic shock which difference is large enough to induce the diffusion of the acidic or basic hyperosmotic buffer into the vesicle internal compartment.

[0078] The aqueous medium is a medium that resembles water (in particular with respect to pH) but that might contain a low concentration of salts or compounds, e.g., for buffering the pH value in a neutral range.

[0079] As indicated above, the aqueous medium has an osmolarity of not more than 400 mOsm / l. In an embodiment, the osmolarity of the aqueous medium is equal to or less than 300 mOsm / l, equal to or less than 250 mOsm / l, equal to or less than 200 mOsm / l, equal to or less than 150 mOsm / l, equal to or less than 100 mOsm / l, equal to or less than 75 mOsm / l, equal to or less than 50 mOsm / l, equal to or less than 25 mOsm / l, equal to or less than 10 mOsm / l, equal to or less than 5 mOsm / l equal to or less than 1 mOsm / l. In an embodiment, the osmolarity is in the range of 1 mOsm / l to 200 mOsm / l or in the range built up from any of the before mentioned osmolarities (such as 10 mOsm / l to 150 mOsm / l etc.). In an embodiment, the osmolarity of the aqueous medium is in a range between 0 mOsm / l and 49 mOsm / l, between OmOsm / l and 45 mOsm / l, between 0 mOsm / l and 40 mOsm / l, in particular between 0 mOsm / l and 35 mOsm / l, in particular between 0 mOsm / l and 30 mOsm / l, between 0 mOsm / l and 25 mOsm / l. In specific embodiments, the liposomes can optionally be extruded or filtered to obtain liposomes having a specific size. The hydration of the lipid bilayer components / lipid blend can be conducted at room temperature (i.e., around 20-25 °C) or while heating (e.g., at a temperature up to 60 °C (e.g., prewarmed aqueous medium), preferably up to 45 °C) and optionally slowly stirring for a period of about 15 minutes to 4 hours, preferably about 2 hours. At that stage, the final concentration of lipids is preferably at about 100 mg / g, if the hydration was performed while heating the mixture is cooled down to room temperature (i.e., around 20-25 °C). The mixture can optionally be degassed (e.g., under vacuum) to remove air bubbles.

[0080] In an embodiment, the hydrated liposomes so prepared are sterilized so as to obtain sterilized liposomes or sterilized suspension containing the liposomes. The sterilization can be carried out by, e.g., sterile filtration or steam sterilized (e.g., autoclaving), e.g., for a period of about 5 minutes to 2 hours, 10 minutes to 1 hour, or 15 minutes, or 30 minutes.

[0081] In another embodiment, the vesicles are stored for a first period of time prior to carrying out the step of mixing the liposomes (or the liposomes-containing suspension) with the acidic buffer. This storage can be optimally accomplished if the liposomes are sterilized after the hydration in aqueous medium step because then no or little degradation processes will occur in the sterilized liposomes suspension. The first period of time can be one day, a few days, one week, several weeks (1, 2, 3 or 4 weeks), one month or even several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months). Sterilized liposomes contained in an aqueous medium are stable entities. Since they do not yet contain any specific basic buffer used to prepare the pH gradient, no buffer loss due to liposomes bilayer degradation or leakage of the liposomes has to be feared. This is also true if the liposomes, in an embodiment, contain low amounts of electrolytes molecules since an according osmolarity within the vesicles would then be in a range of between 0 or 1 mOsm / l to 200 mOsm / l.

[0082] In a specific embodiment, the hydrated lipid blend (or liposome aqueous suspension) is in an enclosure separate from the acidic and hyperosmotic buffer and the neutralizing aqueous solution. In a specific embodiment, the volume of the hydrated lipid blend (or liposome aqueous suspension) in its separate enclosure is between about 40 mL and 400 mL or between about 40 mL and about 300 mL or between about 50 mL and about 100 mL or between 55 mL and about 75 mL. In a very specific embodiment, it is about 68.1 mL or a multiple thereof (2 x or 3 x or 4 x or 5 x or more). In a specific embodiment, the weight of the hydrated lipid blend (or liposome aqueous suspension) in its separate enclosure is between about 40 g and about 400 g or between about 40 g and about 300 g or between about 50 g and about 100 g or between 55 g and about 75 g. In a very specific embodiment, it is about 68.1 g or a multiple thereof (2 x or 3 x or 4 x or 5 x or more).

[0083] In a specific embodiment, the hydrated lipid blend is prepared as described under the Liposome aqueous suspension (LAS) heading below.

[0084] Acidic and hyperosmotic buffer

[0085] Thereafter, the hydrated (and optionally sterilized) liposomes are mixed with an acidic buffer having an osmolarity being at least 200 mOsm / l higher than the osmolarity of the aqueous medium to apply an osmotic shock to the liposomes and to obtain buffer-filled liposomes. In an embodiment, the osmolarity of the acidic buffer is at least 220 mOsm / l higher than the osmolarity of the aqueous medium, at least 250 mOsm / l higher, at least 300 mOsm / l higher, at least 350 mOsm / l higher, at least 400 mOsm / l higher, at least 450 mOsm / l higher, at least 500 mOsm / l higher, at least 550 mOsm / l higher, at least 600 mOsm / l higher, at least 650 mOsm / l higher, at least 700 mOsm / l higher, at least 750 mOsm / l higher, at least 800 mOsm / l higher, at least 850 mOsm / l higher, at least 900 mOsm / l higher, at least 950 mOsm / l higher, at least 1000 mOsm / l higher, at least 1050 mOsm / l higher, at least 1100 mOsm / l higher or at least 1200 mOsm / l higher. In an embodiment, the osmolarity of the acidic buffer is in a range of 200 mOsm / l to 1100 mOsm / l higher than the osmolarity of the aqueous medium or in a range built up from any of the before mentioned osmolarities (such as 220 mOsm / l to 1200 mOsm / l etc.).

[0086] Thus, the acidic buffer is a hyperosmotic buffer with respect to the aqueous medium used in the liposome’s hydration step. In doing so, an osmotic shock is extemporaneously applied to the liposomes. This osmotic shock results in incorporating the acidic buffer within the liposomes. Thus, the osmotic shock serves for a short-term destabilization of the liposomes in order to allow buffer incorporation into the liposomes. Buffer-filled liposomes result. In an embodiment, the hyperosmotic buffer can also contain electrolytes that are used to modulate the osmolarity or have a physiological function.

[0087] A sufficient amount of the acidic buffer is to be added to the liposomes suspended in the aqueous medium since otherwise no osmotic shock will be achieved. A sufficient amount can be, depending on the difference between the osmolarity of the aqueous medium and the osmolarity of the basic buffer, a volume that corresponds to at least 0.1 times the volume of the aqueous medium, at least 0.3 times, at least 0.5 times, at least 0.8 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times or at least 5 times. In an embodiment, the acidic buffer can be added in a volume that equals the volume of the aqueous medium. In an embodiment, the volume of the acidic buffer to be added can be 0.1 times to 5 times the volume of the aqueous liposome suspension or any other range that can be built up from the above-mentioned values (such as 0.3 times to 3 times, etc.).

[0088] In an embodiment, the pH value of the hyperosmotic buffer is in a range of pH 1 to pH 6.9, pH 1.5 to pH 6.5, pH 1.5 to pH 6.0, pH 1.5 to pH 5.5, pH 1.5 to pH 5.0, pH 1.5 to pH 4.5, pH 1.5 to pH 4.0, pH 1.5 to pH 3.5, pH 1.5 to pH 3.0, pH 1.5 to pH 2.5, pH 1 ,8t o pH 2.2, pH 1.5 to pH 2.0, pH 2.0 to pH 6.0, pH 2 to pH 5.5, pH 2.0 to pH 5.0, pH 2.0 to pH 4.5, or pH 2.0 to pH 3.5.

[0089] In specific embodiments, the hyperosmotic buffer can contain additional chemical agents such as a complexing agent or a chelating agent.

[0090] In specific embodiments, the hyperosmotic buffer comprises salts such as but not limited to sodium chloride, sodium hydroxide, and / or magnesium chloride.

[0091] In specific embodiments where a sterile transmembrane pH gradient liposome is preferred, the acidic buffer is sterilized. In such embodiments, where the hydrated liposomes have also been sterilized before loading the acidic buffer, fully sterile buffer-filled liposomes or a fully sterile suspension containing buffer-filled liposomes are prepared. The sterilization can be carried out by, e.g., sterile filtration or autoclaving. In an embodiment, the mixture of the aqueous medium and the basic or acidic buffer in which the buffer-filled liposomes are suspended has an osmolarity of at least 200 mOsm / l, of at least 220 mOsm / l, of at least 250 mOsm / l, of at least 300 mOsm / l, of at least 350 mOsm / l, of at least 400 mOsm / l, of at least 450 mOsm / l, of at least 500 mOsm / l, or of at least 550 mOsm / l. In an embodiment, the osmolarity is in the range of 200 mOsm / l to 550 mOsm / l or in the range built up from any of the before mentioned osmolarities (such as 220 mOsm / l to 500 mOsm / l etc.).

[0092] The liposomes acidic buffer mixture can optionally be incubated. In specific embodiments, the mixture is stirred (e.g., by orbital shaking) at e.g., room temperature.

[0093] In a specific embodiment, the acidic and hyperosmotic buffer is in an enclosure separate from that of the neutralizing aqueous solution and the hydrated lipid blend (or liposome aqueous suspension). In a specific embodiment, the volume of the acidic and hyperosmotic buffer in its separate enclosure is between about 20 mL and about 150 mL or between about 20 mL and about 40 mL. In a very specific embodiment, it is about 32.2 mL or a multiple thereof, (2 x or 3 x or 4 x or 5 x or more). In a specific embodiment, the weight of the acidic and hyperosmotic buffer in its separate enclosure is between about 20 g and about 150 g or between about 20 g and about 40 g. In a very specific embodiment, it is about 34 g or a multiple thereof, (2 x or 3 x or 4 x or 5 x or more). In a specific embodiment, the acidic and hyperosmotic buffer is prepared as described under the Citric acid anhydrous solution (CAS) heading below.

[0094] Citric acid anhydrous / lipids ratio

[0095] The citric acid anhydrous: lipids ratio in the liposomal suspension is optimally of about (3.54 g / L) / (6.49 g / L), wherein the lipids are as defined herein and preferably as defined in the Liposome aqueous suspension (LAS) heading below and the liposomal suspension.

[0096] Neutralizing aqueous solution

[0097] Then, a mixture of the aqueous medium and the acidic buffer containing the buffer-filled liposomes is diluted by adding a neutralizing aqueous solution. The mixture of acidic buffer and neutralizing aqueous solution makes up a suspension buffer. Thus, after dilution, transmembrane pH-gradient liposomes suspended in the suspension buffer result. Thereby, the pH of the suspension buffer differs from the acidic buffer contained in the buffer-filled liposomes. The pH difference is in an embodiment at least 1 pH unit, at least 1.5 pH units, at least 2 pH units, at least 2.5 pH units, at least 3 pH units, at least 3.5 pH units, at least 4 pH units, at least 4.5 pH units, at least 5 pH units, at least 5.5 pH units, at least 6 pH units, at least 6.5 pH units, or at least 7 pH units.

[0098] In an embodiment, the pH value of the neutralizing aqueous solution is in a range of pH 7.1 to pH 14, pH 7.1 to pH 13.5, pH 7.1 to pH 13.0, pH 7.1 to pH 12.5, pH 7.1 to pH 12, pH 7.1 to pH 11.5, pH 7.1 to pH 11.0, pH 7.1 to pH 10.5, pH 7.1 to pH 10, pH 7.1 to pH 9.5, pH 7.1 to pH 9.0, pH 7.1 to pH 8.5, pH 7.2 to pH 14, pH 7.2 to pH 13.5, pH 7.2 to pH 13, pH 7.2 to pH 12.5, pH 7.2 to pH 12, pH 7.2 to pH 11.5, pH 7.2 to pH 11, pH 7.2 to pH 10.5, pH 7.2 to pH 10, pH 7.2 to pH 9.5, pH 7.2 to pH 9, pH 7.2 to pH 8.5, pH 7.3 to pH 14, pH 7.3 to pH 13.5, pH 7.3 to pH 13, pH 7.3 to pH 12.5, pH 7.3 to pH 12, pH 7.3 to pH 11.5, pH 7.3 to pH 11 , pH 7.3 to pH 10.5, pH 7.3 to pH 10, pH 7.3 to pH 9.5, pH 7.3 to pH 9, pH 7.3 to pH 8.5, pH 7.4 to pH 14, pH 7.4 to pH 13.5, pH 7.4 to pH 13, pH 7.4 to pH 12.5, pH 7.4 to pH 12, pH 7.4 to pH 11.5, pH 7.4 to pH 11, pH 7.4 to pH 10.5, pH 7.4 to pH 10, pH 7.4 to pH 9.5, pH 7.4 to pH 9, pH 7.4 to pH 8.5, pH 7.5 to pH 14, pH 7.5 to pH 13.5, pH 7.5 to pH 13, pH 7.5 to pH 12.5, pH 7.5 to pH 12, pH 7.5 to pH 11.5, pH 7.5 to pH 11, pH 7.5 to pH 10.5, pH 7.5 to pH 10, pH 7.5 to pH 9.5, pH 7.5 to pH 9, pH 7.5 to pH 8.5, pH 8.0 to pH 13.0, pH 8.5 to pH 12.5, pH 9.0 to pH 13, pH 9.0 to pH 12.5, pH 9.0 to pH 12.0, pH 9.5 to pH 11.5, pH 10 to pH 13, pH 10 to pH 12.5, pH 10 to pH 12.0, pH 10 to pH 11.5, pH 10 to pH 11, pH 10 to pH 12.5, pH 10.5 to pH 12.0, pH 10.5 to pH 13, pH 10.5 to pH 12.5, pH 10.5 to pH 12.0, pH 10.5 to pH 11.5, or pH 10.5 to pH 11. In a specific embodiment, the pH of the neutralizing aqueous solution is about 12.0 to about 13, about 12.0 to about 12.6 or about 12.6.

[0099] In an embodiment, the neutralizing aqueous solution has a composition designed to avoid disrupting the buffer filled vesicles so as to not destabilize these vesicles. It may contain, in addition to water, neutralizing species (basic or acidic, such as weak bases or weak acids) but also chemical agents used to adjust the osmolarity and / or provide a physiological function. Calcium salts can be added in the preparation process to counteract the anticoagulant effects of some weak acids (e.g., citric acid). This is of particular importance if the vesicles are to be used in in vivo applications. Sodium hydroxide, sodium salts (such as sodium chloride), potassium chloride, calcium chloride, magnesium salts, lactate salts, glycerol, icodextrin, glucose, sorbitol, fructose, amino acids or xylitol can also be used as ingredients of the neutralizing aqueous solution. In specific embodiments, the neutralizing aqueous solution contains, in addition to water, xylitol, sodium chloride, sodium hydroxide, potassium chloride, and calcium chloride.

[0100] In an embodiment, the neutralizing aqueous solution has an osmolarity of between 250 mOsm / l and 550 mOsm / l, of between 270 and 520 mOsm / l, of between 290 and 500 mOsm / l, of between 300 and 480 mOsm / l, of between 320 and 450 mOsm / l, of between 330 and 420 mOsm / l, of between 350 and 400 mOsm / l, of between 375 and 400 mOsm / l, of between 380 and 420 mOsm / l, of between 385 and 400 mOsm / l or of between 390 and 400 mOsm / l.

[0101] In an embodiment, the neutralizing aqueous solution has an osmolarity which is less than 200 mOsm / l higher than or lower than the osmolarity of the mixture containing the buffer-containing vesicles (i.e., the buffercontaining vesicles solution), in particular it is less than 150 mOsm / l higher or lower, in particular it is less than 100 mOsm / l higher or lower, in particular it is less than 50 mOsm / l higher or lower, in particular it is less than 20 mOsm / l higher or lower, or in particular it is less than 10 mOsm / l higher or lower than the osmolarity of the mixture containing the buffer-containing vesicles (i.e., the buffer-containing vesicles solution). In an embodiment, the difference in osmolarity between the neutralizing aqueous solution and the mixture containing the buffercontaining vesicles is between 1 mOsm / to 200 mOsm / l, in particular between 10 mOsm / to 150 mOsm / l, in particular between 20 mOsm / to 100 mOsm / l, in particular between 30 mOsm / to 80 mOsm / l, or in particular between 40 mOsm / to 60 mOsm / l.

[0102] Due to the pH differences between the suspension buffer and the acidic buffer, a transmembrane pH-gradient between the inner part of the liposomes and the surrounding suspension buffer is achieved. The resulting transmembrane pH-gradient can be used in accordance with the present disclosure.

[0103] In an embodiment, the pH value of the suspension buffer containing the transmembrane pH-gradient vesicles is in the range of 5.5 to 8.5, of 6.0 to 8.0, of 6.3 to 7.7, of 6.3 to 7.5, of 6.3 to 7.3, of 6.3 to 7.2, of 6.3 to 7.1, of 6.5 to 7.7, of 6.5 to 7.5, of 6.5 to 7.3, of 6.5 to 7.2, of 6.5 to 7.1, of 6.8 to 7.5, or of 7.0 to 7.4. Thus, the suspension buffer may have a physiological pH value. In a specific embodiment, the pH value of the suspension buffer is about 6.5.

[0104] In a specific embodiment, the neutralizing aqueous solution is in an enclosure separate from that of the acidic and hyperosmotic buffer and the hydrated lipid blend (or liposome aqueous suspension). In a specific embodiment, the volume of the neutralizing aqueous solution in its separate enclosure is between about 500 mL and about 4000 mL or between about 600 mL and about 3000 mL or between about 700 mL and about 2000 mL or between about 800 mL and about 1000 mL or between about 975 mL and about 1115 mL. In a very specific embodiment, it is about 950 mL or a multiple thereof, (2 x or 3 x or 4 x or 5 x or more). In a specific embodiment, the weight of the neutralizing aqueous solution in its separate enclosure is between about 500 g and about 4000 g or between about 600 g and about 3000 g or between about 700 g and about 2000 g or between about 800 g and about 1000 g or between about 975 g and about 1115 g. In a very specific embodiment, it is about 960 g or a multiple thereof, (2 x or 3 x or 4 x or 5 x or more).

[0105] In a specific embodiment, the neutralizing aqueous solution is prepared as described under the Sterile xylitol alkaline solution (XAS) heading below.

[0106] Osmotic shock methods are also described in EP 3 291 797 to Leroux et al., the content of which is herein incorporated by reference.

[0107] Transmembrane pH-gradient liposomes as described herein suspended in the neutralizing aqueous solution are liposomal suspensions as described herein. In specific embodiments, such liposomal suspension has a stability (e.g., at room temperature of 20-25 °C) of at least 48 hours from the time it is prepared (i.e. end of reconstitution). In specific embodiments, the liposomal suspension as described herein is in a sterile container such as an infusion bag. In specific embodiments, the volume of the liposomal suspension in the container is between about 800 mL and about 5000 mL or between about 850 mL and about 4200 mL or between about 900 mL and about 3000 mL or between about 950 mL and about 2000 mL or between about 975 mL and about 1115 mL or between about 1050 mL and about 4200 mL or between about 1050 mL and about 3150 mL. In specific embodiment, its volume is of about 1050 mL or a multiple thereof (e.g., 2 x, 3 x, 4 x, 5 x or more). In specific embodiments, the weight of the liposomal suspension in the container is between about 800 g and about 5000 g or between about 850 g and about 4200 g or between about 900 g and about 3000 g or between about 950 g and about 2000 g or between about 975 g and about 1115 g or between about 1050 g and about 4200 g or between about 1050 g and about 3150 g. In specific embodiment, its volume is of about 1050 mL or a multiple thereof (e.g., 2 x, 3 x, 4 x, 5 x or more). In a specific embodiment, the liposomal suspension is prepared as described under the Liposome reconstitution heading below.

[0108] Alternative methods of preparing liposomes

[0109] In another embodiment, the method of preparing the liposomes (e.g., transmembrane pH-gradient liposomes) includes the film hydration method. For example, liposomes bilayer membrane components are dissolved in an organic solvent (e.g., dichloromethane: methanol), the organic solvent is then removed (e.g., by rotary evaporation) to form a dried lipid film. The dried lipid can be stored for future use (e.g., under vacuum). The dried lipid can thereafter be hydrated directly in the buffer such as the acidic buffer described above, and the external solution exchanged with a neutral solution as described above. Alternatively, the film hydration method can first be used to form a lipid film which is then hydrated in the aqueous medium as described above.

[0110] The aqueous medium loaded liposomes can thereafter be subjected to the osmotic shock step described above to load the acidic buffer therein and be subjected to the neutralization solution step described above to create the transmembrane pH-gradient liposomes suspension.

[0111] Alternatives of methods of preparing liposomes are also described in EP 2882421 to Leroux et al., the content of which is herein incorporated by reference.

[0112] Route of administration and mechanism of action

[0113] The liposomes of the present disclosure are intraperitoneally administered.

[0114] The term “intraperitoneal administration” as used herein is meant to be understood as it is commonly understood by the person skill in the art of peritoneal dialysis treatment. For practicing the disclosure, a pharmaceutically effective amount of the liposome (e.g., transmembrane pH-gradient liposome) suspension or composition of the disclosure is administered into the peritoneal cavity, e.g., by injection as a single bolus, by continuous infusion or by perfusion, e.g., by catheter, such as a catheter commonly used for paracentesis.

[0115] The liposomes within the cavity and the nearby tissues and organs take up ammonia and, the case being, at least one of LPS, hepatic and uremic toxins, including bile acids (e.g., LCA, DCA, CDCA, CA, GCA, GCDCA, TCDCA, TC, UDCA, preferably, CA, GCA, GCDCA) and reduce the concentration thereof in blood.

[0116] The liposomes within the cavity and the nearby tissues and organs will also take up the ammonia based on the pH gradient across the liposome membrane. The acidic buffer contained within the liposomes possesses a lower pH than the physiological pH in the peritoneal cavity (which is about 7.5 to 8). Hence, ammonia can diffuse through the hydrophobic liposome bilayer in its uncharged state and be then trapped in its protonated (ionized) state (e.g., ammonium) in the inner liposome compartment.

[0117] The liposome composition retains ammonia and, the case being, LPS and reduces the toxic concentration of the free compounds. The liposomes in the peritoneal cavity are then removed / extracted from the peritoneal cavity with the fluid present therein (dialysate). Intraperitoneal administration and extraction can be performed subsequently (sequentially) and / or simultaneously. Without being so limited, the dialysate can be extracted by passive drainage through a catheter by gravity or pumped out by suction via a pump such as a peristaltic pump used for infusion. The intraperitoneal treatment and system are further described hereinbelow.

[0118] Diseases

[0119] The present disclosure provides a liposomal suspension comprising transmembrane pH-gradient liposomes for use in the treatment of an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof.

[0120] Ammonia or ammonia methylated analog-associated disease or disorder, or a symptom thereof

[0121] As used herein an “ammonia or ammonia methylated analog-associated disease or disorder, or a symptom thereof” includes hyperammonemia (e.g., induced by impaired liver function or endotoxemia), hepatic encephalopathy (HE), decompensated (i.e., with at least one of ascites, HE, jaundice and variceal bleeding) or compensated liver cirrhosis, acute liver failure, acute-on-chronic liver failure, portosystemic bypass, portosystemic shunting, drug-induced hyperammonemia, inborn deficiency in hepatic ammonia metabolism (primary hyperammonemia), inborn deficiency affecting hepatic ammonia metabolism (secondary hyperammonemia), trimethylaminuria, a trimethylamine (TMA)-associated cardiovascular disease (e.g., atherosclerosis, peripheral artery disease, coronary artery disease, myocardial infarction), a TMA-associated kidney disease (e.g., renal tubulointerstitial fibrosis and dysfunction, renal insufficiency, chronic kidney disease-associated mortality), or a symptom thereof. As used herein in relation to an ammonia or its methylated analog-associated disease or disorder, the terms “a symptom thereof” include malodor (from e.g., skin and / or urine and / or expired air) and ascites.

[0122] Endotoxemia

[0123] Lipopolysaccharides (LPS) are the primary constituents of the outer cell wall of Gram-negative bacteria and are released into the environment during bacterial division or death. Given the ubiquitous presence of bacteria inside and outside the body, a significant and continuous release of LPS molecules occurs, posing particular risks when these molecules breach the epithelial barriers and enter the bloodstream of the host. Endotoxemia is defined herein as the presence of detectable levels of LPS in the blood. It can cause an acute inflammatory response by triggering the release of a vast number of inflammatory cytokines in various cell types. The inflammatory response may become deleterious if circulatory LPS becomes abundant, and lead to fever, tissue damage, sepsis (infection-derived systemic inflammation), multi-organ dysfunction, and eventually death. Endotoxemia and endotoxemia derived inflammation affect blood-brain barrier integrity and cerebral blood flow and modulate the effects of systemic ammonia on cerebral dysfunction. Endotoxemia may originate from one or more bacterial infection(s); gut dysbiosis (i.e. alterations in microbiome diversity or composition which may lead to elevated levels of intestinal LPS-containing bacteria); and increased gut permeability. Bacterial species causing endotoxemia are typically gram-negative bacteria such as Escherichia coli and Klebsiella pneumonia. Without being so limited, other gram-negative bacterial species that may be associated with endotoxemia are listed in Fux, A.C., Casonato Melo 0., Michelin, S., Swartzwelter, B. J., Neusch A., Italian! P., Himly M., (2023) Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy. Int. J. Mol. Sci. 24, 8395. Endotoxemia may thus be associated (caused by) with bacterial infections such as ascites infection or bacterial peritonitis; and may also be associated with other conditions resulting from bacterial infections such as sepsis or systemic inflammation.

[0124] Endotoxemia is also associated with (i.e. causing) hyperammonemia, inflammation (e.g., systemic inflammation), liver impairment (e.g., characterized by elevated AST and / or elevated ALT), kidney impairment (e.g., characterized by elevated creatinine and / or elevated urea), and increased cytokine secretion by macrophages (e.g., TNFa and / or IL-6). Each of these disorders are also referred to herein as “symptoms of endotoxemia”. In specific embodiments, the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure treat(s) liver impairment and / or kidney impairment resulting from the bacterial infection causing endotoxemia. Biomarkers for liver impairment include serum levels of transaminases such as AST and / or ALT higher than those of a healthy subject (e.g., normal AST: 8 to 33 U / L in humans; normal ALT : 4 to 36 U / L.in humans). Biomarkers for kidney impairment include serum levels of creatinine levels higher than those in a healthy subject (e.g., normal creatinine: 0.7 to 1.3 mg / dL (61.9 to 114.9 pimol / L) for men and 0.6 to 1.1 mg / dL (53 to 97.2 pimol / L) for women) and serum urea levels higher than those in a healthy subject (normal urea: 5 to 20 mg / dl, or 1.8 to 7.1 mmol urea per liter).

[0125] In specific embodiments, the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure treat(s) bacterial peritonitis or sepsis resulting from the bacterial infection causing endotoxemia. In specific embodiments, liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure is (are) used to treat endotoxemia caused by Escherichia coli and / or Klebsiella pneumonia. Endotoxemia associated disease or disorder

[0126] As used herein, the term “endotoxemia associated disease or disorder” of a subject being treated for endotoxemia refers to at least one of a bacterial infection (e.g., active) such as an ascites infection; a bacterial peritonitis; sepsis; endotoxemia-induced hyperammonemia; endotoxemia-induced liver impairment; and endotoxemia-induced kidney impairment. As used herein the term “endotoxemia-induced” is used to denote that the disease or disorder is mainly caused by endotoxemia or the gram-negative bacterial infection causing endotoxemia rather than from another (known or unknown, preferably known) underlying disease or disorder in the subject.

[0127] Subjects

[0128] As used herein the terms “subject” or “subject in need thereof” refer to a subject who has an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or an endotoxemia-associated disease or disorder or a symptom thereof. In specific embodiments, the subject has an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; and endotoxemia, or an endotoxemia-associated disease or disorder or a symptom thereof. In specific embodiments, the subject has a cirrhosis (decompensated or compensated) and eventually ACLF. In other specific embodiments, the subject has ascites in addition to cirrhosis and ACLF. The term “subject” refers to an animal, more specifically to a mammal or even more specifically to a human in a specific embodiment. The liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure may also be used for veterinary applications and be used for pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cattle, fishes, swine, poultry, etc.). In specific embodiments, the subject has a healthy liver and / or does not suffer from drug-induced hyperammonemia. In a specific embodiment, the subject is an adult.

[0129] As used herein the term “subject’s dry body weight” in the context of treatment of the present disclosure refers to a person's weight without any excess fluid retention, after complete removal of ascites and emptying urinary bladder.

[0130] In certain embodiments, the methods of the present disclosure encompass a step of diagnosing the subject with ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or an endotoxemia-associated disease or disorder or a symptom thereof.

[0131] Combination therapy

[0132] The present disclosure encompasses combining an intraperitoneal administration of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure with one or more other therapies for acute or chronic treatment of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof (simultaneously or sequentially depending on the nature of the additional treatment).

[0133] For example, the combination can comprise the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure with another anti-hyperammonemia agent or therapy or with a medicament or therapy used for the prevention or treatment of at least one other symptom of a disease or condition of the subject having hyperammonemia. In this context, examples of active ingredients or therapies that may be administered in combination (simultaneously or sequentially) with the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) include lactulose, rifaximin, glycerol phenylbutyrate, lactitol, a branched-chain amino acid, neomycin, metronidazole, probiotic, a glutaminase inhibitor, L-ornithine-L-aspartate, hemodialysis, peritoneal dialysis, sodium phenylbutyrate (e.g., Buphenyl®), sodium phenylacetate, sodium benzoate, a combination of sodium phenylacetate / sodium benzoate (e.g., Ammonul®, Ucephan®), glycerol phenylbutyrate (e.g., Ravicti®) or carglumic acid. The combination can also comprise the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure treat(s) as described herein with antibiotics, prebiotics and / or probiotics (such as but not limited to VSL#3 (Rivera-Flores 2020)) for treating endotoxemia. When used to treat a co-morbidity such as urinary tract infection, the at least one other therapeutic agent can be an antibiotic such as trimethoprim / sulfamethoxazole (Bactrim™, Septra™, others), fosfomycin (Monurol™), nitrofurantoin (Macrodantin™, MacroBID), cephalexin (Keflex™), ceftriaxone, a fluoroquinolone such as ciprofloxacin (Cipro™), levofloxacin and others. When used to treat an ulcer, the at least one other therapeutic agent can be an antibiotic such as amoxicillin (Amoxil™), clarithromycin (Biaxin™), metronidazole (Flagyl™), tinidazole (Tindamax™), tetracycline and levofloxacin; a proton pump inhibitor such as omeprazole (Prilosec™), lansoprazole (Prevacid™), rabeprazole (Aciphex™), esomeprazole (Nexium™) and pantoprazole (Protonix™); an acid blocker such as famotidine (Pepcid AC™), cimetidine (Tagamet HB™) and nizatidine (Axid AR™), an antacid that neutralize stomach acid; and / or cytoprotective agents such as sucralfate (Carafate) and misoprostol (Cytotec™). When used in such combination, the compounds or compositions / formulations of the present disclosure could enable the administration of a lower dose of the other drug or therapy (e.g., anti-hyperammonemia drug such as lactulose) and thereby reduce the side effects associated with such drug or therapy, such as diarrhea, nausea, bloating, and flatulence.

[0134] Treatment and prevention

[0135] As indicated above, the present disclosure encompasses the use of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) as described herein for the treatment of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof in a subject as further described herein.

[0136] The terms “treat / treating / treatment” as used herein, refers to eliciting the desired biological response, i.e., a therapeutic effect. In accordance with the disclosure herein, the therapeutic effect comprises one or more of a decrease / reduction in the frequency, duration and / or severity of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof. It may further comprise one or more of a decrease / reduction of frequency, duration and / or severity of at least one a symptom triggered by endotoxemia, and / or duration of symptom-free periods following administration of the liposomes of the present disclosure as described herein, or of a composition (e.g., suspension) comprising the liposomes of the present disclosure, alone or in combination with another agent for the treatment of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof.

[0137] The terms “prevent / preventing / prevention” as used herein, refers to eliciting the desired biological response, i.e., a prophylactic effect. In accordance with the disclosure provided herein, in some embodiments, a prophylactic effect comprises a complete or partial avoidance / inhibition of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; of endotoxemia; or of an endotoxemia-associated disease or disorder or a symptom thereof following administration of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure, alone or in combination with another agent for the prevention or treatment of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; of endotoxemia; or of an endotoxemia-associated disease or disorder or a symptom thereof.

[0138] In some embodiments, "therapeutically effective amount" or “effective amount” or "therapeutically effective dosage" of a liposome or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure results in a treatment of ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; of endotoxemia; or of an endotoxemia-associated disease or disorder or a symptom thereof in a subject in need thereof.

[0139] As used herein the term “reduce” or “reduction” in reference to the effect of the liposomal suspension or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure on the ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; on endotoxemia; or on the endotoxemia-associated disease or disorder or a symptom thereof in the subject in need thereof, refers, without being so limited, to a reduction of at least one symptom of one or these diseases or disorders such as a reduction of serum (or plasma) level of ammonia and / or serum (or plasma) level of LPS, and / or serum (or plasma) level of AST and / or serum (or plasma) level of ALT and / or serum (or plasma) level of creatinine and / or serum (or plasma) level of urea and / or serum (or plasma) level of TNFa and / or serum (or plasma) level of IL-6 and / or serum (or plasma) level of LPS and / or serum (or plasma) level of bile acid and / or inflammation level in the subject in need thereof after treatment with (i.e. after being administered) the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure as compared to the corresponding level in the subject in need thereof prior to treatment. In other specific embodiments, the comparison to determine the reduction is with a corresponding reference level of untreated subjects that suffer from the ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; from endotoxemia; or from an endotoxemia-associated disease or disorder or a symptom thereof. In specific embodiments, it refers to a reduction of at least 10% compared to the serum (or plasma) level of one or more of these biomarkers in the treated subject prior to the treatment; a reduction of at least 15%; a reduction of at least 20%; a reduction of at least 25%; a reduction of at least 30%; a reduction of at least 35%; a reduction of at least 40%; a reduction of at least 45%; a reduction of at least 50%; a reduction of at least 55%; a reduction of at least 65%; a reduction of at least 70%; a reduction of at least 75%; a reduction of at least 80%; a reduction of at least 85%; a reduction of at least 90%; or a reduction of at least 95% or more, so as to reach the corresponding level(s) found in a corresponding healthy subject.

[0140] Dose and dosing regimen

[0141] The dose of liposomal suspension as defined herein for administration to a subject in need thereof suffering from an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; from endotoxemia; or from an endotoxemia-associated disease or disorder or a symptom thereof as defined herein is, when the subject is an adult for example, preferably as provided in Table I. These doses advantageously provide an amount of transmembrane pH-gradient liposomes as defined herein (e.g., VS-01) sufficient to reduce and preferably normalize plasma ammonia (and / or reduce plasma LPS and / or reduce plasma bile acids (e.g., LCA, DCA, CDCA, CA, GCA, GCDCA, TCDCA, TC, UDCA, preferably, CA, GCA, GCDCA)) while limiting a potential increase in plasma cholesterol (from the liposome) and a plasma calcium loss.

[0142] The liposomal suspension may be administered daily for at least one, two, three, four or more days so as to reduce or normalize plasma ammonia (and / or plasma LPS and / or plasma bile acids (e.g., LCA, DCA, CDCA, CA, GCA, GCDCA, TCDCA, TC, UDCA, preferably, CA, GCA, GCDCA)). In specific embodiments, the dose is administered on consecutive days. The administration is optimally continued as needed to reduce or normalize disease-related metabolites (e.g., ammonia and / or plasma LPS and / or plasma bile acids (e.g., LCA, DCA, CDCA, CA, GCA, GCDCA, TCDCA, TC, UDCA, preferably, CA, GCA, GCDCA)).

[0143] Kits

[0144] The present disclosure also provides a kit (e.g., for preparing a liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution; or for treating an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or an endotoxemia-associated disease or disorder or a symptom thereof), the kit comprising (a) a hydration medium (e.g., acidic buffer) as described herein; or more specifically a citric acid anhydrous solution comprising citric acid anhydrous and water, preferably further comprising at least one of sodium chloride, sodium hydroxide, and magnesium chloride hexahydrate; (b) a liposome aqueous suspension (e.g., containing (optionally sterilized) liposomes in an aqueous medium (e.g., with a pH value of around 7, e.g., in the range of 6.0 to 7.5) as described herein; or more specifically a liposome aqueous suspension comprising lipids (the lipids being preferably as defined herein) and water; and (c) an aqueous solution as described herein (e.g., a neutralizing aqueous solution as described herein); or more specifically a neutralization solution as described herein comprising (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride or (vii) any combination of at least two of (i) to (v), preferably the combination comprises all of (i) to (v), wherein the citric acid anhydrous and lipids are in a ratio of (3.54 g / L) / (6.49 g / L) and wherein each of (a), (b) and (c) are in separate enclosures (e.g., wherein (c) is in an infusion bag). The kit may optionally further comprise (d) (d-i) instructions to use (a) to (c) to prevent or treat (preferably treat) an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; an endotoxemia or a symptom thereof, or an endotoxemia associated disease or disorder as described herein; (d-ii) at least one other drug for the prevention or treatment of an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia or a symptom thereof, or an endotoxemia associated disease or disorder as described herein; or (d-iii) a combination of (d-i) and (d-ii).

[0145] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] In the appended drawings:

[0147] FIG. 1 is a schematic diagram of a system for intraperitoneal administration in a subject of a liposomal suspension, according to a preferred embodiment of the present disclosure.

[0148] FIG. 2 is a schematic diagram of a system for draining the liposomal suspension from the subject and washing the peritoneal space of the subject, according to a preferred embodiment of the present disclosure.

[0149] FIG. 3 is a flow chart of a method for intraperitoneal administration in a subject of a liposomal suspension, according to a preferred embodiment of the present disclosure.

[0150] FIGs. 4A-B is a schematic representation of a clinical assay administering various doses of VS-01 to subjects (FIG. 4A) and the study design timeline thereof (FIG. 4B).

[0151] FIGs. 5A-C graphically show plasma citric acid (FIG. 5A), and cholesterol (FIG. 5B) concentration over time after administration of VS-01 to subjects at doses of 15 mL / kg; 30 mL / kg; and 45 mL / kg; and plasma ammonia (FIG.

[0152] 50) over time after single dose (Part A (the line with circle symbols)) or 4 doses (Part B (the line with square symbols)) administration of VS-01 to subjects. In FIG. 50, each graph presents an average of ammonia concentration at each time point for all subjects included in part A or included in part B.

[0153] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0154] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0155] EXAMPLE 1: Material and Methods

[0156] VS-01 Liposome formulation

[0157] Citric acid anhydrous solution (CAS)

[0158] A citrate buffer 600 mM (pH between 1.8 to 2.2, preferably 2.1, osmolality between 1000 - 1120, preferably 1050 mOsm / l) containing citric acid anhydrous (2-hydroxy-1 ,2,3-propanetricarboxylic acid; EC number: [201-069-1]) (600 mM; 115 g / L), sodium chloride (143 mM; 8.36 g / L), sodium hydroxide (97.5 mM; 3.90 g / L), and magnesium chloride hexahydrate (12 mM; 2.44 g / L) in water for injection (acidic and hyperosmotic buffer) was prepared as follows: Each solid ingredient was accurately weighed. The appropriate amount of water for injection was added and the mixture was stirred at room temperature until complete dissolution of all the salts. The acidic solution was then 0.2 pm filtered before it was filled into 50 mL-COC injection bottles. Bottles of citric acid solution (CAS) (hereinafter called bottle 1) were terminally steam sterilized in their final container utilizing the same reference conditions as the Ph. Eur. Chapter 5.1.1 (^121 °C, &15 min in all units).

[0159] In a specific embodiment, the bottle 1 contained about 34 g of the citric acid solution for a volume of about 32.2 mL. This bottle contained about 3.71 g of citric acid anhydrous, about 0.269 g of sodium chloride, about 0.125 g of sodium hydroxide, about 0.0785 g of magnesium chloride hexahydrate, and about 34 g of purified water (for injection). Liposome aqueous suspension (LAS)

[0160] A lipid blend composed of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Lipoid), cholesterol (Sigma-Aldrich™) and N-(carbonyl-methoxy-polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt of mPEG-DSPE, Lipoid) in a molar ratio of 85.5:14:0.5 (100 mg / g lipids) equivalent to a weight % ratio of 90.2:7.78:2.01, respectively was prepared by the spray drying method. 614 mg of DPPC, 53.0 mg of cholesterol and 14.0 mg of DSPE-PEG were co-dissolved in ethanol 96% (v / v) prior spray drying and heated up to 45°C under stirring until complete dissolution of the lipids. After a filtration step (using a 0.45 pm filter), the warm lipid solution was spray-dried with a high-pressure nozzle using liquid nitrogen as drying gas and a feed rate between 10 and 20 kg / hr and an outlet temperature of 55 - 65°C. Ethanol evaporated during the spraydrying process. The dried lipid blend was hydrated with ultra-pure water (aqueous medium) (lipids concentration = 100 mg / mL;144 pM / mL) while heating up to 60°C to ensure that the product was above the lipid hydration temperature (55°C) and stirred 2 hours. The hydrated blend was then degassed using a vacuum pump and finally sterilized in sealed bottles by autoclaving 20 min at 121°C. Bottles (hereinafter called bottle 2) were stored at 2-8°C protected from light. Liposomes having an average diameter between about 10 pm and 15 pm were obtained.

[0161] In a specific embodiment, the bottle 2 contained about 68.1 g of the liposomal aqueous suspension for a volume of about 68.1 mL. This bottle contained about 6.81 g of lipids (i.e. about 6.14 g of DPPC; about 0.530 g cholesterol; and about 0.137 g of mPEG-DSPE) and about 61.29 g of purified water (for injection).

[0162] Sterile xylitol alkaline solution (XAS)

[0163] A neutralization solution (21.3 mg / mL, pH between 12 and 13, preferably 12.6, 380-420 mOsm / kg or mOsm / l, preferably 420 mOsm / kg or mOsm / l) (neutralizing aqueous solution) in a suitable format (e.g., infusion bag) comprising an osmotic agent, xylitol, in an alkaline salt solution was prepared as follows. Xylitol (152 g / mol; 140 mM; 21.3 g / L), sodium chloride (58.4 g / mol; 92.0 mM; 5.38 g / L), sodium hydroxide (40.0 g / mol; 50.0 mM; 2.00 g / L), potassium chloride (74.6 g / mol; 2.00 mM; 0.149 g / L), and calcium chloride 2H2O (147 g / mol; 0.500 mM; 0.0735 g / L) were added to water for injection. Each solid ingredient was accurately weighed. The appropriate amount of water for injection was added and the mixture stirred at room temperature until complete dissolution of all the salts. The alkaline solution was then 0.2 pm filtered before it was filled into an infusion bag. The bag was terminally steam sterilized in its final container utilizing the same reference conditions as the Ph. Eur. Chapter 5.1.1 (>121 °C, >15 min in all units).

[0164] In a specific embodiment, the infusion bag comprises 960 g of XAS for a volume of about 950 mL (considering a measured density of 1.010 g / mL). This bag contains about 20.3 g xylitol, about 5.11 g sodium chloride; about 1.90 sodium hydroxide; about 0.141 g potassium chloride; about 0.0698 calcium chloride 2H2O; and about 932.48 g purified water (for injection).

[0165] Liposome reconstitution

[0166] The reconstitution was performed in sterile conditions (e.g., laminar airflow, sterile syringe, alcohol swabs used to clean surfaces where needle enters bottles or infusion bags, etc.).

[0167] Osmotic shock

[0168] The content of bottle 1 (containing 34 g of the above-described sterile citric acid anhydrous solution (CAS), i.e. one part by weight of a hyperosmolar citric acid solution) was withdrawn with a sterile syringe and injected into bottle 2 (containing 68.1 g of the above-described liposome aqueous suspension (LAS), two parts by weight of liposomes in water). Bottle 1 was gently inverted 5 times to homogenize the solution. Bottle 2 was then placed on a roller mixer with tilt motion (i.e., Cole Parmer™ RS-200D, model number 51901-22) at 60 rpm for 10 min at room temperature. Upon mixing, the difference in osmolarity between the two solutions (i.e., hyperosmotic citric acid solution versus liposomes in water) resulted in an osmotic pressure that favored the encapsulation of citric acid within the liposomes; citric acid penetrated the vesicle’s core, driven by this osmotic gradient between the inner and outer liposomal environments.

[0169] A liposome composition was thus produced containing in a solution of the citric acid anhydrous solution, the liposomes enclosing the citric acid anhydrous solution.

[0170] Neutralization

[0171] At the end of the agitation, the content of bottle 2 was withdrawn with a syringe and injected into the infusion bag containing 960 g of the XAS (neutralizing aqueous solution) via the injection port.

[0172] The mixing of the neutralizing aqueous solution with the liposome composition resulted in the formation of the liposomal suspension containing transmembrane pH-gradient liposomes, and brought the pH and osmolarity to physiological values (final VS-01 formulation).

[0173] The infusion bag was inverted 5 times to homogenize its content. The infusion bag was then placed on a roller mixer with tilt motion (i.e., Cole Parmer™ RS-200D, model number 51901-22) at 60 rpm for at least 15 min at room temperature. At the end of the mixing, a minimal volume of solution (i.e. one drop) was withdrawn from the infusion bag to measure the pH and ensure that it was within specification (pH= 6-10). The appearance of the solution in the infusion bag was milky white.

[0174] The content of the infusion bag (VS-01) is optimally administered to the subject within about 45 hours after reconstitution has occurred.

[0175] One kit of VS-01 (i.e. one bottle 1, one bottle 2 and one infusion bag) generated 1050 mL of final liposomal suspension in the resulting infusion bag which contained citric acid anhydrous (3.54 g / L) / lipids (6.49 g / L). The liposomal suspension contained about 3.71 g of citric acid anhydrous, about 3.28 g sodium, about 3.39 g chloride, about 0.00940 g magnesium; about 20.3 g xylitol; about 0.0742 g potassium; about 0.0190 g calcium; and about 6.81 g total lipids (about 6.14 g of DPPC; 0 about.530 g cholesterol; and about 0.137 g of PEG-DSPE). The number of kits to be prepared depends on the study subjects dry body weight, and is listed in Table I:

[0176] Table I. VS-01 Kits Relative to Weight and Dose

[0177] > < > < > < > <

[0178]

[0179] *For subjects having dry body weights over 140 kg, one or more additional infusion bags can be added as needed provided the peritoneal space is sufficient for the total volume infused.

[0180] Storage

[0181] VS-01 kits (bottle 1, bottle 2 and infusion bag) are advantageously stored in a secured location between 2-8 °C / 35.6-46.4 °F and taken out of the refrigerator and stored in a secured location at room temperature (not above 27°C / 86.6°F) overnight prior to a potential reconstitution on the next day and are either reconstituted, or returned to the refrigerator the next day.

[0182] Method of administration

[0183] In specific embodiments, the method of administration of the liposomal suspension including transmembrane pH- gradient liposomes of the present disclosure generally comprises four of five steps: (1) drainage of ascites, if any, from the subject; (2) administration of VS-01; (3) draining of dialysate; (4) administration of washing solution; and (5) draining of washing solution.

[0184] Drainage of ascites

[0185] Certain subjects benefiting from the method of the present disclosure have ascites (e.g., subjects suffering from decompensated cirrhosis or ACLF, presenting ascites). The method, when applied to such subjects, includes a step of draining ascites via an intra peritoneal paracentesis catheter; and optionally, a step of emptying the subject’s urinary bladder. This paracentesis step may be followed by weighing the subject to ascertain his / her dry weight, which may then be used for calculating the appropriate liposomal suspension dosage to administer. Administration of VS-01

[0186] The infusion bags are optionally prewarmed to up to 37 °C using dry heat.

[0187] In a specific embodiment, one or more infusion bags depending on the subject’s weight (see Table I above), is (are) each connected to a first tube (space line(s)). The tube, or if there is more than one tube(s), each pair of tubes is connected to (e.g., through an LS-2 connector, an LS-4 connector or a 3-way stop cock) into a single second tube (extension line such as Biegler™ extension line). This extension line (e.g., between 40 and 80 cm) is placed on (e.g., coiled around) an infusion solution warming device (e.g., Biegler™ device such as BW685) to pre-warm VS-01 to body temperature not exceeding 37°C prior to administration thereof to the subject’s peritoneal cavity. If there are two extension line(s) (i.e., if there are 3 or 4 infusion bags), they are connected together (e.g., through an LS-2 connector, an LS-4 connector or a 3-way stop cock) into a single third tube (catheter) which is inserted into the subject’s peritoneal space.

[0188] Referring now to FIGs. 1 to 3, there is shown a system and method for intraperitoneal administration in a subject of a liposomal suspension. The system includes at least one infusion pump 10 for pumping the liposomal suspension toward the subject S. In the illustrated example, there are shown four infusion pumps 10, such as those commercialized by B. BRAUN under the trade name Infusomat® Space® large volume infusion pump. Each infusion bag 12 is linked to an infusion pump 10 and all pumps are active during infusion. Optionally, a warming device 14 is connected to a fluid line 16 for warming the liposomal suspension before administering the liposomal suspension to the subject. As indicated above, the warming device may be a Biegler™ device such as BW685. A catheter 18 is fluidly connectable to the infusion bag 12 for insertion into a peritoneal space of the subject s.

[0189] Prior to connecting the extension tube(s) to the catheter, all tubes are primed with VS-01 to avoid air bubbles prior to administration thereof to the subject.

[0190] The infusion is conducted at a flow rate of about 999 mL / h to about 4000 mL / h (or more if additional VS-01 doses are required) depending on the subject’s dry weight and associated required dose.

[0191] The duration of the infusion is between about 50 minutes and about 2.5 hours, and preferably about 1 hour. The maximum of 2.5 hours is set to allow the last transmembrane pH-gradient liposomes to enter the peritoneal space for a sufficient time (i.e. about 30 minutes) to capture ammonia before the end of the dwell time.

[0192] VS-OTs dwell time in the peritoneal space from the start of infusion is about 3 hours. The estimated time to drain VS-01 from the peritoneal space using gravity is approximately 30 minutes.

[0193] A sample of peritoneal fluid was collected from the paracentesis lock at the end of the dwell time for various measurements useful for clinical trial reporting purposes. In treatment settings, this step is optional.

[0194] The time from reconstitution of VS-01 to the end of dwell time optimally does not exceed 48 hours.

[0195] Drainage of dialysate

[0196] At the end of the dwell time, the VS-01 is drained from the peritoneal space through a paracentesis catheter (i.e. that is already in place if ascites were drained prior to VS-01 infusion). The drainage is achieved using a drainage bag 20. The drainage is preferably achieved by gravity. The drainage flow may be accelerated by means of an aspiration device or syringe.

[0197] Administration of washing solution

[0198] The washing solution infusion bag 24 (e.g., containing 1 L of washing solution) is optionally pre-warmed to body temperature not exceeding 37°C (e.g., using a warming plate or warming box (e.g., Biegler™ BW685)) and connected to a first tube (extension line) and connected to a paracentesis catheter 3-way stopcock 22. A syringe 26 (e.g., 50 ml) is connected to the paracentesis catheter 3-way stopcock and the flow of the washing solution is opened. The syringe 26 is used to support inflow of the washing solution into the subject’s peritoneal cavity while opening and closing the 3-way-stop cock 22 by turning its stopcock as applicable. The warming plate typically used in Europe is preferably the Vaba MediTemp™ warming plate. The warming box is typically used in the United States. The 3-way-stopcock 22 has a first inlet fluidly connectable to the catheter 18, a second inlet fluidly connectable to the washing solution infusion bag 24, and a third inlet connectable to the syringe 26 for injecting the washing solution in the peritoneal space of the subject.

[0199] In specific embodiments, the washing solution may be infused within about 20 min (±10 min).

[0200] The washing solution may be any solution suitable for i.p. rinse as per local standard, for example any peritoneal dialysis solution. Without being so limited, it may be DIANEAL® Low Calcium (2.5 mEq / L) 1.5% Dextrose; or Physioneal® 40 Glucose 1.36 % w / v / 13.6 mg / mL

[0201] At the end of the washing step, a sample (e.g., 50 mL) of the washing solution is collected from the catheter lock.

[0202] Drainage of the washing solution

[0203] Immediately after infusion of the washing solution, i.e. without dwell time, the washing solution is drained out of the subject’s peritoneal space by connecting it to the paracentesis lock until complete drainage into another drainage bag 20. The drainage is preferably achieved by gravity. The drainage flow may be accelerated by means of an aspiration device or syringe.

[0204] EXAMPLE 2: Administration of escalating doses of VS-01 to human subjects suffering from decompensated liver cirrhosis

[0205] Various doses (study part A) and multiple doses (study part B) of VS-01 were administered to 12 cirrhotic patients with ascites and covert hepatic encephalopathy (HE) on top of standard of care.

[0206] Within the context of this trial, standard of care was defined by EASL1 and German (DGVS) guidelines, according to their eligibility criteria:

[0207] 1. “Adult cirrhotic patients presenting with ascites with clinical decision for therapeutic paracentesis up to 8 liters as standard of care” met the definition of refractory ascites (diuretic resistance). Standard of care was large-volume paracentesis plus intravenous albumin (6 - 8 g / l ascites) when more than 5 liters of ascites were removed.

[0208] 2. Covert hepatic encephalopathy (HE) (West Haven Grade: Minimal or I): Treatment of minimal HE and covert HE was not routinely recommended apart from a case-by-case basis. Patients with past episodes of overt HE could have been treated for secondary prophylaxis of HE: lactulose was first choice and rifaximin was only used in cases of intolerance of lactulose.

[0209] Patients in this study followed the recommended dietary measures for the ascites and HE. Both lactulose and rifaximin act locally in the gastrointestinal tract and are practically not absorbed. Their side effects are mainly gastrointestinal and of mild severity. Since VS-01 was administered intra-peritoneally and may have induced local symptoms, it may be challenging to know with a high level of certainty the degree of relatedness of mild gastrointestinal symptoms.

[0210] The reported interactions of lactulose and rifaximin with other medicinal products are only in the inner gastrointestinal compartment (antacids, other laxatives or anti-infective agents acting on colonic bacteria). Since VS-01 was administered intra-peritoneally and was not expected to circulate in the gut, no significant drug interactions were anticipated with lactulose or rifaximin.

[0211] Clinical study parts A and B are schematized in FIGs. 4A-B.

[0212] On study Day 0, a therapeutic paracentesis via an i.p. catheter was performed and followed by plasma volume expansion by infusing albumin (8 g / L of ascites removed) according to the EASL guidelines 2018 and as Part A patient’s standard of care for their ascites. The i.p. catheter was left open overnight.

[0213] Part A

[0214] On study Day 1, VS-01 was infused into the three cohort subjects in the peritoneal space for over 60 minutes using the same paracentesis catheter already in place. At the end of a 2-hour dwell time, the peritoneal fluid was drained out (similarly to paracentesis). A washing step was performed by instilling 15 mL / kg of a commercial peritoneal dialysis fluid (Physioneal® 35 Glucose 1.36 % w / v / 13.6 mg / mL) which was immediately drained out under similar conditions.

[0215] VS-01 was delivered i.p. in Cohort 1 at a volume of 15 mL / kg of dry body weight (3.54 mg / mL citric acid / 6.49 mg / mL lipids); the corresponding dose was 53.1 mg / kg citric acid / 97.4 mg / kg lipids. VS-01 doses were increased to 30 mL / kg and 45 mL / kg i.p., in Cohorts 2 and 3, respectively. The corresponding doses were 106 mg / kg citric acid / 195 mg / kg lipids (Cohort 2) and 159 mg / kg citric acid / 292 mg / kg lipids (Cohort 3). For a 70 kg patient, a volume of 1.05, 2.1 and 3.15 L of VS-01 was infused i.p. in patients in Cohorts 1 , 2 and 3, respectively; this volume was not higher than the drained ascitic fluid during the paracentesis procedure.

[0216] The PK parameters of citric acid, 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol were evaluated in plasma using a validated liquid chromatography - mass spectrometry / mass spectrometry (LC-MS / MS) method.

[0217] Part B

[0218] The part B cohort included 3 evaluable patients (N=3) at a dose of VS-01 within the range 30-42 mL / kg VS-01, namely 1 patient at 41.8 mL / kg with 2 VS-01 kits; 1 patient at 33.6 mL / kg with 3 VS-01 kits; and one patient at 37.3 mL / kg with 3 VS-01 kits.

[0219] The cohort received single daily doses of VS-01 administered over 4 days. VS-01 was infused in the peritoneal space for over 60 minutes using the same paracentesis catheter already in place. The dwell time was 3 hours on Days 1 and 4 and 2 hours on Days 2 and 3. After the dwell time, VS-01 was drained via the paracentesis catheter and a washing step administering 15 m L / kg of a commercial peritoneal dialysis solution (Physioneal® 35 Glucose 1.36 % w / v / 13.6 mg / mL) intraperitoneally, immediately followed by drainage.

[0220] In Parts A and B, ammonia concentrations were measured in plasma and in the peritoneal fluid on a Cobas™ 6000 using NH3L2 Ammonia Assay from Roche Diagnostics. The PK / PD data sets included all patients who received at least a single dose of VS-01 with at least three PK measurements post-dose and had no major protocol deviations. Baseline (BL) - corrected PK and PD parameters were calculated with WinNonLin™ (Version 8.1 or higher) using a non-compartmental analysis or SAS (Version 9.3 or higher).

[0221] Results

[0222] VS-01 was generally well tolerated and showed a favorable safety profile. No serious adverse events were reported during the study period. Most treatment emergent adverse events were mild in severity and short in duration. None of the patients discontinued the study nor VS-01 administration due to adverse events. Preliminary efficacy data showed an improvement in overall liver disease severity as measured by the Child-Pugh and Model of end-stage liver disease scores (data not shown), cognitive tests for HE (data not shown), median plasma ammonia levels, and further, in the majority of patients following VS-01 administration.

[0223] Different metabolites known to be associated with infections and with organ failure, respectively, were identified in plasma, and were shown to be reduced following VS-01 treatment (data not shown). This dynamic was more pronounced after repeated VS-01 administrations in study Part B (data not shown).

[0224] More particularly, following single dose i.p. instillation of VS-01, citric acid, DPPC and cholesterol exposure during the dwell period was higher in peritoneal fluid than in plasma over the dose range investigated.

[0225] Plasma citric acid exposure increased in a dose-proportional manner across the single dose administration over the 15-45 mL / kg dose range (FIG. 5A). Plasma cholesterol exposure returned to base line 6 to 12 hours after VS-01 in the 15 mL / kg and 30 mL / kg single doses but not in the 45 mL / kg single dose where it remained high after 12 hours.

[0226] Patients who received the 45 mL / kg dose also displayed a drop in plasma calcium after Day treatment compared to prior to treatment (data not shown).

[0227] Following single dose administration plasma ammonia Cmin was observed within approximately 1 h of the start of i.p. administration of VS 01 while peritoneal fluid ammonia Cmax was observed within approximately 2 h of the start of i.p. administration of VS 01 , occurring at or near the end of the 2 h dwell period.

[0228] On Dosing Day 1 of the multiple dosing period, plasma ammonia Cmin was observed at approximately 3 h poststart of i.p. administration of VS 01 at or near the end of the dwell period. On Dosing Day 4, plasma ammonia Cmin was observed at approximately 12 h post-start of i.p. administration of VS 01.

[0229] After three hours, the transmembrane pH gradient is anticipated to decrease, in turn lowering capture efficiency while increasing the subject's exposure to lipids including cholesterol. On both Dosing Day 1 and Dosing Day 4, peritoneal fluid ammonia Cmax was observed at approximately 3 h poststart of i.p. administration of VS-01 , at or near the end of the dwell period.

[0230] Plasma ammonia levels gradually decreased after repeated VS-01 treatment and normalized at the end of the study in Part B (FIG. 50).

[0231] EXAMPLE 3: Administration of VS-01 to human subjects suffering from ACLF with ascites

[0232] A multi-center, randomized, controlled, open-label, study of VS-01 in adult patients with ACLF grades 1 and 2 and ascites is conducted to assess the efficacy, safety and tolerability of VS-01 on top of standard of care, compared to standard of care alone. Approximately 60 patients are enrolled. Sample size was calculated to meet the study objectives assuming a 10% drop-out rate.

[0233] Patients with ACLF grade 1 or 2 (according to European Association for the Study of the Liver [EASL]-CLIF criteria as described in the EASL-Clinical Practice Guideline on acute-on-chronic liver failure [EASL Clinical Practice Guidelines, 2023]); organ failures are calculated based on the CLIF-C OF score), and ascites requiring paracentesis are enrolled in the study.

[0234] Patients are admitted to the ward, ICU or other units that have the ability to perform the required patient monitoring. Patients are randomized in a 1:1 ratio to receive either VS-01 on top of standard of care (SOC) (Active Treatment group) or SOC alone (Control group). Randomization is stratified by country and CLIF-C ACLF score (<48 vs. >48) at baseline (BL, i.e., prior to dosing on Day 1). Randomization is centralized and occurs on Day 1. Patients are screened 1 day (24 hours) and up to 4 days (96 hours) prior to BL on Day 1. After randomization, patients receive the allocated treatment and are followed up to Day 90. The patients of Active Treatment group and Control group receive SOC throughout the study and continue to receive SOC as required after End of Treatment (EOT) on Day 4.

[0235] SOC is the standard medical management of patients with decompensated cirrhosis and ACLF as per respective guidelines (EASL Clinical Practice Guidelines, 2023; EASL Clinical Practice Guidelines, 2018; EASL Practice Guideline, 2014; Acute-on-Chronic Liver Failure Clinical Guidelines, 2022; AASLD Practice Guidance, 2021). SOC shall be given considering the overall clinical presentation, precipitating events, and the nature and severity of organ dysfunctions and failures. SOC may include but is not limited to intravenous ( / .v.) fluid therapy, i.v. albumin therapy, antibiotics, nutrition, thromboprophylaxis, the use of terlipressin or norepinephrine for the treatment of HRS-AKI where indicated, laxatives and non-absorbable antibiotics for HE, oxygen for respiratory support, and renal replacement therapy for renal failure. SOC is administered after each treatment session with VS-01, if applicable.

[0236] In all patients (both Active Treatment group and Control group), ascites drainage (therapeutic or diagnostic) via an i.p. paracentesis catheter is performed on Day 0 and followed by plasma volume expansion by infusing albumin (8 g / liter [L] of ascites removed) if large volume paracentesis (LVP) is performed (> 5 L of ascites) (EASL Clinical Practice Guidelines, 2018; AASLD guideline on the treatment of ascites, spontaneous bacterial peritonitis and HRS-AKI [AASLD Practice Guidance, 2021]). Albumin following paracentesis can be administered also in case of < 5 L of ascites are being removed, if this is in line with local SOC. The catheter will be removed for the Control group on Day 1 after randomization. Patients allocated to the Control group are only treated with SOC throughout the study.

[0237] Active Treatment group: Patients receive the VS-01 i.p. infusion over a dwell period of 3 h on study daily over 4 consecutive days on top of SOC. At the end of each VS-01 dosing, VS-01 fluid is removed similarly to paracentesis (duration of about 30 min) and a washing step is performed by infusing 1 L of any solution suitable for i.p. rinse as per local standard. Patients also receive SOC considering respective guidelines (EASL Clinical Practice Guideline on ACLF [EASL Clinical Practice Guidelines, 2023], EASL Clinical Practice Guideline on decompensated cirrhosis [EASL Clinical Practice Guidelines, 2018], the common guideline of the American Association for the Study of Liver Diseases [AASLD] and EASL on HE in chronic liver disease [EASL Practice Guideline, 2014], the American College Gastroenterology [ACG] Guideline on ACLF [Acute-on-Chronic Liver Failure Clinical Guidelines, 2022], and the AASLD guideline on the treatment of ascites, spontaneous bacterial peritonitis and hepatorenal syndrome - acute kidney injury [HRS-AKI], respectively [AASLD Practice Guidance, 2021]). SOC is administered after each treatment session with VS-01, if applicable.

[0238] VS-01 is presented as a kit of three preparations which is to be mixed extemporaneously to give 1 L (per kit) by the pharmacist under aseptic conditions and according to the Pharmacy Manual.

[0239] VS-01 (3.54 mg / mL citric acid / 6.49 mg / mL lipids) is delivered i.p:, the volume infused and corresponding citric acid / li pid dose per dry body weight is shown in T able I above.

[0240] Control group: Patients allocated to the Control group are treated with SOC considering respective guidelines (EASL Clinical Practice Guidelines, 2023, EASL Clinical Practice Guidelines, 2018, EASL Practice Guideline, 2014, Acute-on-Chronic Liver Failure Clinical Guidelines, 2022, AASLD Practice Guidance, 2021).

[0241] The scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

Claims

33CLAIMS:

1. Liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the suspension comprising citric acid anhydrous and lipids in a ratio of (3.54 g / L) / (6.49 g / L).

2. The liposomal suspension of claim 1, for administration to a subject (a) in a daily dose of citric acid anhydrous / lipids of about 71 / 130 mg / kg to about 142 / 260 mg / kg based on the subject’s (dry) body weight, preferably in a daily dose of citric acid anhydrous / lipids of (a-i) about 71 / 130 mg / kg to about 92.9 / 173 mg / kg for a subject having a weight between 40 kg and < 53kg; (a-ii) about 96 / 175 mg / kg to about 142 / 260 mg / kg for a subject having a weight 53 Kg and < 79 kg; and (a-iii) about 106 / 195 mg / kg to about 142 / 260 mg / kg for a subject having a weight 79 Kg and < 140 kg; (b) wherein administration comprises one daily dose or at least two daily doses, or at least three daily doses or four daily doses; or (c) any combination of (a) and (b).

3. The liposomal suspension of claim 2, wherein (a’) the administration comprises one, two or three daily doses; (b’) the doses are administered on consecutive days; or (o’) a combination of (a’) and (b’).

4. The liposomal suspension of any one of claims 1 to 3, wherein (A) the lipids comprise (i) at least one phospholipid as main constituent, wherein the at least one phospholipid preferably comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%; (ii) cholesterol, preferably in a range of 10 to 40 mol%; (iii) 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%; or (iv) any combination of at least two of (i) to (iii), preferably the combination comprise (i) to (iii), and most preferably the lipids contain dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG) at 85.5:14:0.5 mol%; (B) the aqueous solution contains, in addition to water, (i) xylitol, (ii) sodium, (iii) chloride, (iv) magnesium; (v) potassium, (vi) calcium; (vii) citric acid anhydrous, or (viii) any combination of at least two of (i) to (vii), preferably the combination comprises, two of, three of, four of, five of, six of, 7 of or all of (i) to (vii); (C) the liposomes have an average diameter between about 8 pm and 12 pm; or (D) any combination of at least two of (A) to (C).

5. Kit for preparing a liposomal suspension comprising transmembrane pH-gradient liposomes in an aqueous solution, the kit comprising (a) a citric acid anhydrous solution comprising citric acid anhydrous and water; (b) a liposome aqueous suspension comprising lipids and water; and (c) a neutralizing aqueous solution comprising water and (i) at least one sugar alcohol; (ii) at least one salt; or (iii) a combination of (i) and (ii); wherein the citric acid anhydrous and lipids are in a ratio of (3.54 g / L) / (6.49 g / L) and wherein each of (a), (b) and (c) are in separate enclosures.

6. The kit of claim 5, wherein (a) the citric acid anhydrous solution further comprises at least one of sodium chloride, sodium hydroxide, and magnesium chloride hexahydrate, preferably at least two thereof and most preferably all three thereof; (b) the lipids comprise (i) at least one phospholipid as main constituent, wherein the at least one phospholipid preferably comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%; (ii) cholesterol, preferably in a range of 10 to 40 mol%; (iii) 1 ,2-distearoyl-sn-glycero-3-34phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%; or (iv) any combination of at least two of (i) to (iii), preferably the combination comprise (i) to (iii), and most preferably the lipids contain dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG) at 85.5:14:0.5 mol%; (c) the neutralizing aqueous solution comprises (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride; or (vi) any combination of at least two of (i) to (v), preferably the combination comprises two of, or three of, or four of or all five of (i) to (v); (d) (i) the volume of the citric acid anhydrous solution is between about 20 mL and about 40 mL, preferably about 32.2 mL; (ii) the volume of the liposome aqueous suspension is between about 55 mL and about 75 mL, preferably about 68.1 mL; (iii) the volume of the neutralizing aqueous solution is between about 900 mL and about 1000 mL, preferably about 950 mL; (iv) the combined volume of the citric acid anhydrous solution, the liposome aqueous suspension and the neutralizing aqueous solution is between about 975 mL and about 1115 mL, preferably about 1050 mL; or (v) a combination of at least two of (i) to (iv); (e) (i) the enclosure of the citric acid anhydrous solution is a bottle; (ii) the enclosure of the liposome aqueous suspension is a bottle; (iii) the enclosure of the neutralizing aqueous solution is a bag, such as an infusion bag; or (iv) a combination of at least two of (i) to (iii); or (f) a combination of at least two of (a) to (e).

7. The liposomal suspension of any one of claims 1 to 4 or the kit of claim 5 or 6, which is for use in the treatment of an ammonia- or ammonia methylated analog-associated disease or disorder, or a symptom thereof; endotoxemia; or endotoxemia-associated disease or disorder or a symptom thereof, in a subject in need thereof, preferably a human, wherein the treatment preferably includes peritoneal administration of the liposomal suspension to the subject in need thereof.

8. The liposomal suspension or kit for its use of claim 7, wherein the ammonia- or ammonia methylated analog-associated disease or disorder is cirrhosis with ascites or acute-on-chronic liver failure with ascites.

9. System for intraperitoneal administration in a subject of the liposomal suspension defined in any one of claims 1 to 5, comprising:an infusion pump (10) for pumping the liposomal suspension contained in an infusion bag (12) via a fluid line (16);a warming device (14) connectable to the fluid line (16) for warming the liposomal suspension before administering the liposomal suspension to the subject; anda catheter (18) fluidly connectable to the infusion bag (12) for insertion into a peritoneal space of the subject.

10. The system of claim 9, comprising one, two, three or four infusion pumps (10) each respectively being in fluid communication with one, two, three or four infusion bags (12), each infusion bag preferably containing about 1050 mL of the liposomal suspension the infusion pumps (10) being selectively activated depending on the dry body weight of the subject, wherein a flow rate of each pump is preferably set at 1000 mL / hour, and wherein atdwell time of the liposomal suspension in the peritoneal space of the subject preferably lasts for about 3 hours before being drained.

11. The system of claim 9 or 10, further comprising a drainage bag (20) for removing the liposomal suspension from the peritoneal space of the subject.

12. The system of claim 11, further comprising:a washing solution infusion bag (24) containing a washing solution of peritoneal dialysis solution that is optionally warmed before being injected in the peritoneal space of the subject and preferably contains about 1 L of the washing solution; anda 3-way-stop cock (22) having a first inlet fluidly connectable to the catheter (18), a second inlet fluidly connectable to the washing solution infusion bag (24), and a third inlet connectable to a syringe (26) for injecting the washing solution in the peritoneal space of the subject.

13. The system of claim 12, further comprising another drainage bag (20) for removing the washing solution in the peritoneal space of the subject.

Images