Treating endotoxemia with a liposomal suspension

Abstract

The present disclosure provides a liposomal suspension, for use in the treatment of endotoxemia in a subject in need thereof, wherein the treatment comprises intraperitoneally administering the liposomal suspension to the subject and removing a dialysate containing liposomes from the subject.

Description

[0001] METHOD OF TREATING ENDOTOXEMIA

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure is concerned with the treatment of endotoxemia with liposomes.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Bacterial infections can result in endotoxemia, a condition that occurs when lipopolysaccharides (LPSs) released upon the death of bacteria traverse the gastrointestinal barrier and enter the bloodstream which may lead to systemic inflammation. LPS, consisting of a lipid moiety and a polysaccharide moiety, is a component of the cell membrane of Gram-negative bacteria. A significant load of this endotoxin induces an excessive and uncontrolled systemic inflammatory response and lead to sepsis. More particularly, endotoxin-induced systemic inflammation can result in organ (e.g., liver, kidney) dysfunction, organ failure and death, especially in individuals with pre-existing compromised organs. For instance, systemic inflammation is a primary driver of acute-on-chronic liver failure (ACLF), a severe syndrome affecting patients with acutely decompensated cirrhosis. The most common infection causing sepsis- induced ACLF is spontaneous bacterial peritonitis, which is often caused by Gram-negative bacteria that have translocated from the intestinal lumen to ascitic fluid via the systemic circulation.

[0006] There is a need for treating endotoxemia subjects in need thereof.

[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 vitro and in vivo studies showing that liposomes were able to counteract LPS- induced inflammatory response and hepatic / renal damage.

[0010] More specifically, in accordance with the present disclosure, there are provided the following items:

[0011] Item 1. Liposomal suspension, for use in the treatment of endotoxemia in a subject in need thereof, wherein the treatment comprises intraperitoneally administering the liposomal suspension to the subject and removing a dialysate containing liposomes from the subject. In a specific embodiment, the liposomal suspension comprises transmembrane pH gradient liposomes.

[0012] Item 2. The liposomal suspension for use of item 1 , wherein the subject has an endotoxemia associated disease or disorder.

[0013] Item 3. The liposomal suspension for use of item 2, wherein the endotoxemia associated disease or disorder comprises a bacterial infection caused by Escherichia coli.

[0014] Item 4. The liposomal suspension for use of item 2 or 3, wherein the endotoxemia associated disease or disorder comprises liver impairment and / or kidney impairment.

[0015] Item 5. The liposomal suspension for use of any one of items 1 to 4, wherein the liposomes contain a hydroxy acid, preferably citric acid, most preferably citric acid anhydrous.

[0016] Item 6. The liposomal suspension for use of item 5, wherein the liposomes contain about 200 nM citric acid anhydrous.

[0017] Item 7. The liposomal suspension for use of any one of items 1 to 6, wherein the liposomes’ lipid bilayer comprises at least one phospholipid as main constituent.

[0018] Item 8. The liposomal suspension for use of item 7, wherein the at least one phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%.

[0019] Item 9. The liposomal suspension for use of any one of items 1 to 8, wherein the liposomes’ lipid bilayer comprises cholesterol, preferably in a range of 10 to 40 mol%.

[0020] Item 10. The liposomal suspension for use of item 9, wherein the liposomes’ lipid bilayer further comprises 1 ,2- distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%.

[0021] Item 1 1. The liposomal suspension for use of any one of items 1 to 6, wherein the liposomes’ bilayer contains 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%, and the liposomes’ inner compartment contains citric acid anhydrous.

[0022] Item 12. The liposomal suspension for use of any one of items 1 to 11 , wherein the liposomes have an average diameter between about 8 pm and 12 pm (e.g., average diameter between about 10 pm and 15 pm).

[0023] Item 13. The liposomal suspension for use of any one of items 1 to 12, wherein the liposomal suspension contains (I) xylitol, (II) sodium chloride, (ill) 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).

[0024] Item 14. The liposomal suspension for use of any one of items 1 to 13, wherein the subject is human.

[0025] DEFINITIONS

[0026] 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.

[0027] 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. 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.

[0028] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0029] 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.

[0030] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0031] Herein, the term "about" has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.

[0032] 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.

[0033] Liposomes

[0034] Liposomes according to the present disclosure comprise a lipid bilayer membrane.

[0035] Liposomes composition

[0036] 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) suspended in an aqueous solution (e.g., neutralizing aqueous solution) of the present disclosure.

[0037] Lipid bilayer membrane

[0038] 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.

[0039] 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 (DMPA); 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.

[0040] 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%, 1 1 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.

[0041] 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.

[0042] 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.

[0043] In other embodiments, the liposome lipid bilayer membrane comprises 30 to 100 mol%, more preferably 40 to 95 mol%, 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.

[0044] 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, or 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.

[0045] Hydration medium

[0046] 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.

[0047] Acidic buffer / acidic solution

[0048] 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.

[0049] 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 (carboxy lie 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).

[0050] In specific embodiments, the concentration of acid used in a liposome preparation 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 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 mOsmol / kg.

[0051] 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 in the core of the liposomes.

[0052] 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.

[0053] In alternative embodiments, liposomes for use in the present disclosure are as described in EP 2 882 421 to Leroux et al., the content of which is herein incorporated by reference.

[0054] Composition

[0055] 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 (e.g., 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). Method of preparing liposomes

[0056] Osmotic shock method

[0057] 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.

[0058] 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.

[0059] The resulting dried lipid blend can then be hydrated in the aqueous medium as further described below.

[0060] Aqueous medium

[0061] 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).

[0062] 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 of an 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.

[0063] 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 acidic or basic (preferably acidic) hyperosmotic buffer provoking the osmotic shock, which difference is large enough to induce the diffusion of the acidic or basic (preferably acidic) hyperosmotic buffer into the vesicle internal compartment.

[0064] 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.

[0065] 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 0 mOsm / l and 45 mOsm / l, or between 0 mOsm / l and 40 mOsm / l; in particular between 0 mOsm / l and 35 mOsm / l; more particularly between 0 mOsm / l and 30 mOsm / l, or between 0 mOsm / l and 25 mOsm / l.

[0066] In specific embodiments, the liposomes can optionally be extruded or filtered to obtain liposomes having a specific size.

[0067] 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 of 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.

[0068] 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 about 15 minutes, or about 30 minutes.

[0069] 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 (e.g., 1 , 2, 3 or 4 days), one week, several weeks (e.g., 1 , 2, 3 or 4 weeks), one month or even several months (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 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.

[0070] Acidic and hyperosmotic buffer

[0071] 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

[0072] 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.).

[0073] 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.

[0074] 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.).

[0075] 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 .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.

[0076] In specific embodiments, the hyperosmotic buffer can contain additional chemical agents such as a complexing agent or chelating agent.

[0077] In specific embodiments, the hyperosmotic buffer comprises salts such as but not limited to sodium chloride, sodium hydroxide, and / or magnesium chloride.

[0078] In specific embodiments where a sterile transmembrane pH gradient liposome is preferred, the acidic buffer is sterilized. In such embodiments, where the hydrated liposomes had 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.

[0079] 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.).

[0080] 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 (i.e., around 20-25 °C).

[0081] Neutralizing aqueous solution

[0082] 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 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.

[0083] In an embodiment, the pH value of the neutralizing 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

[0084] 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 1 1.5, pH 7.2 to pH 1 1 , pH 7.2 to pH 10.5, pH 7.2 to pH 10, pH 7.2 to pH

[0085] 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 1 1.5, pH 7.3 to pH 1 1 , 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 1 1 , 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 1 1.5, pH 7.5 to pH

[0086] 1 1 , 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

[0087] 12.5, pH 10 to pH 12.0, pH 10 to pH 1 1.5, 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 1 1.5, or pH 10.5 to pH 1 1. In a specific embodiment, the pH of the neutralizing solution is about 12.5.

[0088] In an embodiment, the neutralizing solution has a composition designed to avoid disrupting the buffer filled vesicles so as to not destabilize these vesicles. It may contain 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 solution. In specific embodiments, the neutralizing solution contains xylitol, sodium chloride, sodium hydroxide, potassium chloride, and calcium chloride.

[0089] In an embodiment, the neutralizing 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 385 and 400 mOsm / l or of between 390 and 400 mOsm / l.

[0090] In an embodiment, the neutralizing solution has an osmolarity which is less than 200 mOsm / l higher or lower than the osmolarity of the mixture containing the buffer-containing vesicles (i.e., the buffer-containing vesicles solution), in particular less than 150 mOsm / l higher or lower, in particular less than 100 mOsm / l higher or lower, in particular less than 50 mOsm / l higher or lower, in particular less than 20 mOsm / l higher or lower, or in particular less than 10 mOsm / l higher or lower. In an embodiment, the difference in osmolarity between the neutralizing solution and the mixture containing the buffer-containing 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 particular between 40 mOsm / to 60 mOsm / l.

[0091] 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.

[0092] 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.

[0093] Osmotic shock methods are also described in EP 3 291 797 to Leroux et al., the content of which is herein incorporated by reference.

[0094] Alternative methods of preparing liposomes

[0095] 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.

[0096] 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.

[0097] Alternatives of methods of preparing liposomes are also described in EP 2 882 421 to Leroux et al., the content of which is herein incorporated by reference.

[0098] Route of administration and mechanism of action

[0099] The liposomes of the present disclosure are intraperitoneally administered.

[0100] 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.

[0101] The liposomes within the cavity and the nearby tissues and organs take up LPS and reduce the concentration thereof in blood.

[0102] 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.

[0103] The liposome composition retains ammonia and 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.

[0104] Diseases

[0105] The present disclosure provides liposomes (e.g., transmembrane pH-gradient liposomes) for use in treating endotoxemia or a symptom thereof. It is further for use in the treatment of one or more endotoxemia associated disease(s) or disorder(s), as defined herein below, in a subject suffering from endotoxemia. In specific embodiments, it is for further use in the treatment of one or more pre-existing disease(s) or disorder(s) as defined herein below.

[0106] Endotoxemia

[0107] 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 of a bacterial infection; 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. The origins of these bacterial infections are multiple and in the context of liver and / or kidney impairments (e.g., cirrhosis and ACLF), encompass, without being so limited, infections arising from pathologically accumulated intraperitoneal fluids (ascites), and from bacterial peritonitis. Bacterial peritonitis is an infection of the peritoneum which may result from e.g., hygiene defects prior to, during or after peritoneal dialysis (e.g., medical devices contamination such as catheter contamination); ascites in subjects having liver or kidney impairments; or a rupture of organ in the abdomen. Endotoxemia may arise in the context of various other infections such as gastrointestinal tract infection, metritis, pneumonia, urinary infections, etc. 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 et al., 2023. 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.

[0108] 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”.

[0109] 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 1 14.9 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).

[0110] 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.

[0111] 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 or Klebsiella pneumonia.

[0112] Endotoxemia associated disease or disorder

[0113] 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.

[0114] Pre-existing disease or disorder

[0115] As used herein, the term “pre-existing disease or disorder” refers to any disease or disorder that the subject has prior to the onset of endotoxemia or before receiving a diagnosis of endotoxemia. For example, the subject has a preexisting disease or disorder if she / he received a diagnosis of another disease or disorder at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, etc. prior to receiving a diagnosis of endotoxemia. In specific embodiments, the pre-existing disease or disorder is pre-existing ascites, pre-existing hyperammonemia, preexisting liver impairment or pre-existing kidney impairment. In a specific embodiment, the pre-existing liver impairment is compensated or decompensated cirrhosis. Preferably, the pre-existing liver impairment is decompensated cirrhosis. In another specific embodiment, the pre-existing liver impairment is acute-on-chronic liver failure (ACLF). In another specific embodiment, the pre-existing kidney impairment is kidney failure.

[0116] Subjects

[0117] As used herein the terms “subject” or “subject in need thereof’ refer to a subject who has endotoxemia. In specific embodiments, the subject further has an endotoxemia associated disease or disorder (e.g., hyperammonemia, liver impairment and / or kidney impairment). In specific embodiments, the subject further has a pre-existing disease or disorder. In other specific embodiments, the subject does not have a pre-existing disease or disorder. In other specific embodiments, the subject has pre-existing ascites. In other specific embodiments, the subject has pre-existing hyperammonemia. In other specific embodiments, the subject has pre-existing liver impairment (e.g., cirrhosis (compensated or decompensated) or ACLF). In other specific embodiments, the subject has pre-existing kidney impairment. In other specific embodiments, the subject has two of, or three of or all four of pre-existing ascites, preexisting hyperammonemia, pre-existing liver impairment and pre-existing kidney impairment. In other specific embodiments, the subject does not have pre-existing ascites. In other specific embodiments, the subject does not have pre-existing hyperammonemia. In other specific embodiments, the subject does not have pre-existing liver impairment (e.g., cirrhosis or ACLF). In other specific embodiments, the subject does not have pre-existing kidney impairment. In other specific embodiments, the subject does not have two of, or three of or all four of pre-existing ascites, pre-existing hyperammonemia, pre-existing liver impairment and pre-existing kidney impairment. The term subject refers to an animal, to a mammal or 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.

[0118] In certain embodiments, the methods of the present disclosure encompass a step of diagnosing the subject.

[0119] Combination therapy

[0120] 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 endotoxemia, endotoxemia associated disease or disorder or pre-existing diseases or disorders (simultaneously or sequentially depending on the nature of the additional treatment).

[0121] 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 (e.g., VS-01 ) with at least one of antibiotics, prebiotics and / or probiotics for treating the endotoxemia.

[0122] Also, the combination therapy can include a liposome or composition or suspension thereof (e.g., transmembrane pH- gradient liposomes or composition or suspension thereof) of the present disclosure combined with at least one other drug or therapy used for the prevention or treatment of an endotoxemia associated disease or disorder such as hyperammonemia (e.g., resulting from the bacterial infection) or a pre-existing disease or disorder such as a cirrhosis or ACLF. In this context, examples of therapeutic agents 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) of the present disclosure include another liposomes or composition or suspension thereof of the present disclosure and / or at least one other therapeutic agent or therapy. When used to treat hyperammonemia, the at least one other therapeutic agent or therapy can be at least one of nonabsorbable disaccharides such as lactulose or lactilol, rifaximin, a branched-chain amino acid, neomycin, metronidazole, probiotic (such as but not limited to VSL#3 (Rivera-Flores 2020)), 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. When used to treat a 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™).

[0123] When used in such combination, the liposomes or composition or suspension thereof (e.g., transmembrane pH- gradient liposomes or composition or suspension thereof) 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.

[0124] Treatment and prevention

[0125] 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 endotoxemia in a subject as further described herein.

[0126] 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 endotoxemia or of at least one 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 endotoxemia or at least one symptom thereof.

[0127] 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 endotoxemia or at least one 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 endotoxemia or of at least one symptom thereof; of at least one endotoxemia associated disease or disorder; or of at least one pre-existing disease or disorder.

[0128] 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 endotoxemia or of at least one symptom thereof in a subject in need thereof.

[0129] As used herein the term “a symptom of endotoxemia” refers to any of the clinical characteristics of subjects having endotoxemia and corresponds to events resulting at least in part from the endotoxemia per se. For example, it includes one or more of hyperammonemia, liver impairment (e.g., characterized by elevated AST and / or elevated ALT), inflammation (e.g., systemic inflammation), 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).

[0130] As used herein the term “higher” in reference to the effect of endotoxemia on hyperammonemia and / or on serum (or plasma) level of AST and / or on serum (or plasma) level ALT and / or on serum (or plasma) level of creatinine and / or on serum (or plasma) level of urea and / or on serum (or plasma) level TNFa and / or on serum (or plasma) level of IL-6 as compared to the reference levels (e.g., level ranges considered normal according to literature i.e. level ranges in a corresponding (e.g., gender or age matched if levels vary according to sex or age) healthy subject. In specific embodiments it refers to an increase of at least 10% higher than the reference level; at least 15% higher; at least 20% higher; at least 25% higher; at least 30% higher; at least 35% higher; at least 40% higher; at least 45% higher; at least 50% higher; at least 55% higher; at least 65% higher; at least 70% higher; at least 75% higher; at least 80% higher; at least 85% higher; at least 90% higher; at least 95% higher; at least 100% higher; at least 1 10% higher; at least 120% higher; at least 130% higher; at least 140% higher; at least 150% higher; at least 160% higher; at least 170% higher; at least 180% higher; at least 190% higher; at least 200% higher; at least 210% higher; at least 220% higher; or at least 230% higher or more than the reference level.

[0131] As used herein the term “reduce” or “reduction” in reference to the effect of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure on the endotoxemia or symptom thereof in the subject in need thereof, refers, without being so limited, to a reduction of at least one endotoxemia symptom such as a reduction of serum (or plasma) level of (endotoxemia- induced) hyperammonemia 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 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 endotoxemia. In specific embodiments, it refers to a reduction of at least 10% compared to the serum (or plasma) level 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.

[0132] Kits

[0133] The present disclosure also provides a kit comprising (a) 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; (b) a hydration medium (e.g., acidic buffer as described herein; and (c) an aqueous solution as described herein (e.g., a neutralizing aqueous solution as described herein); and optionally (d) (I) instructions to use (a) to (c) to treat endotoxemia or a symptom thereof as described herein; (II) at least one other drug for the prevention or treatment of endotoxemia or a symptom thereof as described herein, an endotoxemia associated disease or disorder as described herein or a pre-existing disease or disorder as described herein; or (ill) a combination of (I) and (II).

[0134] 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.

[0135] BRIEF DESCRIPTION OF THE DRAWINGS

[0136] In the appended drawings:

[0137] FIGs. 1A-B: Efficient capture of ammonia by VS-01 in the peritoneal space leads to a decrease of LPS-induced plasma ammonia level in rats. Sprague-Dawley rats were intraperitoneally injected with 60 mL / kg VS-01 or a control dialysis solution (CTL, PHYSIONEAL™). One hour later, rats were intraperitoneally injected with LPS at 1 mg / kg for 3 hours. After termination, ammonia levels were measured in peritoneal fluid samples (FIG. 1A) and in plasma (FIG. 1 B). NA= Not analyzed. Each bar is the mean ±SD of each group. Group Healthy: n=6; Group PHYSIONEALTM(control): n=7, Group VS-01 : n=7. ###: p<0.001 (Student t test), $$: p<0.01 (Mann Whitney t-test).

[0138] FIGs. 2A-B: VS-01 improves liver injury in a rat model of endotoxemia. Sprague-Dawley rats were intraperitoneally injected with 60 ml_ / kg VS-01 or a control dialysis solution (CTL, PHYSIONEAL™). One hour later, rats were intraperitoneally injected with LPS at 1 mg / kg for 3 hours. After termination, the serum levels of transaminases AST (FIG. 2A) and ALT (FIG. 2B) were measured. Each bar is the mean ±SD of each group. Group Healthy: n=6; Group PHYSIONEAL™ (control): n=7, Group VS-01 : n=7. #. p<0.05, ###: p<0.001 (Student t test).

[0139] FIGs. 3A-B: VS-01 improves renal function in a rat model of endotoxemia. Sprague-Dawley rats were intraperitoneally injected with 60 mL / kg VS-01 or a control dialysis solution (CTL, PHYSIONEAL™). One hour later, rats were intraperitoneally injected with LPS at 1 mg / kg for 3 hours. After termination, the serum levels of creatinine (FIG. 3A) and urea (FIG. 3B) were measured. Each bar is the mean ±SD of each group. Group Healthy: n=6; Group PHYSIONEAL™ (control): n=7, Group VS-01 : n=7. $$: p<0.01 , $$$: p<0.001 (Mann Whitney t-test) #: p<0.05, #. p<0.05; ###: p<0.001 , (Student t test).

[0140] FIGs. 4A-B: VS-01 reduces LPS-induced release of cytokines from macrophages. After differentiation into macrophages, THP-1 cells were incubated with 1 pig / mL LPS. VS-01 liposomal solution was added concomitant to LPS. Four hours later, the concentration of TNF-a (FIG. 4A) and IL-6 (FIG. 4B) cytokines were measured in cell supernatant.

[0141] FIG. 5: Assessment of a range of VS-01 concentrations on macrophages viability. Toxicity was assessed through the CellTox™ Green Cytotoxicity Assay (Promega). RFU= Relative Fluorescent Unit. Triton was used as a positive control for cell death.

[0142] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0143] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0144] EXAMPLE 1 : Material and Methods

[0145] VS-01 Liposome formulation

[0146] Liposome aqueous suspension (LAS)

[0147] 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 spray-drying process. The dried lipid blend was hydrated with ultra-pure water (aqueous medium) (lipids concentration = 100 mg / mL144 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 were stored at 2-8°C. Liposomes having an average diameter between about 10 pm and 15 pm were obtained.

[0148] Citric acid anhydrous solution (CAS)

[0149] A citrate buffer 600 mM (pH 2.1 , 1041 mOsm / l) containing citric acid (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 (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) 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).

[0150] Sterile xylitol alkaline solution (XAS)

[0151] A neutralization solution (21.3 mg / mL, pH = 12.6, 450 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 (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).

[0152] Liposome reconstitution

[0153] 2.104 g of the above-described sterile citric acid anhydrous solution (CAS) were transferred to 4.215 g of the abovedescribed liposomal aqueous suspension (LAS) in a glass vial. The vial was then placed on a roller mixer (Benchmark Scientific, Model: TubeRoller™) (60 rpm) for 10 min at RT. A liposome composition was produced containing in a solution of the citric acid anhydrous solution, the liposomes enclosing the citric acid anhydrous solution.

[0154] The transmembrane pH-gradient was generated by neutralizing the external acid medium of the liposome composition by transferring 4.861 g of the liposome composition into a 50 mL Falcon tube with 45.701 g of the abovedescribed sterile xylitol alkaline solution (XAS). The mixture of liposome composition and xylitol alkaline solution was inverted 5 times and incubated for 15 min on a roller mixer (60 rpm). This procedure was performed 5 times to obtain 250 mL liposomal solution in total. Immediately after, pH was measured and was within specification (pH= 5-8).

[0155] The resulting multilamellar liposomes suspension (including the transmembrane pH-gradient liposomes) containing 18.4 mM citric acid anhydrous, at pH 6.5-6.6 and about 310 mOsm / l (VS-01) (liposomal suspension) was used for the preclinical studies presented herein.

[0156] Final transmembrane pH gradient liposomes concentrations of 2, 5 and 8 mM were tested with the RED system.

[0157] Induction of endotoxemia in rats

[0158] Sprague Dawley male rats (Janvier Labs) were housed in a temperature-controlled room on a 12h:12h light / dark cycle and had free access to standard food and water. After an acclimation period, rats (380-410g) received a single intraperitoneal injection of 1 mg / kg LPS (Escherichia coli O11 1 :B4) to mimic bacterial infection or bacterial translocation occurring in ACLF patients.

[0159] Three hours after LPS injection, blood collection was performed by sublingual vein puncture under light isoflurane anesthesia and rats were euthanized. Equal volumes of peritoneal fluid samples (475 piL) were also collected for ammonia measurement. Ammonia withdrawal from the lipid vesicles was performed according to a protocol previously described. Blood was transferred into serum-gel tubes or heparin lithium tubes for preparation of serum or plasma, respectively.

[0160] Administration to rats and assessment of capture

[0161] After pre-warming at 37°C, transmembrane pH gradient liposomes VS-01 or a control dialysis solution (Physioneal ™) were intraperitoneally injected to rats one hour before LPS injection at 60 mL / kg and left until termination (4 hours dwell time in total).

[0162] To assess efficient capture of ammonia by VS-01 after i.p. injection, peritoneal fluid samples were collected 4 hours after administration.

[0163] Biochemistry analysis

[0164] Serum hepatic and renal markers and plasma ammonia were measured using Randox™ kits for Daytona Plus automate using references described in Table I.

[0165] Table I: Main items

[0166] Cell lines

[0167] The human monocytic cell line THP-1 (TebuBio™) was cultured in RPMI 1640 with L-glutamine medium (Corning™) supplemented with 10% fetal bovine serum (FBS, Gibco), 1 % Penicilline / Streptomycine (Gibco™) and 25mM Hepes (Sigma™) at 37°C and 5% CO2.

[0168] To induce differentiation into macrophages, 1x105THP-1 cells, cultured in a 96-well plate, were treated with 100 ng / ml Phorbol 12-myristate 13-acetate (PMA, Sigma™) for 24h. After 24h serum starvation, cells were treated with 1 pig / ml LPS (Klebsiella, Sigma). Concomitantly, pre-warmed VS-01 preparation (up to 6 mM lipid) was added into cell media. After 4h incubation, cytokines (TNFa and IL6) were measured in cell supernatants by Homogeneous Time Resolved Fluorescence (HTRF, 62HTNFAPEH and 62HIL06PEH). Fluorescence was measured with Spark™ device (#1801002745, Tecan) to determine the concentration of cytokines.

[0169] Toxicity

[0170] To ensure that VS-01 addition in the media did not cause cell death, toxicity was assessed through the CellTox™ Green Cytotoxicity Assay (Promega) and fluorescence was measured with Spark™ (#1801002745, Tecan).

[0171] TABLE II: MAIN ITEMS

[0172] EXAMPLE 2: Liposomes reduce LPS-induced alteration of liver and renal function in a rat model of endotoxemia

[0173] The capacity of liposomes to counteract LPS response was evaluated in a rat model of endotoxemia. The effect of the intraperitoneal administration of the liposomes of Example 1 was measured on LPS-induced liver and renal function markers, in comparison with a control dialysis solution.

[0174] Confirmation of ammonia capture by liposomes (VS-01) in rats with endotoxemia

[0175] In the liposomes group, ammonia levels in the collected peritoneal fluid samples were 2.7-fold higher than in the control group (p<0.001) (FIG. 1A).

[0176] These results indicate that the liposomes of the present disclosure efficiently captured ammonia in the peritoneal space of rats and validated the effectiveness of transmembrane pH-gradient liposomes in this model.

[0177] While blood ammonia level was up-regulated by 2.2-fold by LPS in the control group rats, the liposomes of Example 1 blunted LPS-induced level of plasma ammonia by 101 % (p=0.004) (FIG. 1 B).

[0178] Effect of liposomes administration on LPS-induced liver injury markers in rats

[0179] Hepatic injury was assessed by measurement of serum transaminases, 3h after LPS administration. An increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) was found in this model by 2.4 and 1.7-fold, respectively compared to healthy animals (FIGs. 2A-B). The liposomes of Example 1 significantly alleviated LPS- induced ALT by 79% (p=0.03) (FIG. 2B) and tended to reduce AST (46%, p=0.13) (FIG. 2A) compared to the LPS group treated with a control solution.

[0180] Effect of liposomes administration on LPS-induced kidney function markers in rats

[0181] Renal function was assessed by measurement in the serum of creatinine, a marker of renal function and of urea, a surrogate marker of hepato-renal function. A 1.5-fold increase in serum creatinine was found 3 hours after LPS injection, whereas it was decreased by 90% (p=0.001) with the liposomes of Example 1 , as compared with control rats (FIG. 3A). The liposomes also reduced the LPS-induced serum level of urea by 27% (p=0.02) (FIG. 3B).

[0182] EXAMPLE 3: Liposomes reduce LPS-induced cytokine secretion in macrophages

[0183] Using a cellular system, the direct capacity of the liposomes of Example 1 to remove and deactivate LPS was assessed through the measurement of LPS-induced cytokine secretion in THP-1 macrophages treated with increasing doses of liposomes.

[0184] While LPS treatment of human macrophages led to a strong increase of TNFa and IL-6 by 17.7-fold and 18.4-fold respectively, the addition of the liposomes to the cell media reduced the LPS-induced cytokine secretion in a dosedependent manner (FIGs. 4A-B). The maximum efficacy was observed with 6 mM liposomes for TNFa secretion (- 86%, p<0.001 ) (FIG. 4A) and with 4 mM liposomes for IL-6 secretion (-97% p<0.001 ) (FIG. 4B). IC50 were calculated at 1 .3 mM and 0.5 mM for TNFa and IL-6, respectively.

[0185] Cell viability was assessed 4h after incubation of the liposomes, showing a slight effect for concentration above 2 mM (FIG. 5).

[0186] EXAMPLE 4: Liposomes in ACLF preclinical model

[0187] To determine VS-01 efficacy in an ACLF preclinical model, LPS is used as a precipitating factor in cirrhotic rats to induce systemic inflammation and organ failure, therefore mimicking ACLF pathophysiology (Tripathi et al., 2018).

[0188] For cirrhosis induction, male rats undergo CCI4 inhalation 3 times a week together with phenobarbital (0.3 g / L) in the drinking water for 15 to 16 weeks until ascites development (decompensated cirrhosis stage). After one week of wash-out, LPS is injected intravenously at 0.01-1 mg / kg for 4 hours before assessment of pro-inflammatory cytokines serum levels and organ failure (kidney / liver function markers, brain edema and neurological behavior test, coagulation test, respiration test, portal hypertension, etc.).

[0189] VS-01 is administered i.p. at 60 mL / kg, 30 min after LPS injection, and kept until the end of the experiment (3.5 hours dwell time in total). A control dialysis solution (Physioneal ™) is used as a control group.

[0190] 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. REFERENCES

[0191] Fux, A.C., Casonato Melo C., 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. https: / / doi.org / 10.3390 / ijms24098395.

[0192] Tripathi, D. M., Vilaseca, M., Lafoz, E., Garcia-Caldero, H., Viegas Haute, G., Fernandez-Iglesias, A., . . . Gracia- Sancho, J. (2018). Simvastatin Prevents Progression of Acute on Chronic Liver Failure in Rats With Cirrhosis and Portal Hypertension. Gastroenterology, 155(5), 1564-1577. doi: 10.1053 / j.gastro.2018.07.022.

Claims

CLAIMS:

1. Liposomal suspension, for use in the treatment of endotoxemia in a subject in need thereof, wherein the treatment comprises intraperitoneally administering the liposomal suspension to the subject and removing a dialysate containing liposomes from the subject.

2. The liposomal suspension for use of claim 1 , wherein the subject has an endotoxemia associated disease or disorder.

3. The liposomal suspension for use of claim 2, wherein the endotoxemia associated disease or disorder comprises a bacterial infection caused by Escherichia coli.

4. The liposomal suspension for use of claim 2 or 3, wherein the endotoxemia associated disease or disorder comprises liver impairment and / or kidney impairment.

5. The liposomal suspension for use of any one of claims 1 to 4, wherein the liposomes contain a hydroxy acid, preferably citric acid, most preferably citric acid anhydrous.

6. The liposomal suspension for use of claim 5, wherein the liposomes contain about 200 nM citric acid anhydrous.

7. The liposomal suspension for use of any one of claims 1 to 6, wherein the liposomes’ lipid bilayer comprises at least one phospholipid as main constituent.

8. The liposomal suspension for use of claim 7, wherein the at least one phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol % to 90 mol%.

9. The liposomal suspension for use of any one of claims 1 to 8, wherein the liposomes’ lipid bilayer comprises cholesterol, preferably in a range of 10 to 40 mol%.

10. The liposomal suspension for use of claim 9, wherein the liposomes’ lipid bilayer further comprises 1 ,2- distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%.

11. The liposomal suspension for use of any one of claims 1 to 6, wherein the liposomes’ bilayer contains 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%, and the liposomes’ inner compartment contains citric acid anhydrous.

12. The liposomal suspension for use of any one of claims 1 to 1 1 , wherein the liposomes have an average diameter between about 8 pm and 12 pm.

13. The liposomal suspension for use of any one of claims 1 to 12, wherein the liposomal suspension contains (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).

14. The liposomal suspension for use of any one of claims 1 to 13, wherein the subject is human.

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