Use of transmembrane pH gradient liposomes to treat hyperammonemic crisis associated with inborn errors of metabolism

Abstract

The present disclosure provides use of a liposomal suspension comprising transmembrane pH gradient liposomes for the treatment of acute hyperammonemic episodes (HAC) associated with an inborn error of metabolism (IEM) in a subject, the treatment comprising intraperitoneally administering the liposomal suspension to the subject and removing dialysate containing ammoniated liposomes from the subject.

Description

[Technical Field]

[0001] The present disclosure relates to the use of transmembrane pH gradient liposomes for the treatment of hyperammonemic crisis associated with inborn errors of metabolism in a subject in need thereof, more particularly, a subject in need thereof having a urea cycle disorder or organic acidemia. [Background technology]

[0002] Hyperammonemic crisis (HAC) is manifested by acute hyperammonemia, a life-threatening condition that can be a substantial cause of brain damage and death if not treated early and effectively [Savy 2018].

[0003] HAC can be associated with inborn errors of metabolism (IEM) or acquired clinical conditions distinct from IEM based on their etiology, clinical manifestations, and treatment. In IEM, along with HAC, its direct neurological consequences are the primary concern; in most other clinical conditions, hyperammonemia is caused by liver failure, other organ dysfunction, and can be drug-induced.

[0004] More specifically, HAC associated with IEM is associated with genetic deficiencies in genes encoding enzymes that affect the urea cycle (physiological removal of ammonia), either directly (primary hyperammonemia) in urea cycle disorders (UCDs) or indirectly (secondary hyperammonemia) in organic acidemias (OAs). The entire urea cycle is present only in the liver, where it is expressed in hepatocytes in the periportal region, in an otherwise healthy liver.

[0005] The biological and clinical manifestations of disease, outcomes, and prognosis in IEM subjects are all driven solely by the genetic defect leading to hyperammonemia. In fact, the duration of coma and the level of blood ammonia concentration are the main factors determining mortality and neurological outcomes. Ammonia levels are unique drivers of the treatment decision tree.

[0006] All other clinical conditions that cause HAC are acquired diseases and conditions, 95.7% of which are related to liver disease. In liver disease, hyperammonemia results from reduced ammonia removal, which occurs when the number of functional periportal hepatocytes falls below the threshold required for adequate ammonia detoxification. The neurological signs of liver failure are called "hepatic encephalopathy." In contrast to hyperammonemia in IEM, the pathogenesis of hepatic encephalopathy is thought to involve multiple factors, including the role of neurotoxins other than ammonia, impaired neurotransmission due to metabolic changes in liver failure, altered cerebral energy metabolism, systemic inflammatory responses, and alterations in the blood-brain barrier. In liver failure, the clinical signs, prognosis, and treatment of the disease are primarily caused by the destruction of the liver itself, ultimately leading to multiple organ failure. Treatment of hepatic encephalopathy in chronic liver disease relies on controlling the trigger and reducing intestinal ammonia production with lactulose, with or without rifaximin, to prevent recurrence. The remaining 4.3% of cases of acquired hyperammonemia are rare and associated with serious conditions leading to increased ammonia production. In these non-hepatic conditions, the clinical signs, outcomes, and prognosis of the disease are often associated with multiple organ dysfunction associated with conditions of increased catabolic metabolism, such as hematologic-oncological disorders, organ transplants, and severe gastrointestinal infections. Drug-induced hyperammonemia can also result from disruption of the urea cycle or enhanced renal release of ammonia into the systemic circulation. Valproic acid is the most well-known causative agent. Treatment of non-hepatic acquired hyperammonemia is primarily based on removal of the causative event and organ support, such as hemodialysis.

[0007] In conclusion, HAC in IEM represents a unique and distinct condition involving a genetic disorder affecting the metabolism of ammonia via the urea cycle in an otherwise healthy liver.

[0008] The prevalence of these genetic disorders may be higher than current estimates (1 / 35,000–1 / 69,000 live births, considering all UCDs) due to unreliable newborn screening and underdiagnosis of fatal cases. UCDs account for 23% of HACs in children admitted to pediatric intensive care units. The prevalence of all underlying genetic disorders leading to HAC is below the 200,000 threshold in the United States.

[0009] Current research is aimed at correcting the underlying defect in each IEM that causes HAC, but results have so far been inconclusive. Gene transfer trials using adenoviral vectors for ornithine transcarbamylase (described further below) deficiency did not result in a useful increase in enzyme activity [Leonard 2004]. These studies were discontinued due to severe complications and the death of one patient [Raper 2003]. Gene transfer in a bovine model of citrullinemia showed positive results but has not yet been tested in humans [Leonard 2004]. An open-label clinical trial in adults with late-onset OTCD is ongoing (ClinicalTrials.gov Identifier: NCT02991144). Hepatocyte infusion is an alternative therapy currently under investigation. Hepatocytes infused into the liver may provide an alternative enzyme source. However, biochemical correction to date appears to be transient, and the benefit to patients remains unclear [Horslen 2003].

[0010] The clinical manifestations of HAC are similar in all IEMs, regardless of their specific underlying genetic defect (defined further below). Elevated ammonia blood levels are the sole cause of neurological clinical signs, and liver failure is not associated with hyperammonemia. In view of the similar clinical symptoms, common treatments, and outcomes if untreated, HAC associated with IEMs, regardless of the underlying genetic defect, is identified as a single condition with a high unmet medical need.

[0011] Current initial management of HAC does not consider the specific diagnosis of a genetic disorder, which typically takes several days. The medical goal is to rapidly reduce ammonia levels across the board. To reduce the high mortality and morbidity associated with HAC in IEM, immediate initiation of treatment is believed to reduce mortality and morbidity [Enns 2007, Hediger 2018, Savy 2018]. Treatment includes dietary modification, ammonium scavengers, and immediate transfer of the patient to a tertiary care center for emergency detoxification of ammonia through acute renal replacement therapy (RRT), the fastest method of removing ammonium from the bloodstream.

[0012] Specifically, current treatments for HAC caused by IEM in neonates include hemodialysis (HD), peritoneal dialysis (PD), or continuous RRT to reduce plasma levels. PD can be initiated immediately, but its rate of ammonia removal is slow, making it difficult to balance ammonia formation. HD is the most effective method for removing ammonia, but it is often unavailable, highly invasive, and hypotension is a frequent complication, leading to ammonia levels returning to normal after HD is discontinued. CRRT, more specifically, continuous intravenous HD using high dialysate flow rates, appears to be the best available option. In practice, pediatric patients with HAC must be transferred to highly specialized tertiary care centers with CRRT devices adapted to pediatric sizes.

[0013] Disadvantages of CRRT in neonates include difficulty establishing vascular access lines, difficulty with fluid balance, and a lack of equipment suitable for neonates. The relatively large circuit volume (60 mL) required for blood priming carries several risks. Detailed fluid balance is not possible, and current equipment used for CRRT is not approved or cleared for infants weighing less than 8 kg. Therefore, dialysis in IEM HAC is often initiated late when ammonia levels exceed 1000 μmol / L, which may contribute to poor outcomes [Hediger 2018]. No acute treatment is available for early-onset crisis.

[0014] In addition, a significant proportion of late-onset HAC (7% previously reported in 299 patients representing 1181 episodes of acute HA) still requires prompt dialysis to avoid further impairment of neurological function [Enns 2007].

[0015] There is a need to develop alternative therapeutic interventions for the treatment of HAC associated with IEM, particularly for the treatment of pediatric patients (e.g., neonates) presenting with HAC associated with IEM.

[0016] This description makes reference to several documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0017] The present disclosure provides an alternative method for treating HAC associated with IEM, providing liposomes that confer optimized ammonia clearance to the peritoneal fluid, allowing for efficient initiation of PD immediately after HAC is confirmed and before transferring the patient to a tertiary care center. While any patient suffering from this disease can benefit from the methods presented herein, they are particularly useful in pediatric patients. The treatment provided by the present disclosure reduces the burden on patients and parents and reduces costs to the healthcare system.

[0018] More specifically, the present disclosure provides the following:

[0019] Item 1. Use of a liposome suspension comprising transmembrane pH gradient liposomes for treating acute hyperammonemic episodes (HAC) associated with an inborn error of metabolism (IEM) in a subject, the treatment comprising intraperitoneally administering the liposome suspension to the subject and removing dialysate containing ammoniated liposomes from the subject.

[0020] Item 2. The use according to Item 1, wherein the subject is a pediatric subject.

[0021] Item 3. The use according to any one of Items 1 to 3, wherein the subject has a urea cycle disorder (UCD).

[0022] Item 4. The use according to Item 3, wherein the UCD is ornithine transcarbamylase deficiency.

[0023] Item 5. The use according to any one of Items 1 to 5, wherein the liposome contains a hydroxy acid, preferably citric acid, most preferably anhydrous citric acid.

[0024] Item 6. The use according to Item 5, wherein the liposome contains about 200 nM anhydrous citric acid and preferably has an internal pH of about 2. Item 7. The use according to any one of Items 1 to 6, wherein the lipid bilayer of the liposome comprises at least one phospholipid as a main component.

[0025] Item 8. The use according to Item 7, wherein the at least one phospholipid contains dipalmitoylphosphatidylcholine (DPPC), preferably in the range of 60 mol% to 90 mol%.

[0026] Item 9. The use according to any one of Items 1 to 8, wherein the lipid bilayer of the liposome contains cholesterol, preferably in the range of 10 to 40 mol %.

[0027] Item 10. The use according to Item 9, wherein the lipid bilayer of the liposome further contains 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in the range of 0.2 to 5 mol %.

[0028] Item 11. The use according to any one of Items 1 to 6, wherein the bilayer of the liposome contains dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)-2000] (DSPE-PEG) in a ratio of 85.5:14:0.5 mol%, and the inner compartment of the liposome contains anhydrous citric acid.

[0029] Item 12. The use according to any one of Items 1 to 11, wherein the liposomes have an average diameter of about 8 μm to 12 μm.

[0030] Item 13. The use according to any one of Items 1 to 12, wherein the liposome 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 includes (i) to (v).

[0031] definition Liposomes Liposome Composition Liposomes according to the present disclosure comprise a lipid bilayer membrane surrounding an acidic buffer (acidic solution).

[0032] lipid bilayer membrane In a preferred embodiment, the liposomal lipid bilayer membrane comprises at least one natural or synthetic phospholipid. Preferred phospholipids are long saturated phospholipids, e.g., those having alkyl groups of more than 12, preferably more than 14, more preferably more than 16, and most preferably more than 18 carbon atoms.

[0033] In specific embodiments, the natural or synthetic phospholipid is 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-phosphoelanoyl Phosphoelhanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoelanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoelanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoelanolamine (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)] (DMPG), The liposome lipid bilayer membrane may comprise at least one of the following: 1,2-dioleoyl-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), and natural L-α-phosphatidylcholine (from chicken eggs, EPC, or from soybeans, SPC). In a specific embodiment, the natural or synthetic phospholipid is DPPC. In a specific embodiment, the main component of the liposome lipid bilayer membrane is 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, hi specific embodiments, the natural or synthetic phospholipid forms about 85.5 mol% of the liposome bilayer membrane.

[0034] In other embodiments, the liposomal 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 comprises 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% of the liposomal bilayer membrane. 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 liposomal bilayer membrane. In a specific embodiment, the at least one ammonia retention-enhancing compound forms at least 10 mol% of the liposomal bilayer membrane. In a specific embodiment, the at least one ammonia retention-enhancing compound forms about 14 mol% of the liposomal bilayer membrane.

[0035] 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.

[0036] In another embodiment, the liposome lipid bilayer membrane comprises 10 to 100 mol %, more preferably 25 to 75 mol %, more preferably 40 to 70 mol %, and most preferably 50 to 60 mol % of at least one sphingolipid, preferably sphingomyelin.

[0037] In another embodiment, the liposome lipid bilayer membrane comprises 30 to 100, more preferably 40 to 95, and most preferably 45 to 60 mol % of at least one surfactant. In a specific embodiment, the at least one surfactant comprises a hydrophobic alkyl ether (e.g., Brij), alkyl ester, polysorbate, sorbitan ester, and / or alkyl amide.

[0038] In other embodiments, the average diameter size of the liposomes is greater than 900 nm, greater than 1000 nm, greater than 2000 nm, greater than 3000 nm, greater than 4000 nm, greater than 5000 nm, greater than 6000 nm, greater than 7000 nm, 3000 nm to 15 μm, 4000 nm to 15 μm, 5000 nm to 15 μm, etc. to avoid too rapid drainage from the peritoneal space. , 6000 nm to 15 μm, 7000 nm to 15 μm, 8000 nm to 15 μm, 3000 nm to 14 μm, 4000 nm to 14 μm, 5000 nm to 14 μm, 6000 nm to 14 μm, 7000 nm to 14 μm, 8000 nm to 14 μm, 3000 nm to 13 μm, 4000 nm to 13 μm, 5000 nm to 13 μm, 6000 nm to 13 μm, 7000 nm to 13 μm, 8000 nm to 13 μm. In a specific embodiment, the average diameter of the liposomes is about 8 μm to about 12 μm.

[0039] Acidic buffer / acidic solution The acidic buffer in the inner compartment of the liposome preferably has a high buffering capacity at low pH due to high retention of basic compounds (e.g., ammonia). The acid is non-toxic to animals and permeates (or only weakly) through the liposome membrane.

[0040] Although not limited thereto, the acid encapsulated in the liposome core may be (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 polymer (polycarboxylic 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 a specific embodiment, the acid is a hydroxy acid, such as citric acid (e.g., anhydrous citric acid).

[0041] In specific embodiments, the concentration of acid used in methods such as osmotic shock may vary between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of about 100 mM to 900 mM, or about 100 mM to 900 mM, or about 300 mM to 800 mM, or about 400 mM to 750 mM, or about 500 mM to 750 mM, or about 500 mM to 650 mM, or about 600 mM is optimally used. Osmolalities of 500-1500 mOsmol / kg, 600-1400 mOsmol / kg, 700-1400 mOsmol / kg, 800-1400 mOsmol / kg, 800-1350 mOsmol / kg, 900-1350 mOsmol / kg, 950-1300 mOsmol / kg, 950-1250 mOsmol / kg, or 1000-1200 mOsmol / kg are optimally used. In another specific embodiment, the concentration of citric acid (e.g., anhydrous) used in the present method may vary between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of approximately 600 mM is used, and an osmolality of 1000-1200 mOsmol / kg is used in the osmotic shock method. In a preferred embodiment, the transmembrane pH gradient liposomes produced by the methods described herein have an internal concentration of about 200 nM anhydrous citric acid and an internal osmolality that is physiological, ie, approximately 350 mOsm / kg.

[0042] The acid in the core (the inner compartment of the liposome) is present at a concentration that results in a pH of 1 to 6, in a specific embodiment, a pH of 1.5 to 3, and in a more specific embodiment, a pH of about 2 in the core of the liposome.

[0043] In a specific embodiment, the liposome contains 200 nM citric acid (anhydrous) in its internal compartment / core, which has a pH of about 2.

[0044] In an alternative embodiment, the liposomes for use in the present disclosure are as described in EP 2 882 421 to Leroux et al.

[0045] composition According to another aspect of the present invention, 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 present invention may comprise a pharmaceutically acceptable carrier / excipient, including, but not limited to, an aqueous or non-aqueous solution. Pharmaceutically acceptable carriers may also include physiologically acceptable aqueous vehicles (e.g., sugar solutions, saline), neutralizing species (basic or acidic, e.g., weak bases or weak acids), as well as chemical agents used to adjust osmolality and / or provide physiological function. Excipients encompassed by the present disclosure include, but are not limited to, glycerol, tris((hydroxymethyl)aminomethane) (TRIS), agents that counteract the 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 suitable for the intraperitoneal route. In specific embodiments, the liposome composition (e.g., 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)-(v), preferably, the combination includes all of (i)-(v).

[0046] Methods for preparing liposomes Osmotic shock method In a specific embodiment, the lipid blend can be prepared by mixing the components of the lipid bilayer in a solvent such as an alcohol or a mixture of water and an organic solvent (e.g., an alcohol such as ethanol or t-butanol) until completely dissolved to form a homogeneous lipid mixture. Mixing can be performed at room temperature (i.e., approximately 20-25°C) or with heating (e.g., at temperatures up to 60°C, preferably up to 45°C), optionally with slow mixing.

[0047] The mixture can optionally be filtered (e.g., through a 0.2 μm filter), and the organic solvent is then removed, for example, by freeze drying, spray drying (e.g., using liquid nitrogen as the drying gas), rotary evaporation, or other methods.

[0048] The resulting dried lipid blend can then be hydrated in an aqueous medium, as further described below.

[0049] aqueous medium In a preferred embodiment, the components of the lipid bilayer can be mixed directly in an aqueous medium with an osmolality of 400 mOsm / l or less (direct lipid hydration method).

[0050] In certain embodiments, the aqueous medium has a pH value of approximately 7, e.g., in the range of 6.0-7.5, 6.1-7.4, 6.2-7.3, 6.3-7.2, 6.4-7.1, 6.5-7.3, 6.6-7.3, 6.7-7.3, 6.8-7.3, 6.9-7.1, 6.95-7.01, or about 7.0. In certain embodiments, the aqueous medium is selected from the group consisting of water (e.g., distilled water, deionized water, ultrapure water, or any other type of purified water), a mixture of water and an organic solvent (e.g., alcohol) as defined above, an aqueous solution of an organic salt, an aqueous solution of an inorganic salt, an aqueous solution of an organic substance, and combinations thereof. In certain embodiments, the aqueous medium is selected from the group consisting of an aqueous solution of an organic salt having a pH value of approximately 7, an aqueous solution of an inorganic salt having a pH value of approximately 7, an aqueous solution of an organic substance having a pH value of approximately 7, water, and combinations thereof.

[0051] When organic or inorganic salts or other organic compounds are used, these salts or compounds are present in low concentrations in the aqueous medium so as to maintain an osmolality difference between the aqueous medium and the hyperosmotic buffer that causes the osmotic shock, which difference is sufficient to induce diffusion of the acidic or basic hyperosmotic buffer into the vesicle interior compartment.

[0052] An aqueous medium is a medium that resembles water (especially with respect to pH), but may contain low concentrations of salts or compounds, for example, to buffer the pH value in the neutral range.

[0053] As indicated above, the aqueous medium has an osmolality of 400 mOsm / L or less. In some embodiments, the osmolality of the aqueous medium is 300 mOsm / L or less, 250 mOsm / L or less, 200 mOsm / L or less, 150 mOsm / L or less, 100 mOsm / L or less, 75 mOsm / L or less, 50 mOsm / L or less, 25 mOsm / L or less, 10 mOsm / L or less, 5 mOsm / L or less, or 1 mOsm / L or less. In some embodiments, the osmolality is in the range of 1 mOsm / L to 200 mOsm / L, or a range constructed from any of the foregoing osmolalities (e.g., 10 mOsm / L to 150 mOsm / L, etc.). In certain embodiments, the osmolality of the aqueous medium is in the range of 0 mOsm / l to 49 mOsm / l, 0 mOsm / l to 45 mOsm / l, 0 mOsm / l to 40 mOsm / l, particularly 0 mOsm / l to 35 mOsm / l, in particular 0 mOsm / l to 30 mOsm / l, 0 mOsm / l to 25 mOsm / l.

[0054] In specific embodiments, the liposomes can optionally be extruded or filtered to obtain liposomes having a particular size.

[0055] Hydration of the lipid bilayer component / lipid blend can be carried out at room temperature (i.e., approximately 20-25°C) or with heating (e.g., at temperatures up to 60°C (e.g., in a preheated aqueous medium), preferably up to 45°C), optionally with slow stirring for about 15 minutes to 4 hours, preferably about 2 hours. At this stage, if hydration is carried out with heating, the mixture, the final concentration of lipids is preferably about 100 mg / g, is cooled to room temperature (i.e., approximately 20-25°C). The mixture can optionally be degassed (e.g., under high vacuum) to remove air bubbles.

[0056] In some embodiments, the hydrated liposomes so prepared are sterilized to obtain sterile liposomes or a sterile suspension containing liposomes. Sterilization can be carried out, for example, by sterile filtration or steam sterilization (e.g., autoclaving), for example, for about 5 minutes to 2 hours, 10 minutes to 1 hour, 15 minutes, or 30 minutes.

[0057] In another embodiment, the vesicles are stored for a first period of time before performing the step of mixing the liposomes (or a suspension containing liposomes) with an acidic buffer. This storage can be optimally achieved if the liposomes are sterilized after the step of hydration in an aqueous medium. This is because no or very little degradation processes occur in a sterilized liposome suspension. The first period of time can be one day, several 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). The sterilized liposomes contained in an aqueous medium are stable entities. Because they do not yet contain any specific basic buffer used to prepare the pH gradient, there is no need to worry about loss of the buffer due to liposomal bilayer degradation or liposomal leakage. This is also true in certain embodiments when the liposomes contain low amounts of electrolyte molecules, in the range of 0 or 1 mOsm / l to 200 mOsm / l depending on the osmolality within the vesicle.

[0058] Acidic and hypertonic buffers The hydrated (and optionally sterilized) liposomes are then mixed with an acidic buffer having an osmolality at least 200 mOsm / L higher than the osmolality of the aqueous medium to osmotically shock the liposomes and obtain buffer-filled liposomes. In certain embodiments, the osmolality of the acidic buffer is at least 220 mOsm / L higher, 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, or at least In some embodiments, the osmolality of the acidic buffer is in the range of 200 mOsm / l to 1100 mOsm / l higher than the osmolality of the aqueous medium, or a range constructed from any of the foregoing osmolalities (e.g., 220 mOsm / l to 1200 mOsm / l).

[0059] Therefore, the acidic buffer is a hyperosmolar buffer with respect to the aqueous medium used in the liposome hydration step. In this way, an osmotic shock is immediately applied to the liposomes. This osmotic shock incorporates the acidic buffer into the liposomes. Therefore, the osmotic shock serves to destabilize the liposomes for a short period of time to allow the incorporation of the buffer into the liposomes. Buffer-filled liposomes are obtained. In some embodiments, the hyperosmolar buffer may also be used to adjust the osmolality or contain electrolytes with physiological function.

[0060] Because osmotic shock cannot be achieved otherwise, a sufficient amount of acidic buffer is added to the liposomes suspended in the aqueous medium. A sufficient amount can be a volume corresponding to at least 0.1 times, 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 the volume of the aqueous medium, depending on the difference between the osmolality of the aqueous medium and the osmolality of the basic buffer. In some embodiments, the acidic buffer can be added in a volume equal to the volume of the aqueous medium. In some embodiments, the volume of the acidic buffer added can be 0.1 to 5 times the volume of the aqueous liposome suspension, or any other range (e.g., 0.3 to 3 times) that can be constructed from the aforementioned values.

[0061] In certain embodiments, the pH value of the hypertonic buffer solution is in the 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.

[0062] In specific embodiments, the hypertonic buffer may contain additional chemical agents, such as complexing or chelating agents.

[0063] In specific embodiments, the hypertonic buffer comprises a salt such as, but not limited to, sodium chloride, sodium hydroxide, and / or magnesium chloride.

[0064] In a specific embodiment in which sterile transmembrane pH gradient liposomes are preferred, the acidic buffer is sterilized.In such an embodiment, if the hydrated liposomes are also sterilized before adding the acidic buffer, a completely sterile buffer-filled liposome or a completely sterile suspension containing the buffer-filled liposome is prepared.Sterilization can be carried out, for example, by sterile filtration or autoclaving.

[0065] In some embodiments, the mixture of aqueous medium and basic or acidic buffer in which the buffer-filled liposomes are suspended has an osmolality of at least 200 mOsm / L, at least 220 mOsm / L, at least 250 mOsm / L, at least 300 mOsm / L, at least 350 mOsm / L, at least 400 mOsm / L, at least 450 mOsm / L, at least 500 mOsm / L, or at least 550 mOsm / L. In some embodiments, the osmolality ranges from 200 mOsm / L to 550 mOsm / L, or a range derived from any of the foregoing osmolalities (e.g., 220 mOsm / L to 500 mOsm / L).

[0066] The liposome-acidic buffer mixture can optionally be incubated. In a specific embodiment, the mixture is agitated (e.g., by orbital shaking), for example, at room temperature.

[0067] Neutralize the aqueous solution The mixture of the aqueous medium and the acidic buffer solution containing the buffer-filled liposomes is then diluted by adding a neutralizing aqueous solution. The mixture of the acidic buffer solution and the neutralizing solution constitutes a suspension buffer. Thus, after dilution, transmembrane pH gradient liposomes suspended in the suspension buffer are obtained. Therefore, the pH of the suspension buffer is different from that of the acidic buffer solution contained in the buffer-filled liposomes. In some embodiments, the pH difference is 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.

[0068] In one embodiment, the pH value of the neutralization solution is 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~pH11, pH7.2~pH10.5, pH7.2~pH10, pH7.2~pH9.5, pH7.2~pH9, pH7.2~pH8.5, pH7.3~pH14, pH7.3~pH13.5, pH7.3~pH13, pH7.3~pH12.5, pH7.3 ~pH12, pH7.3~pH11.5, pH7.3~pH11, pH7.3~pH10.5, pH7.3~pH10, pH7.3~pH9.5, pH7.3~pH9, pH7.3~pH8.5, pH7.4~pH14, pH7.4~pH13.5, pH7.4~pH1 3, pH7.4~pH12.5, pH7.4~pH12, pH7.4~pH11.5, pH7.4~pH11, pH7.4~pH10.5, pH7.4~pH10, pH7.4~pH9.5, pH7.4~pH9, pH7.4~pH8.5, pH7.5~pH14, p H7.5~pH13.5, pH7.5~pH13, pH7.5~pH12.5, pH7.5~pH12, pH7.5~pH11.5, pH7.5~pH11, pH7.5~pH10.5, pH7.5~pH10, pH7.5~pH9.5, pH7.5~pH9, pH7 The neutralization solution may have a pH range of 0.5 to 8.5, pH 8.0 to 13.0, pH 8.5 to 12.5, pH 9.0 to 13, pH 9.0 to 12.5, pH 9.0 to 12.0, pH 9.5 to 11.5, pH 10 to 13, pH 10 to 12.5, pH 10 to 12.0, pH 10 to 11.5, pH 10 to 11, pH 10 to 12.5, pH 10.5 to 12.0, pH 10.5 to 13, pH 10.5 to 12.5, pH 10.5 to 12.0, pH 10.5 to 11.5, or pH 10.5 to 11. In a specific embodiment, the pH of the neutralization solution is about 12.5.

[0069] In some embodiments, the neutralization solution has a composition that helps prevent the buffer-filled vesicles from being destabilized and disruptive to these vesicles. The neutralization solution may contain not only neutralizing species (basic or acidic, e.g., weak bases or weak acids), but also chemical agents used to adjust osmolality and / or provide physiological function. Calcium salts can be added to the preparation process to counteract the anticoagulant effect of some weak acids (e.g., citric acid). Of particular importance is that the vesicles are used in in vivo applications. Sodium hydroxide, sodium salts (such as sodium chloride), potassium chloride, calcium magnesium chloride salts, lactate, glycerol, icodextrin, glucose, sorbitol, fructose, amino acids, or xylitol can also be used as components of the neutralization solution. In specific embodiments, the neutralization solution contains

[0070] In some embodiments, the neutralization solution has an osmolality of 250 mOsm / l to 550 mOsm / l, 270 to 520 mOsm / l, 290 to 500 mOsm / l, 300 to 480 mOsm / l, 320 to 450 mOsm / l, 330 to 420 mOsm / l, 350 to 400 mOsm / l, 375 to 400 mOsm / l, 385 to 400 mOsm / l, or 390 to 400 mOsm / l.

[0071] In one embodiment, the neutralization solution has an osmolality that is less than 200 mOsm / l higher or lower than the osmolality of the vesicle-containing mixture containing the buffer (i.e., the vesicle solution containing the buffer), particularly less than 150 mOsm / l higher or lower, particularly less than 100 mOsm / l higher or lower, particularly less than 50 mOsm / l higher or lower, particularly less than 20 mOsm / l higher or lower, particularly less than 10 mOsm / l higher or lower. In one embodiment, the difference in osmolality between the neutralization solution and the mixture containing vesicles containing a buffer is 1 mOsm / ~200 mOsm / l, in particular 10 mOsm / ~150 mOsm / l, in particular 20 mOsm / ~100 mOsm / l, in particular 30 mOsm / ~80 mOsm / l, in particular 40 mOsm / ~60 mOsm / l.

[0072] Due to the pH difference between the suspension buffer and the acidic buffer, a transmembrane pH gradient is achieved between the interior portion of the liposome and the surrounding suspension buffer, and the resulting transmembrane pH gradient can be used in accordance with the present disclosure.

[0073] In certain embodiments, the pH value of the suspension buffer containing the transmembrane pH gradient vesicles is in the range of 5.5 to 8.5, 6.0 to 8.0, 6.3 to 7.7, 6.3 to 7.5, 6.3 to 7.3, 6.3 to 7.2, 6.3 to 7.1, 6.5 to 7.7, 6.5 to 7.5, 6.5 to 7.3, 6.5 to 7.2, 6.5 to 7.1, 6.8 to 7.5, or 7.0 to 7.4. Thus, the suspension buffer can have a physiological pH value. In a specific embodiment, the pH value of the suspension buffer is about 6.5.

[0074] The osmotic shock method is also described in EP 3 291 797 to Leroux et al.

[0075] Alternative methods for preparing liposomes In another embodiment, the method for preparing transmembrane pH gradient liposomes includes a film hydration method. For example, the components of the liposome bilayer membrane are dissolved in an organic solvent (e.g., dichlorometham-methanol), and then the organic solvent is removed (e.g., by rotary evaporation) to form a dried lipid film. The dried lipid can be stored (e.g., under high vacuum) for future use. The dried lipid can then be directly hydrated in the above-mentioned acidic buffer solution, or the external solution can be exchanged with the above-mentioned neutral solution. Alternatively, the film hydration method can be used first to form a lipid film, and then hydrated in an aqueous medium as described above.

[0076] The liposomes to which an aqueous medium has been added can then be subjected to the above-mentioned osmotic shock step, to which an acidic buffer solution is added, and the above-mentioned neutralization solution step can be carried out to produce a transmembrane pH gradient liposome suspension.

[0077] An alternative method for preparing liposomes is also described in EP 2 882 421 to Leroux et al.

[0078] Route of administration and mechanism of action The liposomes of the present disclosure are administered intraperitoneally.

[0079] As used herein, the term "intraperitoneal administration" is meant to be understood as commonly understood by those skilled in the art of peritoneal dialysis therapy. To practice the present invention, a pharmaceutically effective amount of a liposome suspension of the present disclosure is administered into the peritoneal cavity, for example, by injection as a single bolus, by continuous infusion or perfusion, for example, by a catheter such as a catheter commonly used for paracentesis.

[0080] The liposomes within the cavity and nearby tissues and organs take up ammonia based on the pH gradient across the liposome membrane. The acidic buffer contained within the liposome has a pH lower than the physiological pH in the peritoneal cavity (approximately 7.5-8). Therefore, ammonia can diffuse through the hydrophobic liposome bilayer in an uncharged state and then become trapped in the inner liposome compartment in a protonated (ionized) state (e.g., ammonium).

[0081] The liposomes sequester ammonia for an extended period of time, reducing the toxic concentration of the free compound. The ammonia-loaded liposomes in the peritoneal cavity are removed / extracted from the peritoneal cavity along with the fluid (dialysate) present therein. Intraperitoneal administration and extraction can occur subsequently and / or simultaneously. While not limited thereto, the dialysate can be extracted by passively draining it through a catheter by gravity, or by pumping it by suction through a pump, such as a peristaltic pump used for infusion.

[0082] disease Acute hyperammonemia in the context of IEM is referred to as "HAC" or "acute hyperammonemic episode," which refer to the same medical condition. HAC is defined as a plasma ammonia level greater than 80 μmol / L to 100 μmol / L in neonates up to 1 year of age and greater than 55 μmol / L in older children and adults [Haeberle 2013]. In published longitudinal studies of IEM, HAC was defined as "compatible clinical symptoms associated with a plasma ammonia level greater than 100 μmol / L" [Kent 2017] or "an acute hyperammonemic episode defined as a single hospitalization for hyperammonemia" [Enns 2007].

[0083] In mammals, the hepatic urea cycle is the primary pathway for detoxifying ammonia. Ammonia is continuously produced by the breakdown of proteins and other nitrogen-containing molecules. Hyperammonemic crisis occurs whenever the waste nitrogen load exceeds the body's detoxification capacity.

[0084] Hyperammonemia rapidly leads to cerebral edema and associated symptoms of lethargy, anorexia, hyperventilation or hypoventilation, hypothermia, seizures, neurological posturing, and coma. These events occur through various mechanisms, primarily resulting in astrocyte swelling due to increased glutamine. While the specific roles of ammonia, glutamate, and glutamine in cerebral edema are still under investigation, recent data indicate that excessive ammonia exposure alters several amino acid pathways and neurotransmitter systems, brain energy, nitric oxide synthesis, axonal and dendritic growth, signaling pathways, and K+ and water channels. All of these effects can ultimately lead to energy insufficiency, oxidative stress, and cell death.

[0085] etiology As indicated above, the IEMs that cause HAC comprise a group of inherited disorders in which a single genetic defect causes a clinically significant block in the urea cycle, which is responsible for the metabolic clearance of ammonia from the bloodstream.

[0086] As noted above, HAC associated with IEM is designated "primary hyperammonemia" when it is secondary to a genetic deficiency in any of the enzymes or transporters involved in the urea cycle that define UCB, and "secondary hyperammonemia" when urea cycle enzymes are inhibited due to accumulated metabolic products or substrate deficiencies. The most related group of disorders associated with secondary hyperammonemia is called organic acidemias (Oas). Regardless of the underlying genetic disorder, the clinical features, outcome, prognosis, and treatment of HAC associated with IEM are similar.

[0087] Genetic defects underlying IEM UCD UCDs are the most common genetic cause of HAC in infants and children. They result from inherited deficiencies in genes encoding any one of five enzymes, cofactors, and two transporters in the Krebs-Henseleit cycle / urea cycle [Ah Mew 2013]. The five catalytic enzymes are carbamyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG1). Deficiencies are abbreviated as CPS1D, OTCD, ASSD, ASLD, and ARG1D, respectively, and are assigned MIM numbers 237300, 311250, 215700, and 20700, respectively. 900, 207800), the cofactor-generating enzyme is N-acetylglutamate synthetase (NAGS) (MIM number 237310), which is the main activator of CPS1, and two amino acid transporters are ornithine translocase (ORNT1), which induces hyperornithineemia-hyperammonemia-homocitrullinuria syndrome (Triple H syndrome (HHH)) (MIM number 238970), and citrin.

[0088] The urea cycle, as a nitrogen clearance system, occurs primarily in the human liver and intestine, and CPS1 and OTC are exclusively restricted to these tissues. The downstream enzymes that process citrulline to arginine are ubiquitously distributed. As the rate-limiting enzyme in the urea cycle, alterations in the function of the enzyme CPS1 are expected to have the greatest impact on cycle function.

[0089] OTC deficiency is inherited in an X-linked manner. The other seven urea cycle disorders (deficiencies of CPS1, ASS, ASL, ARG1, NAGS, ORNT1, and citrin) are inherited in an autosomal recessive manner.

[0090] Organic aciduria / acidemia Organic acidurias (Oas) are genetic disorders that primarily affect the breakdown of branched-chain amino acids. Important Oas that cause HAC include propionic aciduria / acidemia (PA), methylmalonic aciduria / acidemia (MMA), isovaleric aciduria / acidemia (IVA), maple syrup urine disease (MSUD), glutaric aciduria type I, and multiple carboxylase deficiency. PA, MMA, and MSUD Oas are sometimes referred to as "classical organic acidurias" because they involve the most common organic acids.

[0091] Hyperammonemia in Oas results from a decrease in acetyl-coenzyme A (CoA) and the inhibition of NAGS and CPS1 activity resulting from the toxic accumulation of organic acid metabolites. In these disorders, HAC is accompanied by severe metabolic acidosis with a high anion gap and ketonuria. Organic aciduria induces long-term complications in many organs (including the brain, kidneys, heart, bone, and pancreas), but primarily results in a neurological phenotype.

[0092] Most OA is an autosomal recessive disorder, although some are X-linked. The diagnosis of OA-associated hyperammonemia is made by plasma ammonia levels, urine organic acid chromatography, and plasma acylcarnitine profiles [Savy 2018].

[0093] Regardless of the underlying genetic disorder, the clinical features, outcome, prognosis, and current treatment of primary and secondary HAC associated with IEM are similar.

[0094] Clinical characteristics of HAC associated with IEM As noted above, the clinical features / presentations (symptoms) of primary and secondary HAC associated with IEM are similar, regardless of the underlying genetic disorder.

[0095] Clinical symptoms in patients with HAC caused by IEM can begin as early as the first day of life or as late as adulthood, with acute or chronic symptoms. Severity tends to be inversely related to the age of the subject.

[0096] Subjects with HAC caused by IEM are clinically characterized by their neurological, psychiatric, or hepatic / gastrointestinal symptoms, or a combination of these three categories of symptoms. Without treatment or delayed diagnosis, survivors suffer from severe neurological impairment and seizures. Even in cases of partial deficits, the symptoms are more variable and appear later (at any age), and there is a risk of sequelae of neurological symptoms and death associated with hyperammonemia. There is a strong correlation between the duration and severity of hyperammonemia and brain damage, making prompt diagnosis and appropriate treatment essential to optimize patient outcomes.

[0097] The severity of symptoms is related to the residual enzyme activity and the position of the defective enzyme in the urea cycle. Thus, severe enzyme deficiency or complete absence of enzyme activity leads to the accumulation of ammonia and other metabolites from the first day of life, whereas partial deficiency, regardless of age, can cause hyperammonemia of varying severity, often caused by catabolic events, protein overload, or certain medications. Careful review of the medical and family history should be systematic. This includes addressing the following issues: unexplained death, presence of psychiatric or neurological disorders in the family, inbreeding (which is frequently present in all UCDs except OTCD, which are X-linked), documented voluntary avoidance of proteins by the patient and / or family members, and medications taken by the patient.

[0098] Laboratory data generally useful in diagnosing hyperammonemia associated with UCD include plasma ammonia levels, pH, CO2, anion gap, plasma amino acids, and urine organic acid analysis.

[0099] Common acute symptoms: altered level of consciousness (from drowsiness / lethargy to coma) reminiscent of encephalitis or drug intoxication; acute encephalopathy; seizures (usually accompanied by altered level of consciousness, not isolated); ataxia (usually accompanied by decreased level of consciousness); pseudo-stroke episodes; temporary loss of vision; vomiting and progressive anorexia; liver failure; failure of several organs; peripheral circulatory disorders; postnatal psychosis; dysarthria; asterixis (in adults); learning disabilities, neurodevelopmental delay, mental retardation; chorea, cerebral palsy; long-term cortical blindness; progressive spastic diplegia or quadriplegia (AR) described in GD1 or triple H syndrome); protein aversion, spontaneous protein eating; abdominal pain, vomiting; growth retardation; hepatomegaly, elevated liver enzymes; migraine-like headaches, tremors, psychiatric symptoms (hyperactivity, mood swings, behavioral changes, aggression, hallucinations, paranoia, manic episodes, affective disturbances and personality changes); self-endangerment; pseudo-autistic symptoms; brittle hair (characteristic of ASLD); specific neuropsychological phenotype in heterozygous OTC patients; episodic nature of signs and symptoms.

[0100] Neonatal manifestations. Affected neonates are usually asymptomatic at birth. After an interval of 24 hours to several days, neonates quickly present with lethargy / somnia, anorexia / refusal to feed and vomiting, loss of thermoregulation, neurologic posturing, cerebral edema causing lethargy, hyperventilation followed by hypoventilation, hypothermia, hypotonia or hypertonia, convulsions / seizures, coma, multiple organ failure, and irritability that progress to death [Ah Mew 2013, Summar 2008, Haeberle 2013]. These symptoms may mimic a sepsis continuum, which may cause delayed diagnosis. Clinical manifestations of HAC associated with milder forms of IEM with residual enzyme activity usually occur later in life (late onset almost anywhere from infancy to adulthood), with recurrent episodes of mild to moderately severe hyperammonemia that can be triggered by illness, stress, or excessive protein intake [Summar 2008]. In late-onset IEM, HAC is usually less severe and symptoms are more subtle.

[0101] Only 27% of 678 UCD patients enrolled in the UCDC Natural History Study were reported to have had neonatal HAC. Similarly, Summar

[2008] reported that the majority (66%) of 260 patients with UCD presented with HAC beyond the neonatal period (>30 days) (Figure 1A).

[0102] Adult manifestations. Adults (over 16 years of age) with UCD deficiency may be from the childhood cohort or may be diagnosed later (they may be younger or older). In both cases, the clinical manifestations are the same as those of the infantile or juvenile forms.

[0103] The diagnosis of UCD in adults is usually made during acute decompensation. These are primarily OTC deficiencies and occur predominantly in women. Clinical signs are primarily neurological, with impaired consciousness and psychotic symptoms predominating. A small proportion of patients do not show any prior signs suggesting decompensation, and two-thirds have an aversion to animal protein and are voluntary vegetarians.

[0104] Predisposing factors for HAC in UCD patients include infections, fever, vomiting, diarrhea, internal or gastrointestinal bleeding, reduced protein or energy intake (e.g., preoperative fasting, major weight loss in the newborn), uterine catabolism and involution during the postpartum period (mainly in over-the-counter patients), chemotherapy, high-dose glucocorticoids, strenuous or prolonged physical exercise, surgery under general anesthesia, and excessive protein intake (e.g., protein-rich foods: meat, fish, eggs, dairy products; inadequate artificial feeding). Medications, primarily valproate and L-asparaginase / pegaspargase, have also been associated with hyperammonemia decompensation. In adults, more specific factors include rapid weight loss (weight-loss diets, bariatric surgery), anorexia, and postpartum.

[0105] Outcome and prognosis An early age at onset (Figure 1B) and severe clinical symptoms (coma and elevated plasma ammonium levels >1000 μmol / L) (Figure 1C) correlate with high mortality and poor neurological outcome [Krivitzky 2009, Enns 2007]. Until recently, it was estimated that approximately 25% of newborns with high exposure to ammonia caused by UCD died early, and this proportion continues to increase (Figure 1D) [Hediger 2018].

[0106] Historically, most children with early-onset neonatal HAC due to severe UCD or OA enzyme deficiency died as newborns, and few survived infancy. Late-onset HAC associated with IEM is usually associated with better outcomes [Enns 2007, Hediger 2018], but neurological morbidity remains high, with a significant proportion of patients exhibiting poor neurocognitive outcomes and behavioral disorders [Krivitzky 2009, Ah Mew 2013]. Krivitzky

[2009] reported that approximately half of children with neonatal-onset HAC exhibited intellectual disability, 30% of whom exhibited severe impairment. In comparison, only one-quarter of the late-onset group suffered from intellectual disability, despite evidence of neurocognitive and behavioral impairments related to attention and executive function (Figure 1B).

[0107] Published data from 103 subjects with neonatal onset of HAC derived from a longitudinal study of UCD showed that across all types of enzyme deficiency, 47%–68% of pediatric patients had poor outcomes (IQ / Developmental Quotient <70). This was observed in both patients under 4 years of age and those aged 4 years or older, and there was no difference between patients with proximal UCD (Figure 1E) and those with distal UCD (i.e., ASS and ASL).

[0108] Combination therapy The present disclosure encompasses combining intraperitoneal administration of the transmembrane pH gradient liposomes described herein with other therapies to treat HAC associated with IEM for acute or chronic treatment, either simultaneously or sequentially (e.g., acute treatment as disclosed herein followed by at least one other treatment) depending on the nature of the additional treatment.

[0109] Treatment and Prevention The present disclosure encompasses the use of liposomes / liposomal suspensions or compositions described herein for the treatment of acute hyperammonemic episodes (HAC) associated with inborn errors of metabolism (IEM) in a subject. The present disclosure may further include the treatment or prevention of any symptoms of HAC associated with IEM downstream of the acute hyperammonia itself, as further described herein.

[0110] As used herein, the term "treat / treating / treatment" refers to eliciting a desired biological response, i.e., a therapeutic effect. According to the disclosure herein, a therapeutic effect includes one or more of a decrease / reduction in the frequency, duration, and / or severity of HAC associated with IEM. It may further include one or more of a decrease / reduction in the frequency, duration, and / or severity of at least one symptom triggered by HAC and / or the duration of a symptom-free period following administration of a liposome of the present disclosure or a composition (e.g., a suspension) comprising a liposome of the present disclosure described herein, alone or in combination with another agent for the treatment of HAC associated with IEM or at least one symptom thereof.

[0111] As used herein, the term "prevent / preventing / prevention" refers to eliciting a desired biological response, i.e., a preventative effect. In accordance with the disclosure provided herein, in some embodiments, the preventative effect comprises complete or partial avoidance / inhibition of at least one symptom of HAC associated with IEM following administration of a liposome or liposomal suspension or composition of the present disclosure, alone or in combination with another agent for the prevention or treatment of muscular dystrophy or at least one symptom thereof.

[0112] In some embodiments, a "therapeutically effective amount" or "effective amount" or "therapeutically effective dosage" of a liposome or liposomal suspension of the present disclosure provided herein results in treatment of HAC associated with IEM in a subject in need thereof, and may also result in treatment or prevention of at least one symptom thereof in a subject.

[0113] As used herein, the term "at least one symptom of HAC associated with IEM" refers to any of the clinical features in pediatric or adult subjects described under the headings "Common Acute Symptoms," "Neonatal Symptoms," or "Adult Symptoms," and corresponds to events attributable at least in part to acute hyperammonemia itself.

[0114] subject As used herein, the term "subject" or "subject in need thereof" refers to a subject who would benefit from receiving an effective amount of liposomes and liposome suspensions. In specific embodiments, this refers to animals, mammals, and humans. The compositions of the present invention can also be used for veterinary purposes and can be used on pets or other animals (e.g., pets such as cats, dogs, and horses, as well as cattle, fish, pigs, poultry, etc.). In a specific embodiment, the subject suffers from HAC associated with IEM. In a specific embodiment, the subject has a healthy liver and / or does not suffer from drug-induced hyperammonemia. In a specific embodiment, the subject is a pediatric subject. As used herein, the term "pediatric subject" refers to a subject who is 21 years of age or younger at the time of diagnosis or treatment, i.e., a newborn (i.e., from birth to 28 days after birth), an infant (from about 29 days to less than 2 years of age), a child (from 2 years to less than 12 years of age), or an adolescent (from about 12 to 21 years of age) with recurrent HAC. In another more specific embodiment, the subject is a newborn, an infant, or a child. In another specific embodiment, the subject is an adult.

[0115] A subject in need thereof is previously diagnosed with HAC associated with IEM. In certain embodiments, the methods of the present disclosure include diagnosing a subject. The use of the terms "a," "an," and "the," and similar referents in the context of describing the disclosure (particularly in the context of the claims that follow) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0116] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0117] Recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually referred to herein. Every subset of values ​​within a range is also incorporated herein as if it were individually listed herein.

[0118] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0119] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better clarify the disclosure and does not limit the scope of the disclosure unless otherwise specified.

[0120] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0121] As used herein, the term "about" has its ordinary meaning. In embodiments, "about" may mean plus or minus 10% of the qualified numerical value.

[0122] 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.

[0123] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. In the accompanying drawings: [Brief explanation of the drawings]

[0124] [Figure 1A]Number of patients by diagnosis and age at first episode. Each patient is counted once at the age at which the first reported episode of hyperammonemia occurred [Summar 2008]. [Figure 1B] Cognitive range across UCD subjects aged 3–6 years: neonatal (NO) versus late onset (LO) [Krivitzky 2009]. [Figure 1C] The proportion of hyperammonemic episodes that survived according to peak ammonium levels was based on the number of episodes for which data on plasma ammonium levels were available [Enns 2007]. [Figure 1D] The relationship between the primary outcome parameters, classified as "normal outcome," "died," "disabled," and "alive but no further information" (no survival information), is specified for the single and UCD groups. Absolute values ​​are listed above each column [Hediger 2018]. [Figure 1E] Neurodevelopmental outcomes for subjects aged 4 years and older by diagnosis [Ah Mew 2013]. Each diagnostic cohort, proximal UCD (CPSD / OTCD), ASD, ALD, and the total neonatal UCD cohort, were stratified by neurodevelopmental outcome. When the Full-Scale Intelligence Quotient (FSIQ) was unavailable, Verbal IQ or Performance IQ was used to determine categorization. For severe / profound impairment, subjects could not be assessed by traditional IQ testing for their age range. Instead, the Bayley Scales were administered to derive DQ; for mild to moderate impairment, FSIQ scores of 45–69; for low average / borderline functioning, FSIQ scores of 70–89; for roughly average, FSIQ scores of 90–109; and for above average, FSIQ scores of 110 (no patients met this criterion). [Figure 2] Linear regression model for estimation of the relationship between ammonia clearance in peritoneal fluid (mL / min) and the volume of VS-01 infused (mL). [Figure 3]Mean peritoneal fluid ammonia concentrations in B6EiC3Sn a / A-Otcspf-ash / J mice after a single session with VS-01, Sidak's multiple comparison test. [Figure 4A] Mean blood ammonia concentrations in B6EiC3Sn a / A-Otcspf-ash / J mice after a single session with VS-01. Dunnett's multiple comparison test. [Figure 4B] Mean blood ammonia concentrations in B6EiC3Sn a / A-Otcspf-ash / J mice after a single session with VS-01. Graph reproduces Figure 4A, but includes the ammonia concentrations achieved by VS-01 1 hour after administration for each of four mice separately (i.e., 2005 (outlier), 2006, 2007, and 2008) in addition to the average value for all mice in the VS-01 group. Dunnett's multiple comparison test. DETAILED DESCRIPTION OF THE INVENTION

[0125] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0126] Example 1: Materials and Methods mouse Hemizygous male B6EiC3Sn a / A-Otc spf- ash / J mice, strain number 002343. RRID:IMSR_JAX:002343 (generic name: sparse coat) were used for the assay. These mice are characterized by slow and patchy hair development and harbor a spontaneous Otc gene on the X chromosome. spf These individuals carry the sparse coat mutation, a C-to-A missense mutation (H117N) in exon 4 that changes a histidine to an asparagine, creating a hypomorphic allele. This mutation results in only 5-10% of normal hepatic ornithine transcarbamylase (OTC) activity, ultimately resulting in higher plasma ammonia concentrations. This hepatic OTC deficiency is similar to that seen in congenital hyperammonemia type II, which is seen in children and is characterized by life-threatening episodes of acute metabolic failure accompanied by hyperammonemia.

[0127] Liposomal solution formulation Liposomes composed of dipalmitoylphosphatidylcholine (DPPC, Lipoid), cholesterol (Sigma-Aldrich), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)-2000] (DSPE-PEG, Lipoid) in a ratio of 85.5:14:0.5 mol% were prepared by the direct lipid hydration method. 595.4 mg of DPPC, 51.3 mg of cholesterol, and 13.3 mg of DSPE-PEG were co-dissolved in a 70:30 (w / w) t-butanol:water mixture to form a homogeneous mixture. The organic solvent was then removed by lyophilization. The dried lipid blend was hydrated with ultrapure water (lipid concentration = 100 mg / mL) by heating at 60 °C for 2 h with gentle mixing, and finally sterilized in a sealed bottle by autoclaving at 121 °C for 15 min. Liposomes with an average diameter of about 8 μm-12 μm were obtained.

[0128] 68.1 mL of the liposomes thus obtained was incubated with 32.2 mL of 600 mM citrate buffer (pH 2.1, 1041 mOsm / L) containing citric acid, sodium chloride, sodium hydroxide, and magnesium chloride for 30 minutes. The incubation was carried out at room temperature with shaking on an orbital shaker. The resulting liposomes contained 200 mM citric acid and had an internal pH of approximately 2.

[0129] A transmembrane pH gradient was generated by neutralizing the external acid medium with 950 mL of a neutralization solution (pH = 12.5, 392 mOsm / L) made from xylitol, sodium chloride, sodium hydroxide, potassium chloride, and calcium chloride. The resulting multilamellar liposome suspension containing 18.4 mM anhydrous citric acid at pH 6.5 and approximately 310 mOsm / L was used in preclinical studies.

[0130] Study design: This non-clinical study was conducted in accordance with good scientific principles and internal standard operating procedures at Charles River Laboratories, Inc., MA 01545 USA.

[0131] 22 males B6EiC3Sn a / A-Otc spf-ash 1 / J mice (approximately 7-8 weeks old at the start of treatment) were received from The Jackson Laboratory. Animals were housed singly in polycarbonate cages containing appropriate bedding. Upon arrival, all animals were fed LabDiet™ 5LG4 and hydrogel pellets ad libitum, except during designated procedures. Municipal tap water treated with reverse osmosis and ultraviolet irradiation was available ad libitum to all animals. On day -1, all animals were weighed, blood ammonia readings were obtained using a handheld meter (ARKRAY PocketChem BA), and assigned to treatment groups in a manner that produced groups with no significant differences in body weight and blood ammonia. Treatment began on day 1 under fasting conditions.

[0132] On the day of the experiment, the liposome solution VS-01 was reconstituted under a laminar flow hood using clean procedures. A commercially available dialysis solution (Dianeal) was used as a control article.

[0133] A volume of 100 mL / kg of both Dianeal (control) and liposome solution (VS-01) (354 mg / kg / day citric acid) was administered via intraperitoneal injection to animals in Groups 1 and 2, respectively. The solutions remained in the peritoneal cavity for dwell times of 0.5, 1, and 2 hours according to the assigned sampling time points as described below.

[0134] On day 1, after a 5-hour fast, whole blood from submandibular blood draws was tested for ammonia levels at the following time points: pre-dose for all groups, then alternating post-dose: 3 animals from Group 1 were tested 0.5, 1, and 2 hours post-dose, 4 animals from Group 2 were tested 0.5 and 1 hour post-dose, and 5 animals from Group 2 were tested 2 hours post-dose.

[0135] Peritoneal dialysate samples were collected ±10 min via intraperitoneal suction (21 g needle / 1 mL syringe) according to the same schedule, transferred to uniquely labeled cryotubes, and frozen on dry ice. All samples were stored for no more than 1 h in a freezer set to maintain -80 °C pending analysis using a validated ammonia method [Cobas 6000 / NH3L2 Ammonia Assay from Roche Diagnostics (MLM-VAL-1954)]. Study parameters included mortality, cage-side observations, and body weight measurements. Animals were euthanized by CO2 asphyxiation on day 1.

[0136] Example 2: Ammonia clearance in peritoneal fluid of a UCD model increased significantly with the volume of VS-01 administered All animals tolerated the medication well with no signs of adverse reactions to the treatment.

[0137] Ammonia extracted from the blood into the peritoneal cavity was significantly higher (p<0.0006) at all time points during the residence time after a single intraperitoneal injection of the liposome suspension of Example 1 (354 mg / kg / day of citric acid) (VS-01) compared to the control solution (Figure 3).

[0138] This resulted in a significant decrease in blood ammonia at 0.5 hours (p<0.0089) and 2 hours (p<0.0007) post-dose (Figure 4A). At 1 hour post-dose, mean blood ammonia was skewed by an outlier in animal 2005, likely related to the injection procedure, which may have increased systemic exposure to VS-01 (Figure 4B).

[0139] Peritoneal fluid ammonia clearance in OTC mice was calculated from blood exposure to ammonia (AUC0-2) over a 2-hour residence time and the amount of ammonia extracted 2 hours after administration (amount = concentration in peritoneal fluid × volume of injected fluid). The mean ammonia clearance in the peritoneal fluid was 0.3 mL / min compared to 0.1 mL / min after VS-01 and Dianeal treatment, respectively.

[0140] The above shows that VS-01 is useful for treating HAC associated with IEM.

[0141] The scope of the claims should not be limited by the embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0142] References Ah Mew 2013 Clinical Outcomes of Neonatal Onset Proximal versus Distal Urea Cycle Disorders Do Not Differ.The Journal of Pediatrics.

[0143] Enns 2007 Survival after Treatment with Phenylacetate and Benzoate for Urea-Cycle Disorders N Engl J Med 2007;356:2282-92.

[0144] Haeberle 2013 Clinical and biochemical aspects of primary and secondary hyperammonemic disorders Archives of Biochemistry and Biophysics 536(2013)101-108.

[0145] Hediger 2018 The impact of ammonia levels and dialysis on outcome in 202 patients with neonatal onset urea cycle disorders J Inherit Metab Dis.

[0146] Horslen 2003 Isolated Hepatocyte Transplantation in an Infant With a Severe Urea Cycle Disorder Pediatrics 2003; 111:1262-1267.

[0147] Kent 2017 Hyperammonemic crises in patients with urea cycle disorders on chronic nitrogen scavenger therapy with either sodium phenylbutyrate or glycerol phenylbutyrateNeuropsychiatry(London)(2017)7(2),131-136.

[0148] Krivitzky 2009 Intellectual,Adaptive,and Behavioral Functioning in Children with Urea Cycle Disorders Pediatr Res.2009 July ; 66(1):96-101.

[0149] Leonard 2004 The role of liver transplantation in urea cycle disorders Molecular Genetics and Metabolism 81(2004)S74-S78.

[0150] Raper 2003 Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer.Molecular Genetics and Metabolism 80(2003)148-158.

[0151] Savy 2018 Acute pediatric hyperammonemia: current diagnosis and management strategies.Hepatic Medicine: Evidence and Research 2018:10 105-115.

[0152] Summar 2008 Diagnosis,Symptoms,Frequency and Mortality of 260 Patients with Urea Cycle Disorders from a 21-Year,Multicentre Study of Acute Hyperammonaemic Episodes Acta Paediatr.2008 October ; 97(10):1420-1425.

Claims

1. 1. Use of a liposome suspension comprising transmembrane pH gradient liposomes for the treatment of acute hyperammonemic episodes (HAC) associated with an inborn error of metabolism (IEM) in a subject, the treatment comprising intraperitoneally administering the liposome suspension to the subject and removing dialysate containing ammoniated liposomes from the subject.

2. The use according to claim 1, wherein the subject is a pediatric subject.

3. The use according to any one of claims 1 to 3, wherein the subject has a urea cycle disorder (UCD).

4. 4. The use according to claim 3, wherein the UCD is ornithine transcarbamylase deficiency.

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

6. The use of claim 5, wherein the liposome contains about 200 nM anhydrous citric acid.

7. The use according to any one of claims 1 to 6, wherein the lipid bilayer of the liposome comprises at least one phospholipid as a main component.

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

9. The use according to any one of claims 1 to 8, wherein the lipid bilayer of the liposome comprises cholesterol, preferably in the range of 10 to 40 mol %.

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

11. 7. The use according to any one of claims 1 to 6, wherein the bilayer of the liposome contains dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG) in a ratio of 85.5:14:0.5 mol % and the inner compartment of the liposome contains anhydrous citric acid.

12. The use according to any one of claims 1 to 11, wherein the liposomes have an average diameter of about 8 μm to 12 μm.

13. 13. The use according to any one of claims 1 to 12, wherein the liposome 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 comprising all of (i) to (v).

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