Use of transmembrane ph gradient liposomes in treatment of hyperammonemia crises associated with innate metabolic errors

Abstract

The present disclosure provides the use of a liposome suspension comprising transmembrane pH gradient liposomes in the treatment of acute hyperamminemia crisis (HAC) associated with innate metabolic error (IEM) in a subject, wherein the treatment comprises intraperitoneal administration of the liposome suspension to the subject and removal of a dialysate containing ammonia-loaded liposomes from the subject.

Description

Technical Field

[0001] The present disclosure relates to the use of transmembrane pH gradient liposomes in treating hyperammonemic crisis associated with inborn errors of metabolism in a subject in need thereof. More specifically, the subject in need has a urea cycle disorder or an organic acidemia. Background Art

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

[0003] Depending on its pathogenesis, clinical presentation, and treatment, HAC may be associated with inborn errors of metabolism (IEM) or with acquired clinical conditions different from IEM. In IEM, HAC and its immediate neurological consequences are the main issues, while in most other clinical presentations, hyperammonemia is caused by liver failure, other organ dysfunctions, and may be drug-induced.

[0004] More specifically, HAC associated with IEM is related to genetic defects in the genes encoding enzymes that affect the urea cycle (the physiological elimination of ammonia), which may directly lead to urea cycle disorders (UCD) (primary hyperammonemia) or may indirectly lead to organic acidemias (OA) (secondary hyperammonemia). The entire urea cycle exists only in the liver, which is expressed in periportal hepatocytes; the liver is otherwise healthy.

[0005] The biology, clinical presentation, outcomes, and prognosis of IEM patients are all driven entirely by the genetic defects that cause hyperammonemia. In fact, the duration of coma and the level of blood ammonia concentration are the main factors determining mortality and neurological outcomes. The ammonia level is the only driver of the treatment decision tree.

[0006] All other clinical conditions causing HAC are acquired diseases and disorders, 95.7% of which are related to liver diseases. Among liver diseases, hyperammonemia is caused by a decline in ammonia elimination capacity when the number of functional periportal hepatocytes is below the threshold required for adequate ammonia detoxification. The neurological manifestations of liver failure are referred to as "hepatic encephalopathy". In contrast to hyperammonemia in IEM, the pathogenesis of hepatic encephalopathy is thought to involve multiple factors, including the effects of neurotoxins other than ammonia, impaired neurotransmission due to metabolic changes in liver failure, changes in brain energy metabolism, systemic inflammatory responses, and alterations in the blood-brain barrier. In liver failure, the clinical manifestations, prognosis, and treatment of the disease are mainly driven by the destruction of the liver itself, ultimately inducing multiple organ failure. The treatment of hepatic encephalopathy in chronic liver disease relies on controlling the precipitating factors and using lactulose with or without rifaximin to reduce intestinal ammonia production to prevent recurrence. The remaining 4.3% of acquired hyperammonemia due to non-liver causes is rare and is associated with severe diseases that cause increased ammonia production. In these non-liver conditions, the clinical manifestations, outcomes, and prognosis of the disease involve multiple organ dysfunction associated with an increased catabolic state, such as hematological malignancies, organ transplantation, and severe gastrointestinal infections. Drug-induced hyperammonemia may also be due to interference with the urea cycle or increased release of ammonia from the kidneys into the systemic circulation. Valproic acid is the most well-known pathogen. The treatment of non-liver disease-acquired hyperammonemia is mainly based on eliminating the cause and organ support, such as hemodialysis.

[0007] In summary, the hyperammonemic crisis (HAC) in IEM represents a distinct condition involving a genetic disorder that affects ammonia metabolism by the urea cycle in a healthy liver.

[0008] Due to the lack of reliability of newborn screening and underdiagnosis of death cases, the prevalence of these genetic diseases may be higher than the current estimates (prevalence is 1 / 35,000–1 / 69,000 considering all UCDs). Among children admitted to the pediatric intensive care unit, UCDs account for 23% of HAC. In the United States, the prevalence of all potential genetic diseases causing the hyperammonemic crisis (HAC) is below the threshold of 200,000 people.

[0009] The current research goal is to correct the defects underlying each IEM that causes HAC; however, so far, the results have been inconclusive. Gene transfer trials using adenoviral vectors for ornithine transcarbamylase deficiency (described further below) did not result in a significant increase in enzyme activity [Leonard 2004]. These studies were terminated due to serious complications and the death of one patient [Raper 2003]. Gene transfer in a bovine model of citrullinemia showed positive results but has not been tested in humans [Leonard 2004]. An open-label clinical trial for adults with late-onset OTCD is currently underway (ClinicalTrials.gov identifier: NCT02991144). Hepatocyte infusion is an alternative therapy currently under investigation. Hepatocytes infused into the liver can provide an alternative source of enzyme. However, any biochemical correction so far seems to be temporary, and it remains unclear whether patients benefit [Horslen 2003].

[0010] Regardless of the specific underlying genetic defect (defined further below), the clinical manifestations of hyperammonemic crisis (HAC) in all IEMs are similar; elevated blood ammonia levels are the sole cause of the clinical manifestations in the nervous system and do not involve liver failure as a cause of hyperammonemia. Given the similar clinical manifestations, common treatments, and outcomes of non-treatment, hyperammonemic crisis (HAC) associated with IEMs has been identified as a single condition with a high unmet medical need, regardless of the presence of an underlying genetic defect.

[0011] Currently, the initial treatment of hyperammonemic crisis (HAC) does not take into account the specific diagnosis of the genetic disorder, which usually takes several days; the medical goal is always to rapidly reduce ammonia levels. To reduce the high mortality and morbidity associated with hyperammonemic crisis (HAC) in IEMs, immediate initiation of treatment is thought to reduce mortality and morbidity [Enns 2007; Hediger 2018; Savy 2018]. Treatment includes dietary measures, ammonia scavengers, and immediate transfer of the patient to a tertiary center for urgent detoxification of ammonia by acute renal replacement therapy (RRT), which is the fastest way to remove ammonia from the blood.

[0012] Current treatments for hyperammonemic crisis (HAC) caused by neonatal IEM include hemodialysis (HD), peritoneal dialysis (PD), or continuous RRT to reduce plasma levels. Although PD can be initiated immediately, its ammonia removal rate is slow, making it difficult to balance ammonia formation. Although HD is the most effective method for ammonia removal, it is often unavailable, highly invasive, hypotension is a common complication, and ammonia levels tend to rebound after HD termination. CRRT, more specifically continuous venovenous HD with a high dialysate flow rate, seems to be the best option currently. In fact, pediatric patients with hyperammonemic crisis (HAC) must be transferred to highly specialized tertiary centers with CRRT devices suitable for their body size.

[0013] Disadvantages of neonatal CRRT include difficulty in establishing vascular access, difficulty in maintaining fluid balance, and lack of devices suitable for neonates. The circuit volume required for blood priming is relatively large (60 mL), which poses multiple risks. Detailed fluid balance cannot be performed, and current instruments used for CRRT have not been approved or are not approved for infants weighing less than 8 kg. Therefore, dialysis for IEM hyperammonemic crisis (HAC) usually starts late when ammonia levels are higher than 1000 μmol / L, which may lead to adverse outcomes [Hediger 2018]. For early-onset crises, there is no effective acute treatment.

[0014] In addition, a significant proportion of late-onset hyperammonemic crises (HAC) (7% of 299 previously reported patients, representing 1181 acute HAs) still require rapid dialysis to avoid further neurological impairment [Enns 2007].

[0015] Alternative therapeutic interventions for treating hyperammonemic crisis (HAC) associated with IEM are needed, especially for treating pediatric patients (such as neonates) presenting with hyperammonemic crisis (HAC) associated with IEM.

[0016] This specification references multiple documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0017] The present disclosure provides alternative methods for treating hyperammonemic crisis associated with IEM. It provides liposomes that can optimize the ammonia clearance rate of peritoneal fluid, thereby enabling effective PD to be initiated immediately after confirmation of hyperammonemic crisis and before transferring the patient to a tertiary center. Any patient affected by this disease can benefit from the methods proposed herein, but it is particularly useful in pediatric patients. The treatment provided by the present disclosure alleviates the burden on patients and parents and reduces healthcare system costs.

[0018] More specifically, according to the present disclosure, the following items are provided:

[0019] Item 1. Use of a liposome suspension comprising transmembrane pH gradient liposomes in the treatment of an acute hyperammonemic crisis (HAC) associated with inborn errors of metabolism (IEM) in a subject, wherein the treatment comprises intraperitoneal administration of the liposome suspension to the subject and removal of a dialysis fluid containing ammonia-carrying 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 urea cycle disorder is ornithine transcarbamylase deficiency.

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

[0024] Item 6. The use according to Item 5, wherein the liposomes contain about 200 nM anhydrous citric acid and preferably have an internal pH of about 2.

[0025] Item 7. The use according to any one of Items 1 to 6, wherein the lipid bilayer of the liposomes comprises at least one phospholipid as a main component.

[0026] Item 8. The use according to Item 7, wherein the at least one phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), preferably in an amount of 60 mol% to 90 mol%.

[0027] Item 9. The use according to any one of Items 1 to 8, wherein the lipid bilayer of the liposomes comprises cholesterol, preferably in an amount of 10 to 40 mol%.

[0028] Item 10. The use according to Item 9, wherein the lipid bilayer of the liposomes further comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), preferably in an amount of 0.2 to 5 mol%.

[0029] Item 11. The use according to any one of Items 1 to 6, wherein the bilayer of the liposomes contains 85.5:14:0.5 mol% of dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and the internal compartment of the liposomes contains anhydrous citric acid.

[0030] Item 12. The use according to any one of Items 1 to 11, wherein the average diameter of the liposome is about 8 μm to 12 μm.

[0031] 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).

[0032] Definition

[0033] Liposome

[0034] Liposome composition

[0035] The liposome according to the present invention includes a lipid bilayer membrane encapsulating an acidic buffer (acidic solution).

[0036] Lipid bilayer membrane

[0037] In a preferred embodiment, the liposome lipid bilayer membrane contains at least one natural or synthetic phospholipid. Preferred phospholipids are long saturated phospholipids, such as those having an alkyl chain with more than 12, preferably more than 14, more preferably more than 16, and most preferably more than 18 carbon atoms.

[0038] In a specific embodiment, the natural or synthetic phospholipid comprises at least one of the following: 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-phosphoethanolamine (DMPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (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-α-phosphatidylcholine (from eggs, EPC, or from soybeans, SPC). In a specific embodiment, the natural or synthetic phospholipid is DPPC. In a specific embodiment, the major component of the liposome lipid bilayer is at least one natural or synthetic phospholipid. In a specific embodiment, 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 a specific embodiment, the natural or synthetic phospholipid accounts for approximately 85.5 mol% of the liposome bilayer membrane.

[0039] In other embodiments, the liposome lipid bilayer membrane further comprises a compound that enhances ammonia retention. In a specific embodiment, the compound that enhances ammonia retention comprises a sterol derivative. In other specific embodiments, the sterol derivative is cholesterol. In a specific embodiment, the at least one compound that enhances ammonia retention forms at least 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol% or 50 mol% of the liposome bilayer membrane. In a specific embodiment, the at least one compound that enhances ammonia retention forms at least 10 mol% of the liposome bilayer membrane. In a specific embodiment, the at least one compound that enhances ammonia retention forms about 14% of the liposome bilayer membrane.

[0040] 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 a specific embodiment, 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 a specific embodiment, the at least one steric stabilizer forms about 0.5% of the liposome bilayer membrane.

[0041] 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%, and most preferably 50 to 60 mol% of at least one sphingolipid, preferably sphingomyelin.

[0042] In other embodiments, the liposomal 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 includes hydrophobic alkyl ethers (e.g., Brij), alkyl esters, polysorbates, sorbitan esters, and / or alkylamides.

[0043] 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; between 3000 nm and 15 μm, between 4000 nm and 15 μm, between 5000 nm and 15 μm, between 6000 nm and 15 μm, between 7000 nm and 15 μm, between 8000 nm and 15 μm, between 3000 nm and 14 μm, between 4000 nm and 14 μm, between 5000 nm and 14 μm, between 6000 nm and 14 μm, between 7000 nm and 14 μm, between 8000 nm and 14 μm, between 3000 nm and 13 μm, between 4000 nm and 13 μm, between 5000 nm and 13 μm, between 6000 nm and 13 μm, between 7000 nm and 13 μm, between 8000 nm and 13 μm to avoid too rapid drainage from the peritoneal space. In a specific embodiment, the average diameter size of the liposomes is between about 8 μm and about 12 μm.

[0044] Acidic buffer / acidic solution

[0045] The acidic buffer in the internal compartment of the liposome preferably has a high buffering capacity at low pH to highly retain basic compounds (e.g., ammonia). This acid is non-toxic to animals and does not (or only weakly) permeate out of the liposomal membrane.

[0046] Without being limited thereto, the acid encapsulated in the liposomal 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 glucuronic 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, 1,2,3,4,5-butane tetracarboxylic acid; (vii) a pentacarboxylic acid such as 1,2,3,4,5-pentane pentacarboxylic acid; (viii) a polymeric poly(carboxylic acid) such as poly(acrylic acid) or poly(methacrylic acid); (ix) a polyaminocarboxylic acid such as ethylenediaminetetraacetic acid; or (x) a combination of at least two of them. In a specific embodiment, the acid is a hydroxy acid, such as citric acid (e.g., anhydrous citric acid).

[0047] In a specific embodiment, for example, the acid concentration used in the osmotic shock method can vary between 50 and 1000 mM. When using a hydroxy acid such as citric acid, the optimal concentration for use is 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 of a citric acid solution; the optimal osmotic concentration for use is between 500 and 1500 mOsmol / kg, or between 600 and 1400 mOsmol / kg, or between 700 and 1400 mOsmol / kg, or 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. In another specific embodiment, the concentration of citric acid (e.g., anhydrous) used in the method can vary between 50 and 1000 mM. When using a hydroxy acid such as citric acid, a citric acid solution of about 600 mM is used in the osmotic shock method, and its osmotic concentration is between 1000 and 1200 mOsmol / kg. In a preferred embodiment, the transmembrane pH gradient liposomes produced by the method described herein have an internal concentration of anhydrous citric acid of about 200 nM and a physiological internal osmotic pressure, i.e., about 350 mOsm / kg.

[0048] The concentration of acid within the core (the internal compartment of the liposome) can bring the pH of the liposome core to be between 1 and 6, in a specific embodiment, the pH is between 1.5 and 3, and in a more specific embodiment, the pH is about 2.

[0049] In a specific embodiment, the internal compartment / core of the liposome contains 200 nM citric acid (anhydrous), and the pH of this core is about 2.

[0050] In an alternative embodiment, the liposomes for use in the present disclosure are as described in EP 2 882 421 by Leroux et al.

[0051] Composition

[0052] According to another aspect of the present invention, there is provided a composition (in the form of a suspension or other form) comprising the liposomes of the present disclosure and at least one pharmaceutically acceptable excipient or carrier. The compositions of the present invention may contain pharmaceutically acceptable carriers / excipients, including but not limited to aqueous or non-aqueous solutions. Pharmaceutically acceptable carriers may also include physiologically acceptable aqueous carriers (e.g., sugar solutions, saline), neutralizing substances (basic or acidic, e.g., weak bases or weak acids), and chemical agents for adjusting osmotic pressure and / or providing physiological functions. Excipients covered 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 a specific embodiment, 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) to (v), preferably the combination contains all of (i) to (v).

[0053] Method for preparing liposomes

[0054] Osmotic shock method

[0055] In a specific embodiment, the lipid blend can be prepared by mixing the lipid bilayer components in a solvent such as a mixture of an alcohol or water and an organic solvent (such as an alcohol like ethanol or tert-butanol) until completely dissolved to form a homogeneous lipid mixture. The mixing can be carried out at room temperature (i.e., about 20 - 25 °C) or when heated (e.g., at a temperature up to 60 °C, preferably up to 45 °C), and optionally with slow mixing.

[0056] The mixture can be selectively filtered (e.g., through a 0.2 μm filter). Then the organic solvent is removed by, for example, freeze-drying, spray drying (e.g., using liquid nitrogen as the drying gas), rotary evaporation, or other methods.

[0057] Then the resulting dried lipid mixture can be hydrated in an aqueous medium as further described below.

[0058] Aqueous medium

[0059] In a preferred embodiment, the lipid bilayer components can be directly mixed in an aqueous medium with an osmotic pressure not exceeding 400 mOsm / l (direct lipid hydration method).

[0060] In one embodiment, the aqueous medium has a pH value of about 7, such as in the range of 6.0 to 7.5, 6.1 to 7.4, 6.2 to 7.3, 6.3 to 7.2, 6.4 to 7.1, 6.5 to 7.3, 6.6 to 7.3, 6.7 to 7.3, 6.8 to 7.3, 6.9 to 7.1, 6.95 to 7.01, or is about 7.0. In one embodiment, the aqueous medium is selected from the group consisting of water (e.g., distilled water, deionized water, ultrapure water or any other type of pure water), a mixture of water as defined above and an organic solvent (e.g., alcohol), 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 one embodiment, the aqueous medium is selected from the group consisting of an aqueous solution of an organic salt having a pH value of about 7, an aqueous solution of an inorganic salt having a pH value of about 7, an aqueous solution of an organic substance having a pH value of about 7, water, and combinations thereof.

[0061] When using an organic or inorganic salt or other organic compound, these salts or compounds are present in the aqueous medium at a low concentration in one embodiment to maintain an osmotic pressure difference between the aqueous medium and the hypertonic buffer, thereby causing an osmotic shock, and the difference is large enough to induce the diffusion of an acidic or basic hypertonic buffer into the inner compartment of the vesicle.

[0062] The aqueous medium is a water-like medium (especially in terms of pH value), but may contain a low concentration of salts or compounds, such as for buffering the pH value within a neutral range.

[0063] As described above, the osmotic pressure of the aqueous medium does not exceed 400 mOsm / l. In one embodiment, the osmotic pressure 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 one embodiment, the osmotic pressure is in the range of 1 mOsm / l to 200 mOsm / l, or in a range constructed from any of the foregoing osmotic pressures (e.g., 10 mOsm / l to 150 mOsm / l, etc.). In one embodiment, the osmotic pressure of the aqueous medium is between 0 mOsm / l and 49 mOsm / l, between 0 mOsm / l and 45 mOsm / l, between 0 mOsm / l and 40 mOsm / l, especially between 0 mOsm / l and 35 mOsm / l, especially between 0 mOsm / l and 30 mOsm / l, between 0 mOsm / l and 25 mOsm / l.

[0064] In a specific embodiment, the liposomes can be optionally extruded or filtered to obtain liposomes having a specific size.

[0065] The hydration of the lipid bilayer component / lipid blend can be carried out at room temperature (i.e., about 20 - 25 °C), or while heating (e.g., at a temperature up to 60 °C (e.g., a preheated aqueous medium), preferably up to 45 °C), and optionally with slow stirring for about 15 minutes to 4 hours, preferably about 2 hours. At this stage, if the hydration is carried out while heating the mixture, the final concentration of the lipid is preferably about 100 mg / g, and then cooled to room temperature (i.e., about 20 - 25 °C). The mixture can be optionally degassed (e.g., under vacuum) to remove air bubbles.

[0066] In one embodiment, the hydrated liposomes thus prepared are sterilized to obtain sterilized liposomes or a sterilized suspension containing liposomes. Sterilization can be carried out, for example, by sterile filtration or steam sterilization (e.g., autoclaving), for a time, for example, of about 5 minutes to 2 hours, 10 minutes to 1 hour, or 15 minutes or 30 minutes.

[0067] In another embodiment, the vesicles are stored for a period of time before the step of mixing the liposomes (or the suspension containing liposomes) with an acidic buffer. This storage can be optimally achieved if the liposomes are sterilized after the aqueous medium hydration step, since no or hardly any degradation process occurs in the sterilized liposome suspension. The 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 sterile liposomes contained in the aqueous medium are stable entities. Since they do not yet contain any specific basic buffer for preparing the pH gradient, there is no need to worry about buffer loss due to liposome bilayer degradation or liposome leakage. In one embodiment, this is also true if the liposomes contain a small amount of electrolyte molecules, since the corresponding osmotic pressure inside the vesicles will be in the range between 0 or 1 mOsm / l and 200 mOsm / l.

[0068] Acidic hyperosmotic buffer

[0069] Thereafter, the hydrated (and optionally sterilized) liposomes are mixed with an acidic buffer having an osmotic pressure at least 200 mOsm / l higher than that of the aqueous medium to apply an osmotic shock to the liposomes and obtain buffer-filled liposomes. In one embodiment, the osmotic pressure of the acidic buffer is 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, at least 550 mOsm / l, at least 600 mOsm / l, at least 650 mOsm / l, at least 700 mOsm / l, at least 750 mOsm / l, at least 800 mOsm / l, at least 850 mOsm / l, at least 900 mOsm / l, at least 950 mOsm / l, at least 1000 mOsm / l, at least 1050 mOsm / l, at least 1100 mOsm / l or at least 1200 mOsm / l higher than that of the aqueous medium. In one embodiment, the osmotic pressure of the acidic buffer is in the range of 200 mOsm / l to 1100 mOsm / l higher than that of the aqueous medium, or in a range constructed from any of the foregoing osmotic pressures (e.g., 220 mOsm / l to 1200 mOsm / l, etc.).

[0070] Thus, the acidic buffer is a hypertonic buffer relative to the aqueous medium used in the liposome hydration step. In doing so, an osmotic shock is temporarily applied to the liposomes. This osmotic shock causes the acidic buffer to enter the liposomes. Thus, the osmotic shock can render the liposomes short-term unstable to incorporate the buffer into the liposomes. The result is buffer-filled liposomes. In one embodiment, the hypertonic buffer can also contain electrolytes for adjusting the osmotic pressure or having physiological functions.

[0071] An amount of acidic buffer sufficient to cause an osmotic shock if not added to the liposomes suspended in the aqueous medium is added. Depending on the difference between the osmotic pressure of the aqueous medium and the osmotic pressure of the base buffer, the sufficient amount can be 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. In one embodiment, the acidic buffer can be added in a volume equal to the volume of the aqueous medium. In one embodiment, the volume of acidic buffer to be added can be 0.1 times to 5 times the volume of the aqueous liposome suspension or any other range constructed from the foregoing values (e.g., 0.3 times to 3 times, etc.).

[0072] In one embodiment, the pH of the hyperosmotic buffer 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.

[0073] In specific embodiments, the hyperosmotic buffer may contain additional chemical agents, such as complexing or chelating agents.

[0074] In specific embodiments, the hypertonic buffer comprises a salt, such as, but not limited to, sodium chloride, sodium hydroxide, and / or magnesium chloride.

[0075] In the specific embodiment of the preferred sterile transmembrane pH gradient liposomes, the acidic buffer is sterile. In this embodiment, the hydrated liposomes are also sterilized before loading the acidic buffer, and a completely sterile buffer-filled liposome or a completely sterile suspension containing the buffer-filled liposome is prepared. Sterilization can be carried out by, for example, sterile filtration or autoclaving.

[0076] In one embodiment, the mixture of aqueous medium and alkaline or acidic buffer in which the buffer-filled liposomes are suspended has an osmotic pressure of at least 200mOsm / l, at least 220mOsm / l, at least 250mOsm / l, at least 300mOsm / l, at least 350mOsm / l, at least 400mOsm / l, at least 450mOsm / l, at least 500mOsm / l, at least 550mOsm / l. In one embodiment, the osmotic pressure is in the range of 200mOsm / l to 550mOsm / l or in the range established by any of the aforementioned osmotic pressures (such as 220mOsm / l to 500mOsm / l, etc.).

[0077] The liposome acidic buffer mixture may optionally be incubated.In specific embodiments, the mixture is stirred (eg, by orbital shaking), eg, at room temperature.

[0078] Neutralized aqueous solution

[0079] Then, the mixture of the aqueous medium containing buffer liposomes and the acidic buffer is diluted by adding a neutralizing aqueous solution. The mixture of the acidic buffer and the neutralizing solution constitutes the suspension buffer. Thus, after dilution, the transmembrane pH gradient liposomes are suspended in the suspension buffer. Accordingly, the pH of the suspension buffer is different from the acidic buffer contained in the buffer-filled liposomes. In one embodiment, 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.

[0080] In one embodiment, the pH of the neutralizing solution is from pH 7.1 to pH 14, from pH 7.1 to pH 13.5, from pH 7.1 to pH 13.0, from pH 7.1 to pH 12.5, from pH 7.1 to pH 12, from pH 7.1 to pH 11.5, from pH 7.1 to pH 11.0, from pH 7.1 to pH 10.5, from pH 7.1 to pH 10, from pH 7.1 to pH 9.5, from pH 7.1 to pH 9.0, from pH 7.1 to pH 8.5, from pH 7.2 to pH 14, from pH 7.2 to pH 13.5, from pH 7.2 to pH 13, from pH 7.2 to pH 12.5, from pH 7.2 to pH 12, from pH 7.2 to pH 11.5, from pH 7.2 to pH 11, from pH 7.2 to pH 10.5, from pH 7.2 to pH 10, from pH 7.2 to pH 9.5, from pH 7.2 to pH 9, from pH 7.2 to pH 8.5, from pH 7.3 to pH 14, from pH 7.3 to pH 13.5, from pH 7.3 to pH 13, from pH 7.3 to pH 12.5, from pH 7.3 to pH 12, from pH 7.3 to pH 11.5, from pH 7.3 to pH 11, from pH 7.3 to pH 10.5, from pH 7.3 to pH 10, from pH 7.3 to pH 9.5, from pH 7.3 to pH 9, from pH 7.3 to pH 8.5, from pH 7.4 to pH 14, from pH 7.4 to pH 13.5, from pH 7.4 to pH 13, from pH 7.4 to pH 12.5, from pH 7.4 to pH 12, from pH 7.4 to pH 11.5, from pH 7.4 to pH 11, from pH 7.4 to pH 10.5, from pH 7.4 to pH 10, from pH 7.4 to pH 9.5, from pH 7.4 to pH 9, from pH 7.4 to pH 8.5, from pH 7.5 to pH 14, from pH 7.5 to pH 13.5, from pH 7.5 to pH 13, from pH 7.5 to pH 12.5, from pH 7.5 to pH 12, from pH 7.5 to pH 11.5, from pH 7.5 to pH 11, from pH 7.5 to pH 10.5, from pH 7.5 to pH 10, from pH 7.5 to pH 9.5, from pH 7.5 to pH 9, from pH 7.5 to pH 8.5, from pH 8.0 to pH 13.0, from pH 8.5 to pH 12.5, from pH 9.0 to pH 13, from pH 9.0 to pH 12.5, from pH 9.0 to pH 12.0, from pH 9.5 to pH 11.5, from pH 10 to pH 13, from pH 10 to pH 12.5, from pH 10 to pH 12.0, from pH 10 to pH 11.5. pH from 10 to 11, pH from 10 to 12.5, pH from 10.5 to 12.0, pH from 10.5 to 13, pH from 10.5 to 12.5, pH from 10.5 to 12.0, pH from 10.5 to 11.5, or pH from 10.5 to 11. In a specific embodiment, the pH of the neutralizing solution is about 12.5.

[0081] In one embodiment, the neutralizing solution has a composition that does not disrupt the buffer-filled vesicles, thus not disrupting the stability of these vesicles. It may contain neutralizing substances (alkaline or acidic, such as weak bases or weak acids), and may also contain chemical agents for regulating osmotic pressure and / or providing physiological functions. Calcium salts can be added during the preparation to counteract the anticoagulant effect of certain weak acids (such as citric acid). This is particularly important when the vesicles are used for in vivo applications. Sodium hydroxide, sodium salts (such as sodium chloride), potassium chloride, calcium magnesium salts, lactate, glycerol, icodextrin, glucose, sorbitol, fructose, amino acids, or xylitol can also be used as components of the neutralizing solution. In a specific embodiment, the neutralizing solution comprises

[0082] In the examples, the osmotic pressure of the neutralizing solution is from 250 mOsm / l to 550 mOsm / l, from 270 to 520 mOsm / l, from 290 to 500 mOsm / l, from 300 to 480 mOsm / l, from 320 to 450 mOsm / l, from 330 to 420 mOsm / l, from 350 to 400 mOsm / l, from 375 to 400 mOsm / l, from 385 to 400 mOsm / l, or from 390 to 400 mOsm / l.

[0083] In one embodiment, the osmotic pressure of the neutralizing solution is higher or lower than the osmotic pressure of the mixture containing buffer-containing vesicles (i.e., the buffer-containing vesicle solution) by less than 200 mOsm / l, particularly less than 150 mOsm / l, particularly less than 100 mOsm / l, particularly less than 50 mOsm / l, particularly less than 20 mOsm / l, particularly less than 10 mOsm / l. In one embodiment, the osmotic pressure difference between the neutralizing solution and the mixture containing buffer-containing vesicles is between 1 mOsm / to 200 mOsm / l, particularly between 10 mOsm / to 150 mOsm / l, particularly between 20 mOsm / to 100 mOsm / l, particularly between 30 mOsm / to 80 mOsm / l, particularly between 40 mOsm / to 60 mOsm / l.

[0084] Due to the pH difference between the suspension buffer and the acidic buffer, a transmembrane pH gradient is formed between the inside of the liposome and the suspension buffer surrounding it. The resulting transmembrane pH gradient can be used according to the present disclosure.

[0085] In one embodiment, the pH 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, 7.0 to 7.4. Thus, the suspension buffer may have a physiological pH value. In a specific embodiment, the pH of the suspension buffer is about 6.5.

[0086] The osmotic shock method is also described in EP 3 291 797 (Leroux et al.).

[0087] Alternative methods for preparing liposomes

[0088] In another embodiment, the method for preparing the transmembrane pH gradient liposomes comprises the thin film hydration method. For example, the liposome bilayer components are dissolved in an organic solvent (such as dichloromethane:methanol), and then the organic solvent is removed (such as by rotary evaporation) to form a dry lipid film. The dry lipid can be stored for future use (such as under vacuum). The dry lipid can then be directly hydrated in the acidic buffer described above, and the external solution can be exchanged with the neutral solution as described above. Alternatively, a lipid film can be first formed using the thin film hydration method and then hydrated in an aqueous medium as described above.

[0089] Subsequently, the liposomes loaded with the aqueous medium can be subjected to the osmotic shock step described above to load the acidic buffer therein, and the neutralization solution step described above can be carried out to produce a suspension of transmembrane pH gradient liposomes.

[0090] Alternative methods for preparing liposomes are also described in EP 2 882 421 of Leroux et al.

[0091] Route of administration and mechanism of action

[0092] The liposomes of the present disclosure are administered intraperitoneally.

[0093] As used herein, the term "intraperitoneal administration" should be understood as what is commonly understood by those skilled in the art of peritoneal dialysis therapy. To practice the present invention, a pharmaceutically effective amount of the 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, through a catheter, such as a catheter commonly used for puncture.

[0094] Liposomes within the cavity and nearby tissues and organs will absorb ammonia according to the pH gradient on the liposome membrane. The pH of the acidic buffer contained within the liposomes is lower than the physiological pH within the peritoneal cavity (which is approximately 7.5 to 8). Thus, ammonia can diffuse through the hydrophobic liposome bilayer in an uncharged state and then be trapped in the inner liposome compartment in a protonated (ionized) state (such as ammonium).

[0095] The liposomes can sequester ammonia for an extended period and reduce the toxic concentration of the free compound. The ammonia-loaded liposomes in the peritoneal cavity are removed / extracted from the peritoneal cavity together with the fluid (dialysate) present therein. Intraperitoneal administration and extraction can be subsequent and / or simultaneous. Without being limited thereto, 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 for infusion.

[0096] Disease

[0097] Acute hyperammonemia in the context of IEM is referred to as "HAC" or "acute hyperammonemic crisis", representing the same medical condition. HAC is defined as a plasma ammonia level higher than 80 - 100 μmol / L in neonates up to 1 month old and higher than 55 μmol / L in older children and adults [Haeberle 2013]. In published longitudinal studies of IEM, HAC has been defined as "compatible clinical symptoms associated with a plasma ammonia level > 100 μmol / L" [Kent 2017] or "defined as an acute hyperammonemic crisis with a single hospitalization due to hyperammonemia" [Enns 2007].

[0098] In mammals, the hepatic urea cycle is the main pathway for ammonia detoxification. Ammonia is continuously produced by the breakdown of proteins and other nitrogen-containing molecules. Whenever the waste nitrogen load exceeds the detoxification capacity, a hyperammonemic crisis occurs.

[0099] Hyperammonemia rapidly leads to cerebral edema and related symptoms such as lethargy, anorexia, hyperventilation or hypoventilation, hypothermia, seizures, abnormal postures, and coma. These events occur through multiple mechanisms, mainly due to increased glutamine causing astrocyte swelling. The specific roles of ammonia, glutamate, and glutamine in cerebral edema are still under investigation, but recent data show that excessive ammonia exposure alters several amino acid pathways and neurotransmitter systems, brain energy, nitric oxide synthesis, axon and dendrite growth, signal transduction pathways, and K+ and water channels. All of these effects may ultimately lead to energy deficiency, oxidative stress, and cell death.

[0100] Etiology

[0101] As described above, the IEMs that cause HAC include a group of genetic diseases in which a single gene defect results in a clinically significant blockage of the urea cycle responsible for the metabolic clearance of ammonia from the bloodstream.

[0102] As described above, when HAC is secondary to a genetic defect in any enzyme or transporter involved in the urea cycle, HAC associated with IEM is called "primary hyperammonemia", which defines UCBs, while when the enzymes of the urea cycle are inhibited due to metabolite accumulation or substrate deficiency, it is called "secondary hyperammonemia". The group of diseases most associated with secondary hyperammonemia is called organic acidemias (Oas). Regardless of the underlying genetic disease, the clinical features, outcomes, prognosis, and treatment of HAC associated with IEM are similar.

[0103] Potential genetic defects in IEM

[0104] UCD

[0105] UCDs are the most common genetic cause of HAC in infants and children. They are caused by genetic defects in any of the genes encoding the five enzymes, cofactors, and two transporters of the Krebs-Henseleit cycle / urea cycle [Ah Mew 2013]. The five catalytic enzymes are carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG1) (the defect abbreviations are as follows: CPS1D, OTCD, ASSD, ASLD, and ARG1D, with their respective MIM numbers: 237300, 311250, 215700, 207900, 207800); the enzyme that produces the cofactor is N-acetylglutamate synthetase (NAGS) (MIM number 237310), which is the main activator of CPS1; the two amino acid transporters are ornithine translocase (ORNT1), which induces hyperornithinemia, hyperammonemia-homocitrullinuria syndrome (triple H syndrome (HHH)) (MIM number 238970), and citrin.

[0106] The urea cycle occurs mainly in the human liver and intestine as a nitrogen clearance system, and CPS1 and OTC are limited to these tissues. The distribution of the downstream enzymes that process citrulline into arginine is widespread. As the rate-limiting enzyme in the urea cycle, functional changes in the enzyme CPS1 are expected to have the greatest impact on the cycle function.

[0107] OTC deficiency is inherited in an X-linked manner. The remaining seven urea cycle disorders (deficiencies of CPS1, ASS, ASL, ARG1, NAGS, ORNT1, and citrin) are inherited in an autosomal recessive manner.

[0108] Organic aciduria / acidemia

[0109] Organic aciduria (Oas) is a genetic disorder that mainly affects 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.

[0110] Hyperammonemia in Oas is due to decreased acetyl coenzyme A (CoA) and inhibition of NAGS and CPS1 activities caused by the toxic accumulation of organic acid metabolites. In these diseases, HAC is accompanied by severe metabolic acidosis, a high anion gap, and ketonuria. Organic aciduria can lead to long-term complications in many organs, including the brain, kidneys, heart, bones, and pancreas, but mainly results in a neurological phenotype.

[0111] Although most osteochondrodysplasias are autosomal recessive genetic disorders, some are X-linked. The diagnosis of hyperammonemia associated with OA is made through plasma ammonia levels, urinary organic acid chromatography, and plasma acylcarnitine profiles [Savy 2018].

[0112] Regardless of the underlying genetic disease, the clinical features, outcomes, and prognosis of primary and secondary HAC associated with IEM, as well as current treatment methods, are similar.

[0113] Clinical features of HAC associated with IEM

[0114] As mentioned above, regardless of the underlying genetic disease, the clinical features / manifestations (symptoms) of primary and secondary HAC associated with IEM are similar.

[0115] The clinical manifestations of patients with HAC caused by IEM may start as early as the first few days of life or as late as adulthood, presenting as acute or chronic. The severity often inversely correlates with the age of the subject.

[0116] The clinical features of subjects with HAC caused by IEM are neurological, psychiatric, or hepatic / gastrointestinal symptoms, or a combination of these three categories of symptoms. Without treatment or a delay in diagnosis, survivors will develop severe neurological disabilities and epilepsy. Even in cases of partial defects, where the manifestations are more variable and occur later (at any age), there is a risk of sequelae neurological symptoms related to hyperammonemia and a risk of death. There is a strong correlation between the duration and severity of hyperammonemia and brain injury, so rapid diagnosis and appropriate treatment are crucial for optimizing patient outcomes.

[0117] The severity of the symptoms is related to the residual enzyme activity and the position of the deficient enzyme in the urea cycle. Thus, severe enzyme deficiencies or complete absence of enzyme activity are responsible for the accumulation of ammonia and other metabolites from the first day of life, while partial deficiencies lead to varying degrees of hyperammonemia, usually triggered by catabolic events, protein overload, or certain medications, regardless of age. The history and family history should be carefully and systematically verified. This includes addressing the following: unexplained deaths; the presence of mental or neurological disorders in the family; consanguinity (common in all UCDs except OTCD related to the X chromosome); evidence that the patient and / or family members spontaneously avoid protein; medications taken by the patient.

[0118] Clinical laboratory data commonly used to diagnose hyperammonemia associated with UCDs include plasma ammonia levels, pH, CO2, anion gap, plasma amino acids, and urine organic acid analysis.

[0119] General acute manifestations. Altered level of consciousness (ranging from lethargy / somnolence to coma), reminiscent of encephalitis or drug poisoning; acute encephalopathy; seizures (usually associated with an altered level of consciousness rather than isolated); ataxia (usually associated with impaired level of consciousness); pseudo-stroke episodes; transient blindness; vomiting and progressive anorexia; liver insufficiency; multi-organ failure; peripheral circulatory disorders; postpartum psychosis; dysarthria; asterixis (in adults); learning disabilities, neurodevelopmental delay, intellectual disability; chorea, cerebral palsy; permanent cortical blindness; progressive spastic diplegia or quadriplegia (described in ARGD1 or tri-H syndrome); protein aversion, voluntary low-protein diet; abdominal pain, vomiting; growth retardation; hepatomegaly, elevated liver enzymes; migraine-like headache, tremors, ataxia, mental symptoms (hyperactivity, mood changes, behavioral changes, aggression, hallucinations, paranoia, manic episodes, mood disorders, and personality changes); self-harm; pseudo-autistic symptoms; brittle hair (characteristic of ASLD); specific neuropsychological phenotypes in heterozygous OTC patients; occasional nature of signs and symptoms.

[0120] Neonatal presentation. Affected neonates are usually asymptomatic at birth. After a period of rest of 24 hours to a few days, they rapidly become irritable and quickly progress to lethargy / somnolence, anorexia / feeding refusal and vomiting, loss of thermoregulation, abnormal neurological postures, cerebral oedema leading to lethargy, hyperventilation then hypoventilation, hypothermia, hypotonia or hypertonia, seizures / epilepsy, coma, multi-organ failure and death [Ah Mew 2013; Summar 2008; Haeberle 2013]. These symptoms may be similar to a range of sepsis, which may lead to a delay in diagnosis. The clinical presentation of HAC associated with the milder forms of IEM with residual enzyme activity usually occurs later in life (any time from infancy to adulthood, late-onset), with recurrent episodes of mild to moderately severe hyperammonaemia, which may be triggered by illness, stress or excessive protein intake [Summar 2008]. In late-onset IEM, HAC is usually less severe and the symptoms are also more insidious.

[0121] It has been reported that only 27% of 678 UCD patients enrolled in the UCDC natural history study had neonatal HAC. Similarly, Summar

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

[0122] Adult presentation. Adults with UCD deficiency (over 16 years) either come from a paediatric cohort or are diagnosed at a later age (any age, young or old). In both cases, the clinical presentation is the same as in the infant or adolescent form.

[0123] The diagnosis of UCD in adulthood is usually made during an acute decompensation. These are mostly OTC deficiencies and mostly affect females. The clinical symptoms are mainly neurological, with disturbances of consciousness and psychiatric symptoms as the main manifestations. Only a small proportion of patients have never shown signs of decompensation before, and two-thirds of patients have an aversion to animal protein and are vegetarians by choice.

[0124] Inducing factors for HAC in UCD patients. Infection; fever; vomiting, diarrhea; internal or gastrointestinal bleeding; reduced protein or energy intake (e.g., preoperative fasting, significant neonatal weight loss); postpartum uterine catabolism and involution (mainly seen in OTC patients); chemotherapy, high-dose glucocorticoids; strenuous or prolonged physical exercise; surgery under general anesthesia; excessive protein intake (e.g., protein-rich foods: meat, fish, eggs, dairy products; inappropriate artificial nutrition). Drugs: mainly valproic acid, L-asparaginase / pegaspargase. Topiramate, carbamazepine, phenobarbital, phenytoin, primidone, furosemide, hydrochlorothiazide, and salicylates are also associated with decompensated hyperammonemia; more specifically for adults: rapid weight loss (weight-loss diet, bariatric surgery), anorexia, postpartum.

[0125] Results and prognosis

[0126] Early age of onset (Figure 1B) and severe clinical manifestations (coma and high plasma ammonia levels > 1000 μmol / L) (Figure 1C) are associated with high mortality and poor neurological prognosis [Krivitzky 2009; Enns 2007]. It is estimated that until recently, approximately 25% of neonates with hyperammonemia exposure due to UCD died prematurely, and this proportion has been continuously increasing (Figure 1D) [Hediger 2018].

[0127] Historically, most children with early-onset neonatal HAC due to severe UCD or OA enzyme deficiency died in the neonatal period, and few children survived into infancy. Although HAC with a later onset associated with IEM usually has a better prognosis [Enns 2007; Hediger 2018], the incidence of neurological diseases is still high, and a significant proportion of patients exhibit poor neurocognitive outcomes and behavioral disorders [Krivitzky 2009; Ah Mew 2013]. Krivitzky

[2009] reported that approximately half of the children with HAC in the neonatal period showed intellectual disability, and 30% of them had severe intellectual impairment. In contrast, although there is evidence of neurocognitive and behavioral disorders related to attention and executive function, only one-quarter of the late-onset group was affected by intellectual disability (Figure 1B).

[0128] Published data from 103 neonate-onset HAC subjects in a UCD longitudinal study showed that among all types of enzyme deficiencies, 47% to 68% of pediatric patients had a poor prognosis (intelligence quotient / development quotient < 70). This phenomenon was observed in both patients younger than 4 years and those 4 years and older, and there was no difference between proximal UCD patients ( Figure 1E ) and distal UCD patients (i.e., ASS and ASL).

[0129] Combined treatment

[0130] The present disclosure includes combining the intraperitoneal administration of the transmembrane pH gradient liposomes described herein with other therapies for treating HAC associated with IEM (simultaneously or sequentially (e.g., first the presently disclosed emergency treatment followed by at least one other treatment), depending on the nature of the additional treatment) for acute or chronic treatment.

[0131] Treatment and prevention

[0132] The present disclosure includes using the liposomes / liposome suspensions or compositions described herein to treat acute hyperammonemic crises (HACs) associated with inborn errors of metabolism (IEMs) in a subject. It may also include treating or preventing any HAC symptoms associated with IEMs downstream of acute hyperammonemia itself, as further described herein.

[0133] As used herein, the term "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 reduction / decrease in the frequency, duration, and / or severity of HAC associated with IEM. It may also include one or more of the following: reducing / decreasing the frequency, duration, and / or severity of at least one symptom caused by HAC; and / or the duration of an asymptomatic period; administering the liposomes of the present disclosure described herein alone or in combination with other agents for treating HAC associated with IEM or at least one of its symptoms; or administering a composition (e.g., a suspension) comprising the liposomes of the present disclosure.

[0134] As used herein, the term "prevention" refers to eliciting a desired biological response, i.e., a preventive effect. According to the disclosure provided herein, in some embodiments, a preventive effect includes completely or partially avoiding / suppressing at least one symptom of HAC associated with IEM after administering the liposomes or liposome suspension or composition of the present disclosure alone or in combination with another drug for preventing or treating muscular dystrophy or at least one of its symptoms.

[0135] In some embodiments, a "therapeutically effective amount" or "effective amount" or "therapeutically effective dose" of the liposomes or liposome suspension of the present disclosure provided herein results in treatment of HAC associated with IEM in a subject in need thereof. It may also result in treating or preventing at least one symptom of the subject.

[0136] As used herein, the term "at least one symptom of HAC associated with IEM" refers to any clinical feature of a pediatric or adult subject described under the headings "General Acute Presentation", "Neonatal Presentation", or "Adult Presentation" and corresponding to an event caused at least in part by acute hyperammonemia itself.

[0137] Subjects

[0138] As used herein, the term "subject" or "subject in need" refers to a subject who would benefit from receiving an effective amount of the liposome and liposome suspension. It refers to animals, mammals, and in certain embodiments, humans. The compositions of the present invention can also be used in veterinary applications and for pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cows, fish, pigs, poultry, etc.). In a specific embodiment, the subject has HAC associated with IEM. In a specific embodiment, the subject has a healthy liver and / or does not have 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 neonate (i.e., from birth to the first 28 days after birth), an infant (about 29 days to less than 2 years), a child (2 years to less than 12 years), or an adolescent (about 12 years to 21 years) with recurrent HAC. In another more specific embodiment, the subject is a neonate, infant, or child. In another specific embodiment, the subject is an adult.

[0139] The subject in need is pre-diagnosed with HAC associated with IEM. In certain embodiments, the methods of the present disclosure include the step of diagnosing the subject.

[0140] In the context of describing the present disclosure (particularly in the context of the following claims), the use of the terms "a," "an," and "the" and similar references should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context.

[0141] Unless otherwise indicated, "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to").

[0142] Unless otherwise indicated herein, the recitation of numerical ranges herein is merely intended as a convenient method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All sub-range values within the range are also incorporated into the specification as if they were individually recited herein.

[0143] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order.

[0144] The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the present disclosure and does not limit the scope of the present disclosure unless otherwise stated.

[0145] No language in the specification should be construed as indicating that any non-claimed element is essential for the practice of the present disclosure.

[0146] The term "about" herein has its ordinary meaning. In embodiments, it may mean plus or minus 10% of a qualified value.

[0147] 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 pertains.

[0148] Other objects, advantages, and features of the present disclosure will become more apparent by reading the following non-limiting description of specific embodiments of the present disclosure given by way of example only with reference to the drawings. Description of the Drawings

[0149] In the drawings:

[0150] Figure 1A: Number of patients by diagnosis and age at first onset. Each patient was counted once at the age of first presentation of hyperammonemia [Summar 2008].

[0151] Figure 1B: Cognitive range of UCD subjects aged 3 - 6 years: Neonatal (NO) vs. late-onset (LO) [Krivitzky 2009].

[0152] Figure 1C: Survival rate of hyperammonemic crises calculated according to peak ammonia levels was calculated based on the number of episodes for which plasma ammonia level data were available [Enns 2007].

[0153] Figure 1D: Relationship between the main outcome parameters of "normal outcome", "death", "disability", and "alive but no further information" (alive-no-info) specified for individual and group UCDs. Absolute numbers are given above each column [Hediger 2018].

[0154] Figure 1E: Neurodevelopmental outcomes of subjects 4 years of age and older as determined by diagnosis [Ah Mew 2013]. Each diagnostic cohort, proximal UCD (CPSD / OTCD), ASD, ALD, and the entire neonatal UCD cohort were stratified according to neurodevelopmental outcomes. When there was no Full Scale IQ (FSIQ), Verbal IQ or Performance IQ was used to determine classification. In the profound / severe disability range, the age range of subjects could not be tested by traditional IQ tests, and the Bayley Scales were used to derive DQ; in the mild - moderate disability range, the FSIQ score was 45 - 69. In the low average / marginal function range, the FSIQ score was 70 - 89; in the approximately average range, the FSIQ score was 90 - 109; in the above - average range, the FSIQ score was 110 (no subjects met this criterion).

[0155] Figure 2 : A linear regression model for estimating the relationship between peritoneal fluid ammonia clearance rate (mL / min) and the volume of VS - 01 infusion (mL).

[0156] Figure 3 : Mean ammonia concentration in peritoneal fluid of B6EiC3Sn a / AN - Otc Spf - ash / J mice after a single use of VS - 01; Sidak multiple comparison test.

[0157] Figure 4A - Mean blood ammonia concentration of B6EiC3Sn a / AN - Otc Spf - ash / J mice after a single VS - 01 treatment. Figure 4B Replicated Figure 4A the graph of, but in addition to the mean of all mice in the VS - 01 group, it also included the ammonia concentrations reached by four mice (i.e., 2005 (outlier), 2006, 2007, and 2008) at 1 hour after dosing. Dunnett's multiple comparison test. Detailed Description of the Invention

[0158] The present disclosure is further elaborated in detail by the following non - limiting examples.

[0159] Example 1 Materials and Methods

[0160] Mouse

[0161] Hemizygous male B6EiC3Sn a / AN-Otc Spf-ash / J mice, strain number: 002343. RRID: IMSR_JAX: 002343 (common name: sparse fur), used for analysis. These mice are characterized by late and uneven hair development and carry a spontaneous Otcspf (sparse hair) mutation on the X chromosome, namely a C to A missense transversion mutation in exon 4, resulting in a change from histidine to asparagine (H117N), thus generating a hypomorphic allele. This mutation causes the activity of liver ornithine transcarbamylase (OTC) to be only 5-10% of the normal value, ultimately leading to an increase in plasma ammonia concentration. This deficiency of liver OTC is similar to that of childhood congenital hyperammonemia type II, which is characterized by life-threatening acute metabolic decompensation episodes accompanied by hyperammonemia.

[0162] Liposome solution preparation

[0163] Liposomes composed of dipalmitoylphosphatidylcholine (DPPC, Lipoid), cholesterol (Sigma-Aldrich), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG, Lipoid) in a molar ratio of 85.5:14:0.5 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 dissolved together in a 70:30 w / w tert-butanol:water mixture to form a homogeneous mixture. Subsequently, the organic solvents were removed by freeze-drying. The dried lipid mixture was mixed with ultrapure water (lipid concentration = 100 mg / mL), heated, and slowly mixed at 60 °C for 2 hours, and finally autoclaved at 121 °C for 15 minutes in a sealed bottle. Liposomes with an average diameter of approximately 8 μm to 12 μm were obtained.

[0164] 68.1 mL of the resulting liposomes 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 orbital shaking. The resulting liposomes contained 200 mM citric acid and had an internal pH value of approximately 2.

[0165] The transmembrane pH gradient was generated by neutralizing the external acidic medium with 950 mL of a neutralizing solution (pH = 12.5, 392 mOsm / l) made of xylitol, sodium chloride, sodium hydroxide, potassium chloride, and calcium chloride. The resulting multilamellar liposome suspension contained 18.4 mM anhydrous citric acid, had a pH value of 6.5, and a concentration of approximately 310 mOsm / l, and was used for preclinical studies.

[0166] Study design:

[0167] This non-clinical study was conducted in accordance with good scientific principles and internal standard operating procedures of Charles River Laboratories, Inc. (01545, Massachusetts, USA).

[0168] We obtained 22 male B6EiC3Sn a / AN-Otc Spf-ash / J mice (aged approximately 7–8 weeks at the start of dosing) from The Jackson Laboratory. The animals were individually housed in polycarbonate cages with appropriate bedding. Upon arrival, all animals had free access to LabDiet TM 5LG4 and hydrogel particles. Municipal tap water treated by reverse osmosis and ultraviolet irradiation was available ad libitum to all animals. On Day -1, all animals were weighed, and blood ammonia readings were taken using a handheld meter (ARKRAY PocketChem BA) and assigned to treatment groups to generate groups with no significant differences in body weight and blood ammonia. Treatment was initiated on Day 1 under fasting conditions.

[0169] On the day of the experiment, the liposome solution VS-01 was reconstituted under a laminar flow hood using a clean procedure. A commercial dialysate (Dianeal) was used as a control.

[0170] Animals in Group 1 and Group 2 were injected intraperitoneally with 100 mL / kg of Dianeal (control) and liposome solution (VS-01) (354 mg / kg / day citric acid), respectively. The solutions were left in the peritoneal cavity for 0.5, 1, and 2 hours according to the sampling time points specified below.

[0171] After a 5-hour fast on Day 1, ammonia levels in whole blood from submandibular tissue were measured at the following time points: all groups were measured before dosing, and then in a staggered manner after dosing. Three animals in Group 1 were measured at 0.5, 1, and 2 hours after dosing, four animals in Group 2 were measured at 0.5 and 1 hours after dosing, and five animals in Group 2 were measured at 2 hours after dosing.

[0172] According to the same schedule, peritoneal dialysis fluid samples were collected by peritoneal aspiration (21g needle / 1 mL syringe) within + / - 10 minutes, transferred to cryotubes with unique labels, and frozen on dry ice. All samples were stored in a refrigerator set at -80 °C within 1 hour 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.

[0173] Example 2 The clearance rate of ammonia in the peritoneal fluid of the UCD model increased significantly with the increase in the dosage of VS-01.

[0174] All animals tolerated the dose well and showed no signs of treatment-related adverse reactions.

[0175] At all time points during the residence time, compared with the control solution, the ammonia extracted from the blood into the peritoneal cavity after a single intraperitoneal injection of the liposome suspension of Example 1 (VS-01) (354 mg / kg / day citric acid) was significantly (p < 0.0006) higher ( Figure 3 ).

[0176] This led to a significant decrease in blood ammonia at 0.5 hours (p < 0.0089) and 2 hours (p < 0.0007) after administration ( Figure 4A ). At 1 hour after administration, an abnormal value of the average blood ammonia of animal 2005 was observed, which may be related to the injection procedure, which may have increased the systemic exposure of VS-01 ( Figure 4B ).

[0177] The ammonia clearance rate in the peritoneal fluid of OTC mice was calculated by the blood exposure to ammonia (AUC0-2) during a 2-hour residence time and the amount of ammonia extracted 2 hours after administration (amount = peritoneal fluid concentration x injected fluid volume). The average ammonia clearance rates in the peritoneal fluid after treatment with VS-01 and Dianeal were 0.3 mL / min and 0.1 mL / min, respectively.

[0178] The above results indicate that VS-01 is helpful in treating HAC related to IEM.

[0179] 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 entire specification.

[0180] References

[0181] Ah Mew 2013 Clinical Outcomes of Neonatal Onset Proximal versus Distal Urea Cycle Disorders Do Not Differ.

[0182] The Journal of Pediatrics.

[0183] Enns 2007 Survival after Treatment with Phenylacetate andBenzoate for Urea-Cycle Disorders N Engl J Med 2007;356:2282-92.

[0184] Haeberle 2013 Clinical and biochemical aspects of primary and secondary hyperammonemic disorders Archives of Biochemistry and Biophysics 536 (2013) 101–108.

[0185] Hediger 2018 The impact of ammonia levels and dialysis on outcome in 202 patients with neonatal onset urea cycle disorders J Inherit Metab Dis.

[0186] Horslen 2003 Isolated Hepatocyte Transplantation in an Infant With a Severe Urea Cycle Disorder Pediatrics 2003;111:1262–1267.

[0187] Kent 2017 Hyperammonemic crises in patients with urea cycle disorders on chronic nitrogen scavenger therapy with either sodium phenylbutyrate or glycerolphenylbutyrate Neuropsychiatry (London) (2017) 7(2), 131–136.

[0188] Krivitzky 2009 Intellectual, Adaptive, and Behavioral Functioning in Children with Urea Cycle Disorders Pediatr Res. 2009 July;66(1):96–101.

[0189] Leonard 2004 The role of liver transplantation in urea cycle disorders Molecular Genetics and Metabolism 81(2004)S74–S78.

[0190] 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).

[0191] Savy 2018 Acute pediatric hyperammonemia: current diagnosis and management strategies, Hepatic Medicine: Evidence and Research 2018:10 105–115 (Savy 2018 Acute pediatric hyperammonemia: current diagnosis and management strategies. Hepatic Medicine: Evidence and Research 2018:10 105–115).

[0192] Summary 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 (Summary 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

Use of a liposome suspension comprising transmembrane pH gradient liposomes in the treatment of an acute hyperammonemic crisis (HAC) associated with inborn errors of metabolism (IEM) in a subject, wherein the treatment comprises intraperitoneal administration of the liposome suspension to the subject and removal of a dialysate containing ammonia-carrying liposomes from the subject.

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

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

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

5. 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 according to claim 5, wherein, The liposomes contain about 200 nM of anhydrous citric acid.

7. Use according to any one of claims 1 to 6, wherein The lipid bilayer of the liposomes contains at least one phospholipid as a major component.

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. Use according to any one of claims 1 to 8, wherein, The lipid bilayer of the liposomes contains cholesterol, preferably in the range of 10 to 40 mol%.

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

11. Use according to any one of claims 1 to 6, wherein The bilayer of the liposomes contains 85.5:14:0.5 mol% of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and the inner compartment of the liposomes contains anhydrous citric acid.

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

13. The use according to any one of claims 1 to 12, wherein, The liposome suspension comprises (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride, or (vii) any combination of at least two of (i) to (v), preferably the combination comprises all of (i) to (v).

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