WO2025184398A1

WO2025184398A1 - Liposomal formulations, and methods of using and preparing thereof

Info

Publication number: WO2025184398A1
Application number: PCT/US2025/017681
Authority: WO
Inventors: Geoffrey Hird; Teppei SHIRAKURA; Drew Steven FOLK; Kevin Patrick HERLIHY
Original assignee: Glycomine Inc
Current assignee: Glycomine Inc
Priority date: 2024-02-29
Filing date: 2025-02-27
Publication date: 2025-09-04
Anticipated expiration: 2026-08-29
Also published as: TW202543582A

Abstract

The disclosure provides phosphorylated carbohydrate replacement therapies (CRT) that include compositions of phosphorylated carbohydrates, such as mannose-1-phosphate (M1P), and phospholipids, as well as methods for preparing such compositions. Such compositions are suitable for pharmaceutical delivery phosphorylated carbohydrates, such as M1P, for treating CDG type I and CDG type II diseases as well as other metabolic disorders.

Description

LIPOSOMAL FORMULATIONS, AND METHODS OF USING AND PREPARING THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/559,841, filed February 29, 2024, which is incorporated herein by reference in its entirety.

FIELD

[0002] The present invention relates to compositions containing carbohydrates encapsulated in a liposome, methods of delivering the encapsulated carbohydrates, and methods of using such compositions for treating diseases and disorders, such as congenital disorders of glycosylation (CDG). For example, the present invention relates to methods for preparation of carbohydrates and liposomes for pharmaceutical uses.

BACKGROUND

[0003] Glycosylation, the enzymatic attachment of carbohydrates (glycans) to proteins and lipids, is a co-translational and post-translational modification (PTM) that is more common than any other PTM as it applies to a majority of proteins synthesized in the rough endoplasmic reticulum (ER). Glycosylation plays a critical role in a variety of biological processes of membrane and secreted proteins. In the ER, glycosylation defines the protein structure and folding and acts as a quality control mechanism that dictates the export of properly folded proteins to Golgi or targets misfolded ones for degradation. Glycan moieties may also act as ligands for cell surface receptors to mediate cell attachment or stimulate signal transduction pathways. Congenital disorders of glycosylation, also known as CDG syndromes, are a group of rare genetic diseases where tissue proteins and/or lipids carry defective glycosylation and/or lack of glycosylation. These diseases are linked to numerous enzymatic deficiencies and often times cause severe, sometimes fatal, impairments of the nervous system, muscles, intestines, and several other organ systems.

[0004] Common clinical symptoms in children with CDG include hypotonia, developmental delay, failure to thrive, hepatic dysfunction, coagulopathy, hypothyroidism, esotropia, abnormal fat pattern and inverted nipples, hypoglycemia, seizure, cerebellar hypoplasia, and stroke-like episodes in a developmentally delayed child. At an older age, in adolescence or adulthood, the presentation may include ataxia, cognitive impairment, the absence of puberty in females, small testes in males, retinitis pigmentosa, scoliosis, joint contractures, and peripheral neuropathy.

[0005] CDG may be classified into two groups: CDG type I and CDG type II. CDG type I is characterized by defects in the initial steps of N-linked protein glycosylation, z.e., biosynthesis of dolichol pyrophosphate linked oligosaccharide (DLO), which occur in the ER, or transfer of the DLO to asparagine residues of nascent polypeptides. CDG type II involves defects in further processing (synthetic or hydrolytic) of the protein-bound glycan. Currently, twenty -two CDG type I and fourteen type II variants have been identified. One of the most common subtypes of CDG is CDG-Ia (approximately 70% of all CDG cases), which is characterized by loss or reduction of phosphomannomutase 2 (PMM2) activity leading to the deficiency or insufficiency in intracellular N-glycosylation (Jaeken et al. J. of Inherit. Met. Disease. 2008, 31 : 669-672). PMM2 is responsible for the conversion of mannose-6- phosphate to mannose- 1 -phosphate.

[0006] Although several different approaches of developing therapies for CDG have been explored, researchers continue their search for a suitable cure or a therapy for mitigating the disease itself. Existing treatments for manifestations include, for example, nutritional supplements, tube feeding, and a wide range of therapies that attempt to treat gastroesophageal reflux, persistent vomiting, developmental delays, ocular abnormalities, and hypothyroidism. Patients also require intravenous (IV) hydration and physical therapy for stroke-like episodes. Adults with orthopedic symptoms often require wheelchairs, transfer devices, and surgical treatment for scoliosis (Sparks et al., Disorders of Glycosylation Overview. 2005 In: Pagon RA, Adam MP, Bird TD, et al., editors. GeneReviews™. Seattle (WA): University of Washington, Seattle; 1993-2013).

[0007] Currently, CDG-Ib is one known CDG for which a treatment is available, namely oral D-mannose administration. However, such therapy may not be as effective in treating CDG-Ia patients and there are currently limited treatment options for other CDG type I subtypes and CDG type II diseases. One of the reasons for the lack in established therapy for CDG-I disorders may be due to the plethora of heterogeneous clinical phenotypes presented that do not show a direct correlation to the PMM2 enzyme activity.

[0008] Patients suffering from a reduction in PMM2 activity have reduced productions of mannose- 1 -phosphate (M1P, which may also be referred to the in art as Man-l-P), associated with symptoms of multivisceral impairments. In order to overcome PMM2 production deficiency, it is important to supply downstream enzymes with the required substrate (z.e., M1P). However, the delivery and maintenance of such a systemic supply of M1P is problematic, as extracellular enzymes within bodily fluids degrade M1P when delivered exogenously by oral or intravenous administration.

[0009] Derivatives of the polar M1P can be synthesized to make M1P more cell- permeable (US Patent Publication No. 2009/0054353). This approach, however, is also problematic, as the cell-permeable M1P derivatives have been shown to be either unstable for clinical use or cytotoxic via the by-products of the M1P derivatives (Eklund et al., Glycobiology 2005, 15: 1084-1093; Rutschow et al. Bioorg Med Chem 2002, 10: 4043-4049; and Hardre et al., Bioorg Med Chem Lett 2007, 17: 152-155).

[0010] Other potential therapies have focused on inhibiting enzymes that catabolize mannose-6-phosphate (M6P), a precursor to M1P, via the inhibition of phosphomannose isomerases (PMI). The approach focuses on forcing the reaction towards optimizing homeostasis, which with the use of PMI inhibitors, would have been skewed toward production of M6P. These approaches, however, are ineffective as clinical treatment options due to their associated toxicity, off-target side effects, and poor selective tissue penetration.

[0011] Another potential solution is to use a delivery vehicle (e.g., lipid particles) to encapsulate and deliver M1P. However, due to the high charge and polarity of phosphorylated carbohydrates in general, the optimal delivery vehicle must overcome challenges of stability, phosphorylated carbohydrates loading rate and concentration, toxicity, and delivery efficiency.

[0012] Accordingly, unmet needs exist for improved compositions and methods for formulating and delivering phosphorylated carbohydrates, such as M1P and M6P, for treating disorders, such as a congenital disorder of glycosylation (CDG), to subjects (including, for example, humans) in need of such treatment.

BRIEF SUMMARY

[0013] In one aspect, the disclosure provides stabilized buffered liposomal formulations comprising a mannose phosphate, including specifically mannose- 1 -phosphate (M1P). The stabilized liposomal formulations are not prone to lipid degradation and are stable for significant periods of time. Additionally, the liposome particles do not form agglomerates upon storage, which would compromise their efficacy.

[0014] In some aspects, provided are compositions that include liposomes having a lipid membrane enclosing an intraliposomal compartment, in which the liposomes encapsulate a mannose phosphate (e.g., M1P) in the intraliposomal compartment. The compositions further include an intraliposomal buffer comprising a buffering agent, and optionally an acid or base. In some variations, the pH of the intraliposomal buffer is from about 6.0 to about 7.9. In some embodiments, the pH of the intraliposomal buffer is from about 6.2 to about 7.4. In some embodiments, the pH of the intraliposomal buffer is from about 6.4 to about 7.5. In some embodiments, the pH of the intraliposomal buffer is from about 6.5 to about 7.2. In some embodiments, the pH of the intraliposomal buffer is about 6.5. In some embodiments, the pH of the intraliposomal buffer is about 6.8. In some embodiments, the pH of the intraliposomal buffer is about 7.0. In some embodiments, the pH of the intraliposomal buffer is about 7.2. In some embodiments, the pH of the intraliposomal buffer is about 7.4.

[0015] In some embodiments, the intraliposomal buffer comprises tri s(hydroxymethyl)aminom ethane (Tris). In some embodiments, the intraliposomal buffer comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES). In some embodiments, the Tris or HEPES buffer maintain the pH of the intraliposomal compartment at from about 6.8 to about 7.6, or from about 7.0 to about 7.4.

[0016] In some embodiments, the intraliposomal buffer comprises a histidine or a citrate buffer. In some such embodiments, the histidine or citrate buffer maintains the pH of the intraliposomal compartment from about 6 to about 7.2, or at a pH of from about 6.2 to about 7.0, or from about 6.4 to about 6.8.

[0017] In some embodiments, the concentration of the buffering agent in the intraliposomal compartment of the liposomes is at least 15 mM, at least 25 mM, at least 35 mM, at least 50 mM, or at least 60 mM. In other embodiments, the concentration of the buffering agent in the intraliposomal compartment of the liposomes is from about 15 mM to about 75 mM, from about 25 mM to about 75 mM, from about 30 mM to about 60 mM, from about 40 mM to about 60 mM, or from about 45 mM to about 55 mM. In other embodiments, the concentration of the buffering agent in the intraliposomal compartment of the liposomes is about 40 mM, or about 50 mM. In some embodiments, the concentration of the buffering agent in the intraliposomal compartment of the liposomes is higher than the concentration of the buffering agent in the extraliposomal compartment of the liposomes.

[0018] In some embodiments, the pH of the intraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 2 years at 5 °C or 6 months at room temperature. In some embodiments, the pH of the intraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the liposomal composition for 2 years at 5°C or 6 months at room temperature. For instance, if the starting pH of the intraliposomal buffer is 7.2, the pH of the intraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for 2 years at 5°C or 6 months at room temperature.

[0019] In some embodiments, the liposomal compositions further include an extraliposomal buffer comprising a buffering agent and a tonicity modifier. In some variations, the pH of the extraliposomal buffering agent is from about 6.5 to about 7.5. In some embodiments, the extraliposomal buffering agent is the same as the intraliposomal buffering agent. In some embodiments, the concentration of the buffering agent in the extraliposomal buffer is from about 10 mM to about 20 mM. In some embodiments, the concentration of the buffering agent in the extraliposomal buffer is from about 15 mM to about 20 mM.

[0020] In some embodiments, the pH of the extraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 2 years at 5 °C or 6 months at room temperature. In some embodiments, the pH of the extraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the liposomal composition for 2 years at 5°C or 6 months at room temperature. For instance, if the starting pH of the extraliposomal buffer is 7.2, the pH of the extraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for 2 years at 5°C or 6 months at room temperature.

[0021] In some variation, the compositions optionally further include a radical scavenging antioxidant, present in the lipid bilayer. In some embodiments, the radical scavenging antioxidant is butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or tocopherol. In one variation, the tocopherol is alpha-tocopherol. In another variation, the radical scavenging antioxidant is BHT. In certain embodiments, any combinations of the radical scavenging antioxidants described herein may be used. For example, in one variation, both BHT and tocopherol are present in the compositions.

[0022] In certain embodiments, trace amounts of one or more radical scavengers may be present. For instance, such components may be present in commercially available lipids purchased, and get incorporated into the composition. In other embodiments, BHT is absent. In other embodiments, both alpha-tocopherol and BHT are absent. In yet one embodiment, the composition has no detectable amount of alpha-tocopherol and BHT.

[0023] In some embodiments, the extraliposomal buffer comprises tri s(hydroxymethyl)aminom ethane (Tris). In some embodiments, the extraliposomal buffer comprises Tris and saline. In other embodiments, the extraliposomal buffer comprises Tris and sugars, such as sucrose.

[0024] In some embodiments, the intraliposomal buffer and/or the extraliposomal buffer comprises a buffering agent that allows a mannose phosphate (e.g., M1P) to remain solubilized in the presence of ethanol. In some embodiments, the intraliposomal buffer and/or the extraliposomal buffer comprises a buffering agent that allows the mannose phosphate (e.g., M1P) to remain solubilized at between about 10 mM and about 20 mM in the presence of ethanol. In some embodiments, the intraliposomal buffer and/or the extraliposomal buffer comprises a buffering agent that allows the mannose phosphate (e.g., M1P) to remain solubilized at between about 15 mM and about 20 mM in the presence of ethanol. In some embodiments, the intraliposomal buffer and/or the extraliposomal buffer comprises a buffering agent that allows the mannose phosphate (e.g., M1P) to remain solubilized at between about 10 mM and about 15 mM in the presence of ethanol. In some embodiments, the buffering agent is Tris or HEPES. In some embodiments, the buffering agent is Tris. In some embodiments, the buffering agent is HEPES.

[0025] In some embodiments, the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG). In certain variations, the lipid membrane comprises DOPE, DOPC and DSPE conjugated to PEG. [0026] In one variation, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment the mannose phosphate (e.g., M1P), and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising a buffering agent that allows the mannose phosphate (e.g., MlP)to remain solubilized in the presence of ethanol; extraliposomal buffer comprising (1) a buffering agent that allows the mannose phosphate (e.g., MlP)to remain solubilized in the presence of ethanol, and (2) saline; and BHT.

[0027] In one variation, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment the mannose phosphate (e.g., M1P), and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising a buffering agent that allows the mannose phosphate (e.g., M1P) to remain solubilized in the presence of ethanol; and extraliposomal buffer comprising (1) a buffering agent that allows the mannose phosphate (e.g., M1P) to remain solubilized in the presence of ethanol, and (2) saline. In some variations of the foregoing, the extraliposomal buffer has no ethanol, or substantially no ethanol, or no detectable amount of ethanol. In other variations of the foregoing, the extraliposomal buffer has no mannose phosphate (e.g., M1P) , or substantially no mannose phosphate (e.g., M1P), or no detectable amount of mannose phosphate (e.g., M1P). It should be understood that this buffer system in the composition helps to maintain pH, rather than solubility of mannose phosphate (e.g., M1P).

[0028] In one variation, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose phosphate (e.g., M1P), and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising Tris; extraliposomal buffer comprising Tris and saline; and BHT.

[0029] In one variation, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose phosphate (e.g., M1P), and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising Tris; and extraliposomal buffer comprising Tris and saline. [0030] In some embodiments, the liposomal compositions of the disclosure show minimal or no degradation of the individual lipids comprising the liposome. As shown in Example 1, the buffered liposomal compositions are far less prone to lipid degradation compared to compositions formulated in unbuffered solutions (e.g., unbuffered saline solutions). In some embodiments, each of the individual lipids of the liposomal composition degrade less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% when stored at 5°C or room temperature for up to 2 years.

[0031] The buffered liposomal compositions of the disclosure show high purity levels upon storage for 6 months or more. For instance, in particular embodiments, the total impurity level observed in the liposomal compositions of the disclosure is less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% following storage at 5°C or at room temperature for up to 2 years.

[0032] The liposomal compositions of the disclosure also display negligible leakage of the mannose phosphate (e.g., MlPjfrom the intraliposomal component, even following storage of the liposomal composition for up to 2 years. For instance, in some embodiments, less than 10% of the intraliposomal mannose phosphate (e.g., MlP)is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature. For instance, in some embodiments, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the intraliposomal mannose phosphate (e.g., MlP)is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.

[0033] In other aspects, provided is a method of treating a congenital disorder of glycosylation in a subject in need thereof, comprising administering to the subject the liposomal compositions described herein. In some embodiments, the congenital disorder of glycosylation is CDG-Ia, CDG-Ie, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG. In some variations, the compositions are administered intravenously, e.g., by intravenous infusion. In one variation of the foregoing, the mannose phosphate used in the methods herein is M1P.

DESCRIPTION OF THE FIGURES

[0034] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures. [0035] FIG. 1 depicts degradation of individual lipids in a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulas 1, 2, 3, 4, 5 and 6) at various temperatures over a period of one month.

[0036] FIG. 2 depicts total lipid degradation of a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6) at various temperatures , wherein the horizontal line indicates the upper specification limit (USL) of 4%, and no impurities were detected at 0 month.

[0037] FIG. 3 depicts pH changes over time of a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6) at various temperatures, , wherein the horizontal “USL” and “LSL” lines indicate the upper and lower limits of pH based on target specifications of Formulation 1-A, while the horizontal “Target” line indicates the target pH of 7.

[0038] FIGS. 4A and 4B depict particulate count over 10 pm and 25 pm, respectively, for a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6) at various temperatures, wherein the horizontal “USL” line show the upper limit of number of particles per guidance of USP <788> and expected vial fill volume.

[0039] FIGS. 5A, 5B and 5C depict Z average, PDI, and population over 200 nm, respectively, for a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6) at various temperatures.

[0040] FIG. 6 depicts leeching of mannose- 1 -phosphate (M1P) over time for a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6). [0041] FIG. 7 shows the osmolality of samples for a liposomal composition formulated without an intraliposomal buffer (Formulation 1-A) and six liposomal compositions formulated with an intraliposomal buffer (Formulations 1, 2, 3, 4, 5 and 6).

DETAILED DESCRIPTION

[0042] The following description sets forth exemplary compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0043] Provided herein are compositions of a mannose phosphate, such as mannose- 1- phosphate (M1P) encapsulated by liposomes. Also provided here are pharmaceutical compositions and kits containing these compositions. Also provided here are methods for delivering a mannose phosphate (e.g., M1P) for treating a congenital disorder of glycosylation (CDG) of a subject in need thereof, by administering to the subject such compositions.

[0044] It is to be understood that, for each of the variations and embodiments described herein, the liposomes may encapsulate a mannose phosphate. In some embodiments, the mannose phosphate is mannose- 1 -phospate (M1P).

[0045] Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present disclosure, the following terms are defined.

[0046] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, “about x” includes and describes “x” per se. In some embodiments, the term “about” when used in association with a measurement, or used to modify a value, a unit, a constant, or a range of values, refers to variations of +/- 5%, 4%, 3%, 2% or 1%.

[0047] Reference to “between” two values or parameters herein includes (and describes) embodiments that include those two values or parameters per se. For example, description referring to “between x and y” includes description of “x” and “y” per se. Further, it should also be understood that “between x and y” can also be expressed as “about x to y” or “about x-y”.

[0048] As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

Liposomal Compositions

[0049] In some aspects, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose phosphate (e.g., M1P); intraliposomal buffer; extraliposomal buffer; and a radical scavenging antioxidant.

[0050] In some aspects, provided is a composition, comprising: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose phosphate (e.g., M1P); intraliposomal buffer; and extraliposomal buffer.

[0051] In some embodiments, the liposomes have an interior surface in contact with the intraliposomal buffer and an external surface in contact with the extraliposomal buffer. In some embodiments the interior and exterior surfaces are both in contact with a buffer solution of neutral pH.

Phospholipids

[0052] In some embodiments, the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG).

[0053] In certain embodiments with respect the phospholipid having an ethanolamine head group in (a) of the lipid membrane, the unsaturated fatty acid tail independently comprises at least one Cio-28 carbon chain. In certain embodiments, the unsaturated fatty acid tail is an optionally substituted Cio-28 alkenyl or an optionally substituted Cio-28 alkynyl. In some embodiments, each of the fatty acid chains is an unsubstituted Cio-28 alkenyl. In some embodiments, the alkenyl is linear or branched. In some embodiments, each of the fatty acid chains has one or more double bonds. In some embodiments, each double bond has cis configuration. In some embodiments, each double bond has trans configuration. In some embodiments, each of the fatty acid chains is a Cio-28 alkenyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl. In some variations, the phospholipid having an ethanolamine head group in (a) of the lipid membrane has oleoyl tail groups.

[0054] In some embodiments, the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or a salt thereof. In one variation, the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is: or a salt thereof.

[0055] In certain embodiments with respect to the phospholipid having a choline group in (b) of the lipid membrane, the unsaturated fatty acid tail independently comprises at least one Cio-28 carbon chain. In certain embodiments, the unsaturated fatty acid tail is an unsubstituted Cio-28 alkenyl. In some embodiments, the alkenyl is linear or branched. In some embodiments, the unsaturated fatty acid tail has one or more double bonds. In some embodiments, each double bond has cis configuration. In some embodiments, each double bond has trans configuration. In some embodiments, the unsaturated fatty acid tail is a Cio-28 alkenyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl. In some variations, the phospholipid having choline group in (b) of the lipid membrane has oleoyl tail groups.

[0056] In some variations, the phospholipid having a choline group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), or a salt thereof. In some variations, the phospholipid having a choline group and at least one unsaturated fatty acid tail is: or a salt thereof.

[0057] In some embodiments with respect to the phospholipid having an ethanolamine head group in (c) of the lipid membrane, the saturated fatty acid tail independently comprises at least one C4-28 carbon chain. In certain embodiments, the saturated fatty acid tail is an optionally substituted alkyl. In some embodiments, the saturated fatty acid tail is an unsubstituted C4-28 alkyl. In some embodiments, the saturated fatty acid tail is a C4-28 alkyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl. In some variations, the phospholipid having an ethanolamine head group in (c) of the lipid membrane has stearoyl tail groups.

[0058] In some variations, the phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), or a salt thereof. In some variations, the phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is: or a salt thereof.

[0059] In certain variations, the phospholipid conjugated to PEG is DSPE or a salt thereof. In some embodiments, PEGylated phospholipid is DSPE-PEG. In some embodiments, PEGylated phospholipid is DSPE-PEG2000. In some embodiments, the DSPE- PEG is further conjugated to a carbohydrate. In certain embodiments, the DSPE-PEG is further conjugated to a monosaccharide. In some embodiments, the DSPE-PEG is further conjugated to a galactose moiety. In certain embodiments, the DSPE-PEG-galactose has the following structure: or a salt thereof.

[0060] In one embodiment, the lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG. In one variation, the lipid membrane comprises DOPC:DOPC:DSPE- PEG2000 at a molar ratio of from about 58 : 38 : 3 to 75 : 22 : 3; or from about 48.5 : 48.5 : 3 to about 80 : 17 : 3. In one embodiment, the lipid membrane comprises DOPC:DOPE:DSPE- PEG2000 at a molar ratio of about 58 : 39 : 3. In one embodiment, the lipid membrane comprises DOPC:DOPE:DSPE-PEG2ooo at a molar ratio of about 67 : 30 : 3.

[0061] In certain variations, PEG is present in the composition at a concentration that ranges from about 0.5 molar percent to about 20 molar percent. In some embodiments, PEG is present in the composition at a concentration of about 0.5 molar percent, about 1 molar percent, about 2 molar percent, about 3 molar percent, about 4 molar percent, about 5 molar percent, about 6 molar percent, about 7 molar percent, about 8 molar percent, about 9 molar percent, about 10 molar percent, about 11 molar percent, about 12 molar percent, about 13 molar percent, about 14 molar percent, about 15 molar percent, about 16 molar percent, about 17 molar percent, about 18 molar percent, about 19 molar percent, or about 20 molar percent. In some embodiments, PEG is present in the composition at a concentration of at least about 0.5 molar percent, at least about 1 molar percent, at least about 2 molar percent, at least about 3 molar percent, at least about 4 molar percent, at least about 5 molar percent, at least about 6 molar percent, at least about 7 molar percent, at least about 8 molar percent, at least about 9 molar percent, at least about 10 molar percent, at least about 11 molar percent, at least about 12 molar percent, at least about 13 molar percent, at least about 14 molar percent, at least about 15 molar percent, at least about 16 molar percent, at least about 17 molar percent, at least about 18 molar percent, at least about 19 molar percent, or at least about 20 molar percent; or between 0.5 molar percent and 50 molar percent, between 0.5 molar percent and 40 molar percent, between 0.5 molar percent and 30 molar percent, or between 0.5 molar percent and 20 molar percent.

[0062] In certain variations, PEG is present in the composition at a molecular weight that ranges from about 200 Da to about 40,000 Da. In some embodiments, PEG is present in the composition at a molecular weight that ranges from about 200 Da to about 10,000 Da. In some embodiments, PEG is present in the composition at a molecular weight of about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 6,500 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 8,500 Da, about 9,000 Da, about 9,500 Da, or about 10,000 Da; or between about 200 Da and about 10,000 Da.

Buffers

[0063] In some aspects, provided are compositions that include liposomes having a lipid membrane enclosing an intraliposomal compartment, in which the liposomes encapsulate a mannose phosphate, such as M1P, in the intraliposomal compartment. The compositions further include an intraliposomal buffer comprising a buffering gent, and optionally an acid or a base. In some variations, the pH of the intraliposomal buffer is from about 6.0 to about 7.9. In some embodiments, the pH of the intraliposomal buffer is from about 6.2 to about

7.4. In some embodiments, the pH of the intraliposomal buffer is from about 6.4 to about

7.5. In some embodiments, the pH of the intraliposomal buffer is from about 6.5 to about

7.2. In some embodiments, the pH of the intraliposomal buffer is from about 6.8 to about

7.2. In some embodiments, the pH of the intraliposomal buffer is about 6.5. In some embodiments, the pH of the intraliposomal buffer is about 6.8. In some embodiments, the pH of the intraliposomal buffer is about 7.0. In some embodiments, the pH of the intraliposomal buffer is about 7.2. In some embodiments, the pH of the intraliposomal buffer is about 7.3. In some embodiments, the pH of the intraliposomal buffer is about 7.4. In some embodiments, the pH of the intraliposomal buffer is about 7.5. [0064] In some embodiments, the intraliposomal buffer comprises tri s(hydroxymethyl)aminom ethane (Tris). In some embodiments, the intraliposomal buffer comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES). In some embodiments, the intraliposomal buffer comprises bicarbonate. In some embodiments, the Tris or HEPES buffer maintain the pH of the intraliposomal compartment at from about 6.8 to about 7.6, or from about 7.0 to about 7.4. In some embodiments, the pH of the intraliposomal buffer is about 7.0. In some embodiments, the pH of the intraliposomal buffer is about 7.2. In some embodiments, the pH of the intraliposomal buffer is about 7.3. In some embodiments, the pH of the intraliposomal buffer is about 7.4. In some embodiments, the pH of the intraliposomal buffer is about 7.5.

[0065] In some embodiments, the intraliposomal buffer comprises a histidine or a citrate buffer. In some such embodiments, the histidine or citrate buffer maintains the pH of the intraliposomal compartment from about 6 to about 7.2, or at a pH of from about 6.2 to about 7.0, or from about 6.4 to about 6.8.

[0066] In some embodiments, the intraliposomal buffer comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate or tromethamine buffering agent..

[0067] In some embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 15 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 35 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 50 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 50 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is from about 15 mM to about 75 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is from about 30 mM to about 60 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is about 40 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is about 50 mM. [0068] In some embodiments, the pH of the intraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 6 months or 2 years at 5°C or at room temperature. In some embodiments, the pH of the intraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the liposomal composition for six month or for 2 years at 5°C or at room temperature. For instance, if the starting pH of the intraliposomal buffer is 7.2, the pH of the intraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for up to 2 years at 5°C or at room temperature.

[0069] In some variations, the buffering agent comprises a buffer salt. In some embodiments, the extraliposomal buffer comprising a buffer salt and a tonicity modifier. In some variations, the pKa of the buffer salt is between 6.5 to 7.5. In some embodiments, the extraliposomal buffer is in a physiological pH range. In one variation, the buffer salt is tri s(hydroxymethyl)aminom ethane (Tris). In other variations, the extraliposomal buffer comprises bicarbonate, Tris, or HEPES, or any combination thereof. In some embodiments, the concentration of the buffering agent in the extraliposomal buffer is from about 10 mM to about 20 mM. In some embodiments, the concentration of the buffering agent in the extraliposomal buffer is from about 15 mM to about 20 mM.

[0070] In some embodiments, the pH of the extraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 6 months or 2 years at 5°C or at room temperature. In some embodiments, the pH of the extraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the extraliposomal composition for six month or for 2 years at 5°C or at room temperature. For instance, if the starting pH of the extraliposomal buffer is 7.2, the pH of the extraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for up to 2 years at 5 °C or at room temperature.

[0071] In some variations, the tonicity modifier comprises sugar or saline, or a combination thereof. Suitable sugars that may be used for tonicity include, for example, sucrose and dextrose.

[0072] In other variations, the tonicity modifier is an ionic tonicity modifier. In one variation, the tonicity modifier comprises saline. In some embodiments, the osmolality of the liposomal compositions is isotonic. In some embodiments, the osmolality of the liposomal compositions is from about 270 to about 320 mOsm/kg. In some embodiments, the osmolality of the liposomal compositions is from about 290 to about 320 mOsm/kg. In concentration of the tonicity modifier in the extraliposomal buffer is from about 5 mM to about 25 mM, from about 5 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 15 mM, or from about 15 mM to about 20 mM.

[0073] In some embodiments, the M1P and Tris, and optionally an acid, are present in the composition at a ratio suitable to maintain a neutral pH.

[0074] In some variations, the buffer capacity of the intraliposomal solution may be increased to maintain a neutral pH in the presence of mannose- 1 -phosphate (M1P). In some embodiments, the concentration of the intraliposomal buffer is increased to maintain the neutral pH.

Radical Scavenging Antioxidant

[0075] In variations, the composition further comprises a radical scavenging antioxidant. Any suitable radical scavenging antioxidants may be used in the liposomal compositions provided herein. In some embodiments, the radical scavenging antioxidant is butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol. In one variation, the radical scavenging antioxidant is BHT. In other embodiments, the compositions comprises more than one radical scavenging agent. Any combinations of the radical scavenging antioxidants described herein may be used. For example, in one variation, the compositions comprise BHT and alpha tocopherol. It should be understood that, in certain variations, certain of these radical scavenging antioxidants may be present in the liposomal compositions due to their presence in the lipids used (that are commercially available).

Properties of the Liposomal Compositions

[0076] The liposomal compositions described herein are optimized for treating diseases and disorders such as congenital disorders of glycosylation (CDG).

[0077] In some embodiments, the composition has a drug-to-lipid (D/L) ratio of at least 0.01, at least 0.1, or at least 0.2. In certain embodiments, the composition has a D/L ratio from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 0.1 to about 0.4, from about 0.1 to about 0.3, from about 0.1 to about 0.2, or from about 0.5 to about 1.5. In some variations, the D/L ratio is about 0.1, 0.2, or 0.3. It should be understood that the drug-to-lipid (D/L) ratio refers to the mass ratio of drug to total lipids in a given sample.

[0078] In some variations of the foregoing, the total mannose phosphate (e.g., MlP)concentration in the sample is between 1 mg/ml and 50 mg/ml, between 1 mg/ml and 25 mg/ml, 1 mg/ml and 15 mg/ml, between 5 mg/ml and 25 mg/ml, between 5 mg/ml and 12 mg/ml, or between 6 mg/ml and 10 mg/ml.

[0079] In certain embodiments, the encapsulated D/L ratio refers to the mass ratio of drug encapsulated in the liposome to total lipids in a given sample. In some variations, the encapsulated D/L ratio does not exceed 0.15. In other variations, the encapsulated D/L ratio is between 0.001 and 0.15, between 0.01 and 0.15, between 0.1 to 0.15, between 0.1 and 0.14, between 0.1 and 0.13, between 0.1 and 0.12, or between 0.1 and 0.11. In other variations, the encapsulated D/L ratio is about 0.1 +/- 0.25%, about 0.1 +/- 0.20%, about 0.1 +/- 15%, or about 0.1 +/- 0.1%.

[0080] In some variations of the foregoing, the total encapsulated mannose phosphate (e.g., MlP)concentration in the sample is between 1 mg/ml and 15 mg/ml, between 5 mg/ml and 12 mg/ml, or between 6 mg/ml and 10 mg/ml.

[0081] In some embodiments, the composition has a mannose phosphate (e.g., MlP)concentration (based on the free acid) from about 1 mg/ml to about 10 mg/ml. In certain embodiments, the mannose phosphate (e.g., M1P) concentration is from about 1 mg/ml to about 10 mg/ml, from about 1 mg/ml to about 9 mg/ml, from about 1 mg/ml to about 8 mg/ml, from about 1 mg/ml to about 7 mg/ml, from about 1 mg/ml to about 6 mg/ml, from about 1 mg/ml to about 5 mg/ml, or from about 1 mg/ml to about 4 mg/ml, from about 1 mg/ml to about 3 mg/ml, or from about 1 mg/ml to about 2 mg/ml. In some variations, the mannose phosphate (e.g., M1P) concentration is at least 1 mg/ml. In some variations, the mannose phosphate (e.g., M1P) concentration is about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, or 10 mg/ml.

[0082] In other embodiments, the composition has a mannose phosphate (e.g., MlP)concentration (based on the free acid) from about Img/mL to 250 mg/ml, between 1 mg/mL and 200 mg/ml, 1 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 75 mg/ml, from about 1 mg/ml to about 50 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 30 mg/ml to about 55 mg/ml, or from about 30 mg/ml to about 50 mg/ml, or from about 40 mg/ml to about 55 mg/ml, or from about 50 mg/ml to about 55 mg/ml. In some variations of the foregoing, the mannose phosphate (e.g., MlP)concentration is the maximum concentration of mannose phosphate (e.g., MlP)in the liposome.

[0083] In some embodiments, the composition minimizes both lipid degradation and liposomal agglomeration. In some variations, the lipid degradation of the composition is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1%.

[0084] In some embodiments, the liposomal compositions of the disclosure show minimal or no degradation of the individual lipids comprising the liposome. As shown in Example 1, the buffered liposomal compositions are far less prone to lipid degradation compared to compositions formulated in unbuffered solutions (e.g., unbuffered saline solutions). In some embodiments, each of the individual lipids of the liposomal composition degrade less than 10% when stored at 5°C or room temperature for up to 2 years. In some embodiments, each of the individual lipids of the liposomal composition degrade less than 1% when stored at 5°C or room temperature for up to 2 years. In some embodiments, each of the individual lipids of the liposomal composition degrade less than 0.5% when stored at 5°C for up to 2 years. In some embodiments, each of the individual lipids of the liposomal composition degrade less than 0.25% when stored for up to 2 years.

[0085] In some embodiments, the composition has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1% of total lipid impurities.

[0086] The buffered liposomal compositions of the disclosure show high purity levels upon storage for 6 months or more. For instance, in particular embodiments, the total impurity level observed in the liposomal compositions of the disclosure is less than 1%, less than 0.5%, less than 0.25%, or less than 1% following storage at 5°C or at room temperature for up to 2 years.

[0087] In some embodiments, the composition maintains a pH range between 6.5 and 7, 7 and 7.4 or between 7.35 and 7.45. In certain embodiments, the composition maintains a physiological pH range. [0088] In some embodiments, the composition has a Z-average between 80 nm and 130 nm, between 80 nm and 120 nm, between 80 nm and 110 nm, between 80 nm and 100 nm, between 90 nm and 130 nm, between 90 nm and 120 nm, between 90 nm and 110 nm, or between 90 nm and 100 nm.

[0089] In some embodiments, the composition has a poly dispersity index of less than 0.2 or less than 0.1.

[0090] In some embodiments, no free mannose phosphate (e.g., MlP)is detected in the composition.

[0091] In some embodiments, percentage (%) of encapsulated M1P is the percent of encapsulated mannose phosphate (e.g., M1P) divided by the total mannose phosphate in the finished drug product. It should be understood that the finished drug product refers to the liposomal composition. In some variations, the percentage of encapsulated mannose phosphate (e.g., M1P) is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or between 90% and 99%, or between about 90% and 95%, or close to 100%.

[0092] In some embodiments, percentage (%) encapsulation efficiency (“%EE”) is the efficiency of mannose phosphate (e.g., M1P) encapsulation in the liposomes e.g., of the finished drug product) relative to the starting amount of mannose phosphate. In some variations, the %EE is between about 5% and 50%, between about 10% and 30%, or between about 10% and 25%.

[0093] The compositions provided herein may have any one or more of the properties described above. For example, in some variations, the composition provided herein has all of the following properties:

(i) a lipid degradation of less than 10%;

(ii) a Z-average between 80 nm and 130 nm;

(iii) a percentage of encapsulated M1P of at least 80%; and

(iv) a pH range between 6.8 and 7.4. [0094] In other variations, the composition provided herein has all of the following properties:

(i) a lipid degradation of less than 10%;

(ii) a Z-average between 80 nm and 130 nm;

(iii) a percentage of encapsulated M1P of at least about 75%, or an encapsulated M1P concentration of at least 5 mg/ml;

(iv) an encapsulated D/L ratio of between 0.05 and 0.15; and

(v) a pH range between 6.8 and 7.4.

[0095] Stability of the liposomal compositions provided herein, as characterized based on for example lipid degradation, total lipid impurities, pH, Z-average, PDI, Total M1P, encapsulation efficiency, and osmolality, may be measured over a time period over a range of temperatures, such as 5°C, 25°C and 40°C. In some variations, the time period is 1-3 months. In other variations, the time period is at least 6 months, at least 1 year, or at least 2 years.

[0096] The liposomal compositions of the disclosure also display negligible leakage of the M1P from the intraliposomal component, even following storage of the liposomal composition for up to 2 years. For instance, in some embodiments, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% of the intraliposomal mannose phosphate (e.g., M1P) is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.

Pharmaceutical Compositions

[0097] Liposomal compositions of the present disclosure can be incorporated into a variety of formulations for therapeutic use (e.g., by administration) or in the manufacture of a medicament (e.g., for delivering M1P to a subject in need thereof, including for treating or preventing a disease or disorder such as a congenital disorder of glycosylation (CDG) in a subject in need thereof) by combining the composition with appropriate carriers (including, for example, pharmaceutically acceptable carriers or diluents), and may be formulated, for example, into preparations in liquid form.

[0098] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants, e.g., at least compendial grade, at least United States Pharmacopeia (USP) grade, at least National Food (NF) grade, at least analytical grade, at least pharmaceutical grade or at least suitable for human use. Moreover, compositions intended for in vivo use are sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0099] Pharmaceutical compositions of the present disclosure may be used in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time.

[0100] Dosages and desired concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles described in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp.42-46.

[0101] For in vivo administration of any of the liposomal compositions provided herein, normal dosage amounts may vary from 10 ng/kg up to 100 mg/kg of a subject’s body weight per day.

[0102] Administration of the liposomal compositions provided herein can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. [0103] It is within the scope of the present disclosure that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0104] Thus, in some variations, the liposomal compositions provided herein may be chronically or intermittently administered to a subject (including, for example, a human) in need thereof. In certain variations, chronic administration is administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. In certain variations, intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Therapeutic Uses

[0105] These compositions are useful for delivering M1P to a subject in need thereof.

[0106] In some embodiments, the subject is a mammal, such as a human, domestic animal, such as a feline or canine subject, farm animal (e.g., bovine, equine, caprine, ovine, and porcine subject), wild animal (whether in the wild or in a zoological garden), research animal, such as mouse, rat, rabbit, goat, sheep, pig, dog, and cat, and birds. In one embodiment, the subject is a human.

[0107] In some variations, the subject may be at risk. For example, in one variation, the subject is an at risk human. A subject at risk of developing a particular disease, disorder, or condition, such as a congenital disorder of glycosylation, may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. In certain variations, an individual “at risk” is an individual having risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. A subject having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition such as a congenital disorder of glycosylation, than a subject without one or more of these risk factors.

[0108] In some embodiments, congenital disorders of glycosylation (CDG) is a group of genetic disorders that result in errors of metabolism in which glycosylation of a variety of tissue proteins and/or lipids is deficient or defective. Congenital disorders of glycosylation may also be known as CDG syndromes. CDG syndromes may often cause serious, occasionally fatal, malfunction of several different organ systems, such as the nervous system, brain, muscles, and intestines, in affected infants. Manifestations of CDG syndromes may range from severe developmental delay and hypotonia beginning in infancy, to hypoglycemia and protein-losing enteropathy with normal development. Developmental delay can be a common initial indication for a CDG diagnosis. One of the most common subtype of CDG syndromes is CDG-Ia (also known as PMM2-CDG) where the genetic defect leads to the loss of phosphomannomutase 2, which is the enzyme responsible for the conversion of mannose-6-phosphate into mannose- 1 -phosphate.

[0109] CDG syndromes may be classified as type I (CDG-I) and type II (CDG-II). Such classification may depend on the nature and location of the biochemical defect in the metabolic pathway relative to the action of oligosaccharyltransferase. Methods for screening for CDG subtype may include the analysis of transferrin glycosylation status by, for example, isoelectric focusing or ESI-MS. CDG type I include, for example, la (PMM2-CDG), lb (MPI- CDG), Ic (ALG6-CDG) , Id (ALG3-CDG), le (DPMI -CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), li (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), IL (ALG9-CDG), Im (DOLK-CDG), In (RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG), Iq (SRD5A3-CDG), Ir (DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS- CDG, and I/IIx. CDG type II include, for example, Ila (MGAT2-CDG), lib (GCS1-CDG), lie (SLC335C1-CDG), lid (B4GALT1-CDG), lie (COG7-CDG), Ilf (SLC35A1-CDG), Ilg (COG1-CDG), Ilh (COG8-CDG), Hi (COG5-CDG), Ilj (COG4-CDG), IIL (COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG.

[0110] Congenital disorders of glycosylation (CDG) that may be treated with the liposomal compositions provided herein include, for example, la (PMM2-CDG), lb (MPI- CDG), Ic (ALG6-CDG) , Id (ALG3-CDG), le (DPMI -CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), li (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), IL (ALG9-CDG), Im (DOLK-CDG), In (RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG), Iq (SRD5A3-CDG), Ir (DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS- CDG, I/IIx, Ila (MGAT2-CDG), lib (GCS1-CDG), lie (SLC335C1-CDG), lid (B4GALT1- CDG), lie (COG7-CDG), Ilf (SLC35A1-CDG), Ilg (COG1-CDG), Ilh (COG8-CDG), Hi (COG5-CDG), Ilj (COG4-CDG), IIL (COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG. In some variations, the compositions and methods described herein are suitable to treat CDG-Ia, CDG-Ib, CDG-Ie, CDG-Ig, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.

[oni] In some embodiments, “treatment” or “treating” includes an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and/or preventing or delaying the spread of the disease or condition); and/or c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.

[0112] In some embodiments, “prevention” or “preventing” includes any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop. Compounds may, in some embodiments, be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.

[0113] In some variations, an “effective amount” is at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations.

[0114] In some variations, a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease, disorder, or condition, such as a congenital disorder of glycosylation. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the lipid compositions of the present disclosure to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the lipid compositions of the present disclosure are outweighed by the therapeutically beneficial effects.

[0115] In one aspect, provided herein is a method for delivering M1P to a subject in need thereof. The methods provided herein comprise administering to the subject any of the liposomal compositions described herein.

[0116] In another aspect, provided herein is a method for treating a congenital disorder of glycosylation (CDG) in a subject in need thereof. In some embodiments, the method comprises administering to the subject any of the compositions described herein. In some embodiments, the congenital disorder of glycosylation (CDG) is a CDG-Ia disorder. In some embodiments, the administration of the composition induces a 0.05-fold to at least a 3-fold increase in cellular production of higher-order lipid-linked oligosaccharides in the human, as compared to cellular production of higher-order lipid-linked oligosaccharides in the human in the absence of administering the composition to the subject.

[0117] In some variations, the compositions are administered at a weekly or biweekly dose of M1P or a pharmaceutically salt thereof from about 10 mg/kg to about 40 mg/kg, from about 20 mg/kg to about 40 mg/kg, from about 30 mg/kg to about 30 mg/kg, from about 25 mg/kg to about 30 mg/kg. In some variations, the compositions are administered parenterally (e.g., intravenously). For instance, the compositions may be administered by intravenous infusion. It will be understood that in all embodiments, the dose of M1P is based on the molecular weight of the free acid form of M1P (chemical formula C6H13O9P; MW = 260.135 g/mol).

Articles of Manufacture and Kits

[0118] The present disclosure also provides articles of manufacture and/or kits containing any of the liposomal compositions described herein. Articles of manufacture and/or kits of the present disclosure may include one or more containers comprising a purified liposomal composition of the present disclosure. Suitable containers may include, for example, bottles, vials, syringes, and IV solution bags. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the articles of manufacture and/or kits further include instructions for use in accordance with any of the methods of the present disclosure. In some embodiments, these instructions comprise a description of administration of any of the liposomal compositions described herein to deliver the mannose phosphate (e.g., M1P) to a subject in need thereof, to treat a congenital disorder of glycosylation (CDG) to a subject in need thereof, according to any of the methods of the present disclosure. In some embodiments, the instructions comprise a description of how to detect a congenital disorder of glycosylation (CDG), for example in a subject, in a tissue sample, or in a cell. The article of manufacture and/or kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether that subject has the disease and the stage of the disease.

[0119] The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the articles of manufacture and/or kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the article of manufacture and/or kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0120] The label or package insert indicates that the composition is used for delivering M1P and/or treating, e.g., a congenital disorder of glycosylation (CDG). Instructions may be provided for practicing any of the methods described herein.

[0121] The articles of manufacture and/or kits of the present disclosure may be in suitable packaging. Suitable packaging includes, for example, vials, pre-filled syringes, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. An article of manufacture and/or kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a carbohydrate such as M1P capable of treating a congenital disorder of glycosylation (CDG) and/or improving one or more symptoms thereof. The container may further comprise a second pharmaceutically active agent. [0122] Articles of manufacture and/or kits may optionally provide additional components such as buffers and interpretive information. Normally, the article of manufacture and/or kit comprises a container and a label or package insert(s) on or associated with the container.

ENUMERATED EMBODIMENTS

[0123] The following enumerated embodiments are representative of some aspects of the invention.

Al . A composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the intraliposomal compartment comprises a buffering agent and optionally an acid or base, wherein the pH of the intraliposomal compartment is from about 6.0 to about 7.9.

A2. The composition of embodiment Al, wherein the pH of the intraliposomal compartment is from about 6.2 to about 7.4.

A3. The composition of embodiment Al or A2, wherein the buffering agent in the intraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris).

A4. The composition of any one of embodiments A1-A3, wherein the buffering agent in the intraliposomal compartment is 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).

A5. The composition of any one of embodiments Al -A3, wherein the buffering agent in the intraliposomal compartment is a histidine or citrate buffer.

A6. The composition of any one of embodiments A1-A3, wherein the intraliposomal buffer comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate or tromethamine buffering agent.

A7. The composition of any one of embodiments A1-A6, wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal compartment comprises a buffering agent and a tonicity modifier, wherein the pH of the extraliposomal compartment is from about 6.0 to about 7.9. A8. The composition of embodiment A7, wherein the pH of the extraliposomal compartment is from about 6.2 to about 7.4.

A9. The composition of embodiment A7 or A8, wherein the buffering agent in the intraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris).

A10. The composition of any one of embodiments A1-A9, wherein the lipid membrane comprises:

(a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail;

(b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and

(c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG).

Al 1. The composition of embodiment A10, wherein at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), or a salt thereof.

A12. The composition of embodiment A10 or Al l, wherein at least one phospholipid having a choline group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), or a salt thereof.

A13. The composition of any one of embodiments A10-A12, wherein at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or a salt thereof.

A14. The composition of any one of embodiments A10-A13, wherein the lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG.

A15. The composition of embodiment A14, wherein the lipid membrane comprises DOPC, DOPE and DSPE-PEG2000. A16. The composition of any one of embodiments A1-A15, wherein the pH of the intraliposomal buffer does not change upon storage of the liposomal composition for up to 2 years at 5°C.

A17. The composition of any one of embodiments A1-A15, wherein the pH of the intraliposomal buffer does not change upon storage of the liposomal composition for up to 6 months at room temperature.

A18. The composition of any one of embodiments A1-A17, wherein all of the lipids comprising the lipid membrane degrade less than 1% when stored at 5°C or room temperature for up to 2 years.

A19. The composition of any one of embodiments A1-A17, wherein less than 10% of the intraliposomal M1P is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.

A20. The composition of any one of embodiments A1-A19 wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a Z-average between 80 nm and 130 nm.

A21. The composition of any one of embodiments A1-A20, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a poly dispersity index of less than 0.2 or less than 0.1.

A22. The composition of any one of embodiments A1-A21, wherein, over a period of time at temperatures between 5°C and 40°C, no free M1P is detected in the composition.

A23. The composition of any one of embodiments A1-A22, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has an encapsulation efficiency of at least 80%.

A24. The composition of any one of embodiments A1-A23, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a mean osmolality within the range of 290 mOsm/kg to 320 mOsm/kg.

A25. The composition of any one of embodiments A1-A24, further comprising at least one radical scavenging antioxidant. A26. The composition of embodiment A25, wherein at least one radical scavenging antioxidant is butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol, or any combination thereof.

A27. The composition of any one of embodiments A1-A26, further comprising BHT.

A28. The composition of embodiment A27, further comprises alpha tocopherol.

A29. The composition of any one of embodiments A7-A28, wherein the tonicity modifier is an ionic tonicity modifier or sugar.

A30. The composition of any one of embodiments A7-A28, wherein the tonicity modifier comprises saline.

A31. A method of treating a congenital disorder of glycosylation in a subject in need thereof, comprising: administering to the subject a composition of any one of embodiments A1-A30.

A32. The method of embodiment A31, wherein the congenital disorder of glycosylation is CDG-Ia, CDG-Ib, CDG-Ie, CDG-Ig, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.

A33. The method of embodiment A31 or A32, wherein the composition is administered intravenously.

Bl. A composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the intraliposomal compartment comprises a buffering agent and optionally an acid or base, wherein the pH of the intraliposomal compartment is from about 6.0 to about 7.9.

B2. The composition of embodiment Bl, wherein the pH of the intraliposomal compartment is from about 6.2 to about 7.4.

B3. The composition of embodiment B 1 or B2, wherein the buffering agent in the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris). B4. The composition of embodiment B 1 or B2, wherein the buffering agent in the intraliposomal compartment comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).

B5. The composition of embodiment B 1 or B2, wherein the buffering agent in the intraliposomal compartment comprises a histidine or citrate buffer.

B6. The composition of embodiment B 1 or B2, wherein the buffering agent in the intraliposomal compartment comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate, or tromethamine.

B7. The composition of any one of embodiments B 1 to B6, wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal compartment comprises a buffering agent and a tonicity modifier, wherein the pH of the extraliposomal compartment is from about 6.0 to about 7.9.

B8. The composition of embodiment B7, wherein the pH of the extraliposomal compartment is from about 6.2 to about 7.4.

B9. The composition of embodiment B7 or B8, wherein the buffering agent in the intraliposomal compartment and extraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris), and optionally, wherein the concentration of the buffering agent in the intraliposomal compartment is higher than the concentration of the buffering agent in the extraliposomal compartment.

BIO. The composition of any one of embodiments B 1 to B9, wherein the lipid membrane comprises:

(c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG). B 11. The composition of embodiment BIO, wherein at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), or a salt thereof.

B12. The composition of embodiment BIO or Bl 1, wherein at least one phospholipid having a choline group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), or a salt thereof.

B13. The composition of any one of embodiments BIO to Bl 2, wherein at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or a salt thereof.

B14. The composition of any one of embodiments BIO to B13, wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG.

Bl 5. The composition of embodiment Bl 4, wherein the lipid membrane comprises DOPC, DOPE, and DSPE-PEG2000.

Bl 6. The composition of any one of embodiments B 1 to Bl 5, wherein all of the lipids comprising the lipid membrane degrade less than about 1% when stored at 5°C or room temperature for up to 2 years.

Bl 7. The composition of any one of embodiments B 1 to Bl 5, wherein less than 10% of the intraliposomal M1P is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.

Bl 8. The composition of any one of embodiments B 1 to B17 wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a Z-average between 80 nm and 130 nm.

Bl 9. The composition of any one of embodiments B 1 to Bl 8, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a poly dispersity index of less than 0.2 or less than 0.1.

B20. The composition of any one of embodiments B 1 to Bl 9, wherein, over a period of time at temperatures between 5°C and 40°C, no free M1P is detected in the composition. B21. The composition of any one of embodiments B 1 to B20, wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a mean osmolality within the range of 290 mOsm/kg to 320 mOsm/kg.

B22. The composition of any one of embodiments Bl to B21, wherein (a) the composition has an encapsulation efficiency between 5% and 50%; or (b) the percentage of encapsulated M1P is at least about 70%, or a combination of (i) and (ii).

B23. The composition of any one of embodiments B 1 to B22, further comprising at least one radical scavenging antioxidant.

B24. The composition of any one of embodiments B7 to B23, wherein the tonicity modifier is an ionic tonicity modifier or sugar.

B25. The composition of any one of embodiments B7 to B24, wherein the tonicity modifier comprises saline.

B26. A composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment a mannose phosphate, and wherein the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris), wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal component comprises Tris and saline, wherein the concentration of Tris in the intraliposomal compartment is higher than the concentration of Tris in the extraliposomal compartment.

B27. The composition of embodiment B26, wherein the mannose phosphate is M1P.

B28. The composition of embodiment B26 or B27, wherein the Tris present in the intraliposomal compartment is between about 40 mM and 60 mM, and the Tris present in the extraliposomal compartment is between about 10 mM and 20 mM.

B29. The composition of any one of embodiments B26 to B28, wherein the molar ratio of Tris in the intraliposomal compartment to extraliposomal compartment is between about 6: 1 and 2 : 1. B30. The composition of any one of embodiments B26 to B29, wherein the saline present in the extraliposomal component is at least about 125 mM.

B31. A method of treating a congenital disorder of glycosylation in a subject in need thereof, comprising: administering to the subject a composition of any one of embodiments Bl to B30.

B32. The method of embodiment B31, wherein the congenital disorder of glycosylation is CDG-Ia, CDG-Ib, CDG-Ie, CDG-Ig, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.

B33. The method of embodiment B31 or B32, wherein the composition is administered intravenously.

EXAMPLES

[0124] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.

[0125] Abbreviations used are those conventional in the art. The following examples are intended to be illustrative only and not limiting in any way.

Abbreviation Description

NaCl Sodium Chloride

PMM2-CDG Phosphomannomutase 2 congenital disorder of glycosylation

PTL Particle technology Labs

BHT Butylated hydroxytoluene

USP United States Pharmacopeia

TRIS Tris(hydroxymethyl)aminomethane

HEPES 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid

PDI Poly dispersity index

DOPE l,2-dioleoyl- w-glycero-3 -phosphoethanolamine

DOPC l,2-dioleoyl- w-glycero-3 -phosphocholine l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene

DSPE-PEG2000 glycol)-2000] NTA Nanoparticle tracking analysis

SPOS Single particle optical sensing

RRT Relative retention time i p Mannose 1 -phosphate

Example 1

Formulation Development Study

A. Preparation of Liposomal Compositions

[0126] Saline Formulation 1-A is a liposomal M1P formulation prepared in 0.9% (w/v) NaCl. Based on stability studies, lipid degradation due to hydrolysis and/or oxidation was observed. To optimize the formulation to stabilize the lipids, a formulation development study was performed leading to the development of Formulation 1.

[0127] Tris Formulation l is a liposomal M1P formulation optimized for treatment of phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG). The examples provided herein describe the formulation development study for Tris Formulation 1, which was found to reduce the degradation of lipids by hydrolysis and oxidation, and to reduce formation of agglomeration. Various buffers, such as TRIS, HEPES and bicarbonate buffers, were tested. A higher concentration of buffer was used in intraliposomal compartment because mannose- 1 -phosphate (M1P) is the main driver for shifting the pH to a higher pH and the higher buffer capacity helps to maintain the neutral pH.

[0128] The lipid excipients which make the liposomes are DOPC, DOPE, and DSPE- PEG2000. Table 1 summarizes the formulations were tested.

Table 1.

[0129] A higher concentration of buffer was used in intraliposomal compartment to help maintain a neutral pH. Lipid type and ratio and the amount of M1P solution mixed with lipids in ethanol were kept constant.

B. Design of Tests and Short-Term Stability Studies

[0130] The lipid stability of the liposomal formulations prepared above were evaluated using the following tests: pH; size (Z-average, DIO, D50, D90, D99, PDI); M1P (total M1P, free M1P, encapsulation efficiency); lipid (DOPE, DOPC, DSPE-PEG2000, total lipid); lipid impurities; D/L ratio; osmolality (only at T=0); and particle population over 200 nm, over 10 pm/ container , and over 25 pm/container.

[0131] The liposomal formulations were tested under the following stability conditions: accelerated (25°C), long-term (5°C) and stress (40°C), at initial concentrations, 0.5 months, 1 month, and 3 months.

[0132] Additional analysis performed to assess stability of the formulations include (1) formulation preparation, pH, size, M1P, lipid, Drug to Lipid (D/L ratio, osmolality; as well as (2) NTA and SPOS at T=0, 0.5, 1, 2, and 3 month(s).

C. Lipid Degradation

[0133] Lipid degradation over time of the liposomal formulations prepared above was evaluated. With reference to FIG. 1, degradation was observed in Saline Formulation 1-A sample both at 25 and 40°C. DOPE in Formulation 5 samples degraded at 40°C within 1 month. Formulation 6 had degradation of DOPC and DOPE within 1 month. Formulation 3 and Formulation 4 had similar pattern of no more than 7% degradation of DOPC and DOPE in 1 month. Tris Formulation 1 and Formulation 2 also observed no more than 7% degradation. However, at the 3 -month time point, the concentration increased in all samples, which may have come from the assay variability.

[0134] As a next step, the growth of lipid impurities due to lipid degradation was evaluated. With reference to FIG. 2, the horizontal line indicates 4%. No impurities were detected at 0 month.

[0135] In this example, the upper threshold of total impurity was set to 4%. Saline Formulation 1-A sample exceeded the set threshold at 25°C and 40°C. Formulation 5 and Formulation 6 samples exceeded the set threshold at 40°C. The rest of the samples did not exceed the threshold.

[0136] Next, individual impurity peaks were evaluated. With reference to FIG. 1, the horizontal “USL” line indicates 1%. No impurities were detected at 0 month. The data from 3-month stability was not included due to difficulty in comparison of the RRT.

[0137] Analysis of individual impurities shows that the impurities at RRT 0.436-0.440 was unique to Formulation 1-A while impurities at RRT 0.116-0.117 was observed in all the formulations as prominent degradation peaks.

[0138] Both lipid degradation analysis and impurity analysis showed that Saline Formulation 1-A had the least lipid stability and bicarbonate samples (Formulation 5 and Formulation 6) had the second least stability. The stability of HEPES samples and Tris samples were similar and better than the other two formulations.

[0139] A lack of ability to maintain a neutral pH, which is where lipids are most stable may explain why Saline Formulation 1-A and bicarbonate formulations degraded more. With reference to FIG. 3 which depicts pH vs. time, the horizontal lines indicate the upper (“USL”) and lower limits (“LSL”) of pH based on target specifications of Formulation 1-A, while the horizontal “Target” line indicates the target pH of 7. [0140] The ideal pH range to minimize the lipid degradation is close to neutral pH. The pHs of Formulation 1-A and bicarbonate samples were further away from the ideal pH ranges than HEPES and Tris samples.

D. Particulate Analysis

[0141] The stability of the liposomal formulations prepared above was evaluated as nanoparticles. The size distribution of liposome should be narrow, and no agglomeration of liposomes should be observed over time.

[0142] First, the population over 10 pm and 25 pm was evaluated using single particle optical sensing (SPOS). Particulate Count over 10 pm and 25 pm are shown in FIGS. 4A and 4B, respectively. Horizontal “USL” line shows the upper limit of number of particles per guidance of USP <788> and expected vial fill volume.

[0143] At initial and 0.5 month time point, no notable increase in particulates were observed. However, at the 1 month time point, Formulation 6 and Formulation 2 showed increased particulate counts.

[0144] Particle size and distribution was analyzed using dynamic light scattering and nanoparticle tracking analysis. Z-average, PDI and population over 200 nm are shown in FIGS. 5A-5C, respectively. The horizontal lines in FIG. 5A indicates the upper and lower limit of z-average based on the target specifications of Formulation 1-A. The lines in FIG. 5C represent population over 200 nm vs time.

[0145] With reference to FIG. 5A, all z-average data were within the target specifications of Saline Formulation 1-A. PDIs of Formulation 6, Formulation 4 and Formulation 2 were classified as moderate while the rest were classified as narrow. The analysis of population between 200 nm and 1000 nm showed that Formulation 2 had relatively higher population over 200 nm. PDI calculated from DLS measurement agreed with the population over 200 nm from NTA measurement.

[0146] With reference to FIG. 5B, formulations with sucrose (Formulation 6, Formulation 4 and Formulation 2) were found to have wider size distributions, which is less desirable from a long-term storage and terminal sterile filtration perspective.

E. Other Stability Analysis [0147] Other factors were evaluated to determine stability of the liposomal formulations prepared above.

[0148] An in-vitro release (IVR) assay was developed to assess the integrity and consistency of the compositions. The assay utilizes LC-MS to quantify both free and total M1P at various timepoints after the drug product has been incubated in a physiologically- relevant matrix at 37°C with gentle agitation. Free M1P vs time is shown in FIG. 6. Free M1P concentration in the Saline Formulation 1-A sample increased in all temperature ranges. No samples other than Saline Formulation 1-A and Formulation 2 exhibited an increase in free M1P. The lower leaching of M1P over time observed in Tris Formulation 1 is an additional benefit over Saline Formulation 1-A.

[0149] The osmolality of samples is shown in FIG. 7. The horizontal lines indicate the upper and lower limit of osmolality (“USL” and “LSL”) based on the target specifications (horizontal “Target” line) of Formulation 1-A.

Example 2

Long Term Assessment of Critical Quality Attributes for Saline Formulation 1-A and Tris Formulation 1

[0150] Based on the analysis in this example, Tris Formulation 1 (Example 1) displays improved long-term stability over Saline Formulation 1-A in terms of stabilization of pH, potential particle distribution/agglomeration and leakage of M1P out of the liposomes. Hence, Tris Formulation 1 was chosen as the drug product for further clinical development. The components of the drug product assessed in long-term stability assays are listed in Table 2.

Table 2 Drug Product Formulations Used for Long-Term Stability Assessment

[0151] The resultant formulations were subjected to long-term stability studies in which vials containing the compositions were stored inverted at various temperatures. The pH, osmolality, particle size, and encapsulation % (M1P) were evaluated for each formulation. pH and Osmolality were evaluated based on known protocols, such as USP <791> and USP <975> respectively. Encapsulation % (M1P) was evaluated by a liquid chromatography- charged aerosol detection (HPLC-CAD) method.

[0152] Particle size was determined by dynamic light scattering using an intensity weighted distribution calculation. Saline Formulation 1-A was evaluated at 5°C at 1 month, 3 months, 6 months, 12 months, 18 months, and 24 months; at 25°C/60% RH (relative humidity) at 1 month, 3 months, and 6 months; and at 40°C/75% RH at 1 month. At the accelerated conditions of 25°C/60% RH and 40°C/75% RH, the pH decreased from 7.4 at initial to 7.1 and 7.0 respectively at the final timepoints. Similarly, the % encapsulation decreased from >95% to 92% and 90% respectively. These decreases were observed to be notable when compared to a similar stability of Tris Formulation 1. Under accelerated conditions, the pH of Tris formulation 1 decreased from 7.2 to 7.0 after 6 months at 25°C/60% RH and did not change at all (7.2 to 7.2) after 1 month at 35°C. Similarly, the % encapsulation of M1P remained at >95% for the duration of the accelerated studies demonstrating the enhanced stability to M1P leakage of the Tris Formulation 1. Both formulations showed no significant change at the long-term condition of 5°C for any quality attribute through 2 years of study.

Example 3

Long-Term Impact of BHT and Buffer on Lipid Stability

[0153] Based on the analysis in this example, the long-term stability studies on Saline Formulation 1-A and Tris Formulation 1 (Examples 1 and 2) also demonstrate that BHT and buffer control are capable of reducing the degree of lipid degradation at both 5°C and room temperature over a period of two years. Lipid degradation is expected to cause the decrease in pH and % encapsulation observed in Saline Formulation 1-A, particularly under accelerated storage conditions.

[0154] Under the same stability conditions referenced in Example 2 above, Saline Formulation 1-A demonstrated an increase in lipid impurities from <1% at initial release to 1.8 % after 24 months at 5°C, 23.0% after 24 months at 25°C and 100% after 24 months at 40°C. Conversely, the lipid impurities in Tris Formulation 1 remained <1% after 24 months at 5°C, and 15.6% after 24 months at 25°C. The 35°C condition was not evaluated at 24 months for Tris Formulation 1. The degree of lipid degradation was measured utilizing HPLC-CAD and is reported as % chromatographic area. The lipid method was validated to meet the levels of accuracy and precision required of the phase of study.

Example 4

Stability Assessment for Formulation Z

[0155] This example explores lipid degradation at either 5°C or room temperature (25°C) over a period of 6 months of another exemplary formulation, referred to in this example as Formulation Z (the components of which are provided in Table 3 below).

Table 3 Drug Product Formulation Used for Stability Assessment

[0156] Formulation Z showed that lipid impurities remained at <0.1% after 6 months at 5°C, and showed an increase in lipid impurities from 0% at initial release to only 1.78% after 6 months at 25°C. By way of comparison, a comparable formulation containing BHT at 20 pg/mL showed an increase in lipid impurities from 0% at initial release to 1.99% after 6 months at 25°C. The degree of lipid degradation was measured utilizing HPLC-CAD and is reported as % chromatographic area. The lipid method was demonstrated to meet the levels of accuracy and precision required of a development study.

[0157] Further, as shown in Table 4 below, the following were observed for Formulation Z: pH increased slightly after 6 months at both 5°C and at 25°C, and % encapsulation of M1P remained at >92% after 6 months at both 5°C and at 25°C.

Table 4 Stability Data for Formulation Z

Claims

CLAIMS What is claimed is:

1. A composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the intraliposomal compartment comprises a buffering agent and optionally an acid or base, wherein the pH of the intraliposomal compartment is from about 6.0 to about 7.9.

2. The composition of claim 1, wherein the pH of the intraliposomal compartment is from about 6.2 to about 7.4.

3. The composition of claim 1 or claim 2, wherein the buffering agent in the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris).

4. The composition of claim 1 or claim 2, wherein the buffering agent in the intraliposomal compartment comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).

5. The composition of claim 1 or claim 2, wherein the buffering agent in the intraliposomal compartment comprises a histidine or citrate buffer.

6. The composition of claim 1 or claim 2, wherein the buffering agent in the intraliposomal compartment comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate or tromethamine.

7. The composition of any one of claims 1 to 6, wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal compartment comprises a buffering agent and a tonicity modifier, wherein the pH of the extraliposomal compartment is from about 6.0 to about 7.9.

8. The composition of claim 7, wherein the pH of the extraliposomal compartment is from about 6.2 to about 7.4.

9. The composition of claim 7 or 8, wherein the buffering agent in the intraliposomal compartment and extraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris), and optionally, wherein the concentration of the buffering agent in the intraliposomal compartment is higher than the concentration of the buffering agent in the extraliposomal compartment.

10. The composition of any one of claims 1 to 9, wherein the lipid membrane comprises:

11. The composition of claim 10, wherein at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), or a salt thereof.

12. The composition of claim 10 or 11, wherein at least one phospholipid having a choline group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), or a salt thereof.

13. The composition of any one of claims 10 to 12, wherein at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is 1,2-distearoyl- sn-glycero-3 -phosphoethanolamine (DSPE), or a salt thereof.

14. The composition of any one of claims 10 to 13, wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG.

15. The composition of claim 14, wherein the lipid membrane comprises DOPC, DOPE, and DSPE-PEG2000.

16. The composition of any one of claims 1 to 15, wherein all of the lipids comprising the lipid membrane degrade less than about 1% when stored at 5°C or room temperature for up to 2 years.

17. The composition of any one of claims 1 to 15, wherein less than 10% of the intraliposomal M1P is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.

18. The composition of any one of claims 1 to 17 wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a Z-average between 80 nm and 130 nm.

19. The composition of any one of claims 1 to 18, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a poly dispersity index of less than 0.2 or less than 0.1.

20. The composition of any one of claims 1 to 19, wherein, over a period of time at temperatures between 5°C and 40°C, no free M1P is detected in the composition.

21. The composition of any one of claims 1 to 20, wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a mean osmolality within the range of 290 mOsm/kg to 320 mOsm/kg.

22. The composition of any one of claims 1 to 21, wherein (a) the composition has an encapsulation efficiency between 5% and 50%; or (b) the percentage of encapsulated M1P is at least about 70%, or a combination of (i) and (ii).

23. The composition of any one of claims 1 to 22, further comprising at least one radical scavenging antioxidant.

24. The composition of any one of claims 7 to 23, wherein the tonicity modifier is an ionic tonicity modifier or sugar.

25. The composition of any one of claims 7 to 24, wherein the tonicity modifier comprises saline.

26. A composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment a mannose phosphate, and wherein the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris), wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal component comprises Tris and saline, wherein the concentration of Tris in the intraliposomal compartment is higher than the concentration of Tris in the extraliposomal compartment.

27. The composition of claim 26, wherein the mannose phosphate is M1P.

28. The composition of claim 26 or 27, wherein the Tris present in the intraliposomal compartment is between about 40 mM and 60 mM, and the Tris present in the extraliposomal compartment is between about 10 mM and 20 mM.

29. The composition of any one of claims 26 to 28, wherein the molar ratio of Tris in the intraliposomal compartment to extraliposomal compartment is between about 6: 1 and 2 : 1.

30. The composition of any one of claims 26 to 29, wherein the saline present in the extraliposomal component is at least about 125 mM.

31. A method of treating a congenital disorder of glycosylation in a subject in need thereof, comprising: administering to the subject a composition of any one of claims 1 to 30.

32. The method of claim 31, wherein the congenital disorder of glycosylation is CDG-Ia, CDG-Ib, CDG-Ie, CDG-Ig, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.

33. The method of claim 31 or 32, wherein the composition is administered intravenously.