Continuous flow process for producing mannose-1-phosphate, polymorphs and compositions of mannose-1-phosphate, and uses relating thereto

Abstract

Provided herein is a method for synthesizing the alpha isomer of mannose-1-phosphate (M1P) and a composition comprising the alpha isomer of mannose-1-phosphate (M1P). For example, such a method uses techniques in flow chemistry to achieve a substantially pure alpha isomer of M1P. Also provided is a composition, including a pharmaceutical composition, comprising the substantially pure alpha isomer of M1P. Also provided herein is a polymorphic form of M1P, including a polymorphic form that can be generated from the substantially pure alpha isomer of M1P.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 343,447, filed May 18, 2022, the entirety of which is hereby incorporated by reference herein.

[0002] Field The present disclosure relates generally to mannose-1-phosphate ("M1P"), and more specifically to continuous flow processes for producing M1P, including certain polymorphs of M1P, and compositions comprising M1P produced using such continuous flow processes. [Background technology]

[0003] background Glycosylation, the enzymatic attachment of carbohydrates (glycans) to proteins and lipids, is a more common co- and post-translational modification (PTM) than any other PTM, as it applies to the majority of proteins synthesized in the rough endoplasmic reticulum (ER). Glycosylation plays a crucial role in various biological processes of membrane and secreted proteins. Glycosylation defines protein structure and folding in the ER, directs the trafficking of properly folded proteins to the Golgi, or acts as a quality control mechanism to target misfolded proteins for degradation. Glycan moieties may also act as ligands for cell surface receptors to mediate cell attachment or stimulate signaling pathways. Congenital disorders of glycosylation, also known as CDG syndromes, are a group of rare genetic disorders in which tissue proteins and / or lipids carry glycosylation defects and / or glycosylation deficiencies. These diseases are associated with numerous enzymatic defects and often cause severe, sometimes fatal, dysfunction of the nervous system, muscles, intestine, and several other organ systems.

[0004] Common clinical manifestations in children with CDG include hypotonia, developmental delay, failure to thrive, hepatic insufficiency, coagulopathy, hypothyroidism, abnormal lipid patterns and inverted nipples, hypoglycemia, seizures, cerebellar hypoplasia, and stroke-like episodes in developmentally delayed children. In older adolescence or adulthood, symptoms may include ataxia, cognitive impairment, absence of puberty in females, underdeveloped testes in males, retinitis pigmentosa, scoliosis, joint contractures, and peripheral neuropathy.

[0005] CDG can be classified into two groups: CDG type I and CDG type II. CDG type I is characterized by defects in the initial step of N-linked protein glycosylation, i.e., the biosynthesis of dolichol pyrophosphate-linked oligosaccharides (DLOs) that occur in the ER, or the transfer of DLOs to asparagine residues of nascent polypeptides. CDG type II involves defects in the further processing (synthetic or hydrolytic) of protein-linked glycans. Currently, 22 CDG type I variants and 14 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 (PMM) activity, which leads to a deficiency or failure of intracellular N-glycosylation (Jaeken et al. J. of Inherit. Met. Disease. 2008, 31: 669-672). PMM is responsible for the conversion of mannose-6-phosphate to mannose-1-phosphate ("M1P").

[0006] Patients suffering from reduced PMM activity have reduced M1P production, which is associated with symptoms of multiple organ dysfunction. To overcome the deficiency of PMM production, it is important to supply the downstream enzymes with the necessary substrate (i.e., M1P). However, the delivery and maintenance of such a systematic supply of M1P is problematic because extracellular enzymes in body fluids degrade M1P when delivered externally by oral or intravenous administration. Another problem with exogenously delivered M1P is that its high polarity prevents it from penetrating the cell interior (i.e., the cytosol) and therefore from treating the deficiency of PMM production. Potential solutions include encapsulating and delivering M1P to the interior of cells using delivery vehicles, such as lipid particles (see WO2015 / 053910 and WO2020 / 205530). However, to make such formulations commercially viable, commercially viable methods for producing M1P on a large scale are required. Current methods for producing M1P are generally limited by the difficulty of obtaining the desired isomer of M1P in pure form and on a commercially viable economic scale. During conventional production of M1P, synthetic intermediates decompose into the beta anomeric form, thereby resulting in a mixture of alpha and beta anomers in the final product composition. Isolating the pure alpha isomer of M1P requires expensive and time-consuming purification methods. Therefore, there is a need in the art for commercially viable methods for the large-scale production of M1P. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 053910 [Patent Document 2] International Publication No. 2020 / 205530 [Non-patent literature]

[0008] [Non-Patent Document 1] Jaeken et al. J. of Inherit. Met. Disease. 2008, 31: 669-672 Summary of the Invention [Means for solving the problem]

[0009] Quick Overview In a first aspect, there is provided a method for generating a continuous flow, comprising the steps of: a) preparing a compound of formula B-α-1 and a nucleophilic catalyst at a temperature between −30° C. and −10° C. to obtain a pre-cooled solution, wherein the compound of formula B-α-1 is [ka] and the pre-cooled solution contains less than 1% of a compound of formula (B-β-1) [ka] and b) combining the pre-cooled solution and the phosphorylating agent continuously in a first reactor at a temperature between -30°C and 5°C for a residence time between 30 seconds and 5 minutes to obtain a compound of formula (C-α-1) [ka] producing an intermediate composition comprising: c) transferring the intermediate composition from the first reactor to a second reactor; d) continuously combining the intermediate composition with an oxidant in a second reactor at a temperature between 0° C. and 30° C. for a residence time between 10 minutes and 30 minutes to produce a compound of formula (D-α-1) [ka] forming a reaction mixture comprising: A method is provided, comprising:

[0010] In some embodiments, the method further comprises deprotecting the compound of formula (D-α-1) to produce a composition comprising the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, wherein less than 2% of the composition is the beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing.

[0011] In another aspect, the composition produced according to the method described above is provided.In some embodiments, the composition is provided that comprises the alpha isomer of mannose-1-phosphate, or its salt, or any of the hydrates of the above, and the purity of the composition is at least 96%.In some embodiments, less than 2% of the composition is the beta isomer of mannose-1-phosphate, or its salt, or any of the hydrates of the above.

[0012] In another aspect, a method of producing crystalline form C of mannose-1-phosphate is provided, the method comprising combining a starting composition comprising (i) a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) crystalline form A of the substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) crystalline form B of the substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii), with a solvent comprising water and alcohol to produce a suspension, and agitating the suspension to produce a product composition comprising crystalline form C of mannose-1-phosphate.

[0013] In another aspect, there is provided a crystalline form C of mannose-1-phosphate produced according to the method described in the previous paragraph. In some embodiments, there is provided a crystalline form C of mannose-1-phosphate that is a substantially pure alpha isomer of the potassium trihydrate salt of mannose-1-phosphate.

[0014] In another embodiment, there is provided a composition comprising at least 98% by weight of crystalline form C according to the above description.

[0015] In another aspect, a composition is provided that includes (i) a liposome comprising one or more phospholipids conjugated to polyethylene glycol (PEG), and (ii) an alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, encapsulated in the liposome.

[0016] In one embodiment, a composition is provided comprising a liposome having a lipid membrane surrounding an intraliposomal compartment, the liposome encapsulating an alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing in the intraliposomal compartment, the lipid membrane comprising: (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 conjugated to polyethylene glycol (PEG) having an ethanolamine head group and at least one saturated fatty acid tail; an intraliposomal buffer comprising a buffer salt and optionally an acid, wherein the buffer salt has a pKa between 6 and 8.5; an extraliposomal buffer comprising a buffer salt and a tonicity adjusting agent, wherein the buffer salt has a pKa between 6 and 8.5; and optionally a radical scavenging antioxidant.

[0017] In yet another aspect, methods are provided for treating congenital disorders of glycosylation (CDG) in a subject in need thereof by administering to the subject any of the M1P compositions (including pharmaceutical compositions) provided herein.

[0018] The present application can be understood by reference to the following description in conjunction with the accompanying figures. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows an exemplary scheme for producing an M1P salt or a hydrate thereof from (2S,3S,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol.

[0020] [Diagram 2] FIG. 2 shows an exemplary method and system for making (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate, which can be deprotected to produce M1P, or a salt or hydrate thereof.

[0021] [Diagram 3] FIG. 3 shows an exemplary XRPD of crystalline form A of M1P, the dihydrate dipotassium salt.

[0022] [Figure 4] FIG. 4 shows an exemplary XRPD of crystalline form B of M1P, the trihydrate dipotassium salt.

[0023] [Diagram 5] FIG. 5 shows an exemplary XRPD of crystalline form C of M1P, the trihydrate dipotassium salt.

[0024] [Figure 6] FIG. 6 shows an exemplary TGA spectrum of crystalline form C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The following description describes exemplary compositions, methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but instead is provided as a description of exemplary embodiments.

[0026] In some aspects, provided herein is a method comprising continuous flow chemistry to produce pure or substantially pure alpha isomer of mannose-1-phosphate (including its salts and hydrates). In some variations, the produced composition has less than 2% of beta isomer of mannose-1-phosphate (including its salts and hydrates). In other aspects, provided herein is a composition comprising pure or substantially pure alpha isomer of mannose-1-phosphate (including its salts and hydrates), as well as liposomal formulations incorporating such pure or substantially pure alpha isomer of mannose-1-phosphate (including its salts and hydrates), and the use of such formulations. In some embodiments, the purity of a target compound is the amount of the compound (including its salts and hydrates) compared to all other components in a sample, expressed as a percentage. In some embodiments, the purity of a sample is measured by HPLC, including, for example, using an HPLC separation method to separate each component into a separate peak on a chromatogram. The area peak of the target compound is compared with the peak areas of all other constituents and the purity is expressed as a percentage value. Continuous flow generation method

[0027] Referring to FIG. 1, process 100 is an exemplary scheme for producing form C of M1P. Form C can be produced starting from (2S,3S,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (compound 102). In the first step of process 100, the alcohol group of compound 102 is protected using a suitable protecting agent, such as benzoyl chloride (reagent 104), under suitable reaction conditions to produce (2R,3S,4S,5R,6R)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetrabenzoate (compound 106). For example, the reaction can be carried out in the presence of a suitable base, such as pyridine, at a temperature between about 10° C. and 30° C. A suitable reaction time can be about 3 hours. In other embodiments, other suitable protecting agents, solvents, and bases can be used. For example, the reaction may be carried out in acetonitrile using a sufficient amount of base, such as diisopropylethylamine.

[0028] Referring again to process 100 (FIG. 1), compound 106 can be reacted with a suitable base, such as DMAPA (reagent 108), in a suitable solvent, such as THF, under suitable reaction conditions to selectively remove the benzoyl group at the oxygen attached to the anomeric center to give (2R,3R,4S,5S,6S)-2-((benzoyloxy)methyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 110). For example, the reaction can be carried out at a temperature between about 60° C. and 65° C. for a sufficient length of time, such as 12 to 14 hours. In other embodiments, other bases and solvents can be used to effect selective deprotection.

[0029] Referring again to process 100 (FIG. 1), compound 110 can be phosphorylated under suitable flow chemistry conditions to produce (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphaneyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 114). For example, compound 110 and a nucleophilic catalyst, such as DCI, can be dissolved in a suitable solvent, such as acetonitrile, at a temperature between about −30° C. and −10° C. to obtain a first pre-cooled solution. This pre-cooled solution can then be treated with a suitable phosphorylating agent, such as (BnO), at a temperature between about 0° C. and 5° C. for a sufficient time to obtain an intermediate composition comprising compound 114. 2 PN i Pr 2 The first solution may be successively combined with a second solution containing (reagent 112). The two solutions may be successively combined for a total time of between about 30 seconds and 5 minutes.

[0030] Referring again to process 100 (FIG. 1), the intermediate composition including compound 114 can be oxidized under suitable flow chemistry conditions to produce (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 118). For example, ... 2 O 2 and the oxidant to afford compound 118. The intermediate composition containing compound 114 can be continuously combined with the oxidant for a total time of between about 10 minutes and 30 minutes.

[0031] Referring again to process 100 (FIG. 1), compound 118 can be subjected to catalytic hydrogenation to selectively remove the benzyl protecting group and produce (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 126). For example, compound 118 can be dissolved in a suitable solvent or combination of solvents, such as EtOAc and MeOH, and combined with Pd / C (reagent 124). This solution can then be heated to 30° C. with gaseous H 2 O at a temperature between 20° C. and 30° C. for a sufficient time, such as 10-12 hours, to yield compound 126. 2 (Reagent 122).

[0032] Referring again to process 100 (FIG. 1), compound 126 can be deprotected under suitable conditions to produce (2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl dihydrogen phosphate (compound 130), or a salt or hydrate thereof. For example, compound 126 can be dissolved in a suitable solvent, such as MeOH, and reacted with a suitable base, such as KOMe (reagent 128), at a temperature between 20° C. and 25° C. for a sufficient time, such as 20-22 hours, to provide compound 130 or a hydrate thereof.

[0033] In process 100, KOMe is used as a base to convert compound 126 to the potassium salt, compound 130. In other variations, other suitable salts may be used. It should be understood that the base used in this step determines the particular salt resulting from compound 126. For example, when NaOMe was used as the base, compound 126 could be converted to the corresponding sodium salt.

[0034] It is desirable to produce M1P or a salt or hydrate thereof as a substantially pure alpha isomer. Cyclic carbohydrates exist in either "alpha" or "beta" stereochemical forms or isomers, depending on the position of the substituents attached to the anomeric center. Such forms are sometimes called "anomers" because they are isomers at the anomeric center. In alpha anomers, the substituents attached to the anomeric carbon are on the opposite side of the substituents attached to the other carbon adjacent to the ring oxygen (i.e., trans). In beta anomers, the situation is reversed, and the substituents attached to the anomeric carbon are on the same side of the substituents attached to the other carbon adjacent to the ring oxygen (i.e., cis). The alpha and beta forms of M1P are shown below in "chair" and "flat" diagrams. [ka]

[0035] In some variations, a composition comprising substantially pure alpha isomer refers to a composition having less than 5%, less than 4%, less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the beta isomer of M1P. In one variation, a composition comprising substantially pure alpha isomer refers to a composition having no detectable amount of the beta isomer of M1P.

[0036] To produce a substantially pure alpha isomer of M1P, in some embodiments, flow chemistry can be applied to convert compound 110 to compound 118, as described above, and then compound 118 can be deprotected to produce an M1P salt or hydrate. Referring to Figure 2, an exemplary system 200 for producing compound 118 from compound 110 in two steps using flow chemistry is illustrated.

[0037] In the first flow chemistry step, compound 110 is converted to compound 114. Tank 202 contains compound 110 and a nucleophilic catalyst, e.g. DCI, in a suitable solvent, e.g. acetonitrile, at a temperature between about -30°C and -10°C. Tank 204 contains reagent 112 in the same solvent. Tanks 202 and 204 continuously feed pre-cooling tubes 206 and 208, respectively, via pumps 203 and 205, which then combine in mixer 210 and pass through reactor tube 212, at a temperature between about 0°C and 5°C maintained by bath 216. The total residence time in mixer 210 and reactor tube 212 (together constituting flow reactor 214) is between about 30 seconds and 5 minutes. A composition containing compound 114 is the product of this step and is subjected to the second flow chemistry step without isolation or purification.

[0038] In the second flow chemistry step, compound 114 is converted to compound 118. A composition including compound 114 and a suitable oxidant 218 is continuously combined in mixer 220 and passed through reactor tube 222 at a temperature between about 0° C. and 30° C. maintained by bath 226. For example, the oxidant may be H 2 O 2 (reagent 116 in FIG. 1). The total residence time in mixer 220 and reactor tube 222 (together comprising flow reactor 224) is between about 10 minutes and 30 minutes. The reaction mixture containing compound 118 is the product of this step and is collected in tank 228.

[0039] After the second flow chemistry step, the reaction mixture containing compound 118 is quenched with an aqueous solution containing a reducing agent. 2 SO 3or another suitable reducing agent, which is transferred via pump 232 to tank 228 containing the reaction mixture containing compound 118. Quenching is carried out at a temperature between about 0° C. and 25° C. for a period of about 1 minute to 15 minutes. After quenching, the reaction mixture is diluted with a suitable organic solvent and compound 118 is isolated by extraction. In some embodiments, the organic solvent used for extraction is toluene.

[0040] Thus, in some aspects, a continuous flow production method is provided, comprising the steps of: a) preparing a compound of formula (B-α-1) and a nucleophilic catalyst at a temperature between -30°C and -14°C to obtain a pre-cooled solution; b) continuously combining the pre-cooled solution and the phosphorylating agent in a first reactor at a temperature between 0°C and 5°C for a residence time between 30 seconds and 2 minutes to produce an intermediate composition comprising a compound of formula (C-α-1); c) transferring the intermediate composition from the first reactor to a second reactor; and d) continuously combining the intermediate composition with an oxidant in a second reactor at a temperature between 20°C and 25°C for a residence time between 10 minutes and 20 minutes to produce a reaction mixture comprising a compound of formula (D-α-1).

[0041] In some variations, the compound of formula (B-α-1) is [ka] It is.

[0042] In some variations, the compound of formula (C-α-1) is [ka] It is.

[0043] In some variations, the compound of (D-α-1) is [ka] It is.

[0044] In some embodiments, in step a) of the continuous production method described above, suitable nucleophilic catalysts may include, for example, 1H-tetrazole, 5-(p-nitrophenyl)-1H-tetrazole, 5-ethylthio-1H-tetrazole, benzimidazolium triflate, a mixture of dimethylaminopyridine and 5-(p-nitrophenyl)-1H-tetrazole, a mixture of N-methylimidazole and 1H-tetrazole. In one variation, the nucleophilic catalyst is 4,5-dicyanoimidazole.

[0045] In some variations, an organic solvent is used in the pre-cooled solution of step a). In one embodiment, the compound of formula (B-α-1) and the nucleophilic catalyst are provided in a suitable solvent, for example, an organic solvent. In some embodiments, the organic solvent is a polar aprotic solvent. In some embodiments, the polar aprotic solvent is acetone, dichloromethane, dimethylformamide, dimethylpropyleneurea, dimethylsulfoxide, ethyl acetate, hexamethylphosphoric triamide, 2-MeTHF, or tetrahydrofuran. In one embodiment, the organic solvent is acetonitrile, THF, or 2-MeTHF. In one variation, the solution comprises acetonitrile.

[0046] In some embodiments, in step a) of the continuous production method, the pre-cooled solution comprising the compound of Formula (B-α-1) and the nucleophilic catalyst is held at a temperature between -30°C and -10°C, between -30°C and -15°C, between -30°C and -20°C, between -20°C and -10°C, between -20°C and -15°C, or between -30°C and -25°C.

[0047] In some embodiments, in step b) of the continuous production method described above, suitable phosphorylating agents may include, for example, 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane, diethyl phosphoramidic dichloride, tris(1-pyrrolidinyl)phosphine, 2-chloro-1,3,2-benzodioxaphosphorin-4-one, dibenzyl N,N-diethyl phosphoramidite, diallyl N,N-diisopropyl phosphoramidite, bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite, 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite, or 2-cyanoethyl N,N-diisopropylchloro phosphoramidite. In one variation, the phosphorylating agent is (BnO) 2 PN i Pr 2 Also referred to as dibenzyl N,N-diisopropyl phosphoramidite. In some embodiments, the phosphorylating agent is at least partially dissolved in an organic solvent. In some embodiments, the organic solvent comprises a polar aprotic solvent. In other embodiments, the organic solvent is acetonitrile, THF, or 2-MeTHF. In one variation, the organic solvent is acetonitrile.

[0048] In some embodiments, in step b) of the continuous production process, the pre-cooled solution and the phosphorylating agent are continuously combined in a reactor at a temperature between -30°C and 5°C, between -30°C and 0°C, between -30°C and -5°C, between -20°C and 0°C, between -20°C and -5°C, or between -20°C and -10°C.

[0049] In some embodiments, in step b) of the continuous production method, the pre-cooled solution and the phosphorylating agent are continuously combined in a reactor for a residence time between 30 seconds and 5 minutes, 30 seconds and 4 minutes, 30 seconds and 3 minutes, 30 seconds and 2 minutes, or 30 seconds and 1 minute, or less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0050] In some embodiments, in step b) of the continuous production method, the pre-cooled solution and the phosphorylating agent are each provided at a flow rate between 0.5 mL / min and 1 L / min. In some embodiments, in step b) of the continuous production method, the pre-cooled solution is provided at a flow rate between 0.5 mL / min and 1 L / min. In some embodiments, the phosphorylating agent is transferred at a flow rate between 0.5 mL / min and 1 L / min.

[0051] The intermediate composition produced in step b) of the continuous production method comprises a compound of formula (C-α-1). In certain embodiments, the intermediate composition comprising the compound of formula (C-α-1) contains less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the compound of formula (B-α-1). In some variations, the compound of formula (B-α-1) is not detectable in the intermediate composition. In certain embodiments, the intermediate composition comprising the compound of formula (C-α-1) contains less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the compound of formula (B-β-1). [ka] Contains:

[0052] In some variations, the compound of formula (B-α-1) is not detectable in the intermediate composition. In one variation, the intermediate composition comprising the compound of formula (C-α-1) contains less than 1% of the compound of formula (B-α-1) and less than 1% of the compound of formula (B-β-1).

[0053] In some embodiments, in step d) of the continuous production method described above, suitable oxidants include organic peroxides, such as hydroperoxides, peroxyacids, peroxyesters, diacyl peroxides, and dialkyl peroxides. In some embodiments, tert-butyl hydroperoxide, cumene hydroperoxide, or dicumyl peroxide may be used. In one variation, H 2 O 2 In some embodiments, between 1 and 3 molar equivalents of H 2 O 2In some embodiments, the oxidant may be dissolved in a suitable organic solvent. In other embodiments, the oxidant is present in an aqueous solution.

[0054] In some embodiments, in step d) of the continuous process, the intermediate composition comprising the oxidant and the compound of Formula (C-α-1) are continuously combined in a reactor at a temperature between 0° C. and 30° C., between 5° C. and 30° C., between 10° C. and 30° C., between 15° C. and 30° C., between 20° C. and 30° C., or between 25° C. and 30° C.

[0055] In some embodiments, in step d) of the continuous production process, the intermediate composition comprising the oxidant and the compound of formula (C-α-1) are continuously combined in a reactor for a residence time ranging between 10 minutes and 30 minutes, 15 minutes and 30 minutes, 20 minutes and 30 minutes, 25 minutes and 30 minutes, or 10 minutes and 15 minutes, or less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, or less than 12 minutes.

[0056] In some embodiments, in step d) of the continuous production method, the oxidant is provided at a flow rate between 0.5 mL / min and 1 L / min.

[0057] In some embodiments, the reaction mixture containing the compound of formula (D-α-1) contains less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01% of the compound of formula (D-β-1). [ka] Contains:

[0058] In one variation, the reaction mixture containing the compound of formula (D-α-1) contains less than 2% of the compound of formula (D-β-1). In some variations, the compound of formula (D-β-1) is not detectable in the reaction mixture.

[0059] In some embodiments, the reaction mixture containing the compound of formula (D-α-1) is quenched. In some embodiments, the reaction mixture is quenched with a solution containing a reducing agent. In some variations, the solution is an aqueous solution. In some embodiments, the reducing agent is a dithionate. In other embodiments, the reducing agent is a thiosulfate. In one embodiment, the reducing agent is Na 2 S 2 O 3 In one variation, the reducing agent is Na 2 SO 3 In some embodiments, the reducing agent is a metal catalyst. In one variation, the reducing agent is manganese dioxide. In another variation, the reducing agent is Fe 2+ ions. In some embodiments, the reducing agent is iodide. In one variation, the reducing agent is potassium iodide. In another variation, the reducing agent is activated charcoal. In some embodiments, the reducing agent is ascorbic acid. In some embodiments, the reaction mixture is quenched at a temperature between 0° C. and 25° C., between 0° C. and 20° C., between 0° C. and 15° C., between 0° C. and 10° C., between 0° C. and 5° C., or between 10° C. and 200° C. In one variation, the reaction mixture is quenched at a temperature between 0° C. and 25° C.

[0060] In another aspect of the invention, the compound of formula (D-α-1) is extracted into an organic solvent. In some embodiments, the organic solvent comprises toluene. In some embodiments, the organic solvent comprises water, followed by Na 2 SO 4 In some embodiments, the compound of formula (D-α-1) is isolated by evaporating at least a portion of the organic solvent. In some embodiments, the compound of formula (D-α-1) is not subjected to further purification.

[0061] In one aspect of the invention, the compound of formula (D-α-1) is deprotected to produce a composition comprising the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, and less than 2% of the composition is the beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing. In some embodiments, less than 5%, less than 4%, less than 3%, or less than 1% of the composition is the beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing.

[0062] Referring again to Figure 1, in some embodiments, the compound of formula (B-α-1) is produced by reacting the compound of formula (A-α-1) with DMAPA to selectively remove the benzoyl (Bz) group of the oxygen attached to the anomeric center. In some variations, the compound of formula (A-α-1) is [ka] It is.

[0063] In some embodiments, the compound of formula (B-α-1) is produced by reacting the compound of formula (A-α-1) with an amine base to selectively remove the benzoyl (Bz) group of the oxygen attached to the anomeric center. In some embodiments, the amine base is DMAPA, N,N'-dimethyl-1,3-propanediamine, N,N-dimethyldipropylenetriamine, N 1 -isopropyldiethylenetriamine, 3-(methylamino)propylamine, N,N,N',N'-tetraethyldiethylenetriamine, 3,3'-iminobis(N,N-dimethylpropylamine), or diethylenetriamine. In one variation, the amine base is DMAPA.

[0064] In some embodiments, the compound of formula (B-α-1) is produced by reacting the compound of formula (A-α-1) with an amine base at a temperature between 40° C. and 70° C., between 50° C. and 75° C., between 60° C. and 75° C., or between 60° C. and 65° C. In one variation, the reaction is carried out between 60° C. and 65° C.

[0065] In some embodiments, the compound of formula (B-α-1) is produced by reacting the compound of formula (A-α-1) with an amine base over a reaction time of between 6 hours and 15 hours, 8 hours and 15 hours, 9 hours and 15 hours, 10 hours and 14 hours, 11 hours and 14 hours, or 12 hours and 14 hours, or less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, or less than 13 hours. In one variation, the reaction time is between 12 and 14 hours.

[0066] Referring again to Figure 1, in some embodiments, the compound of formula (A-α-1) is produced by reacting the compound of formula (SM-α-1) with benzoyl chloride to attach a benzoyl group to each hydroxyl group. In some variations, the compound of formula (SM-α-1) is [ka] It is.

[0067] In some embodiments, the compound of formula (A-α-1) is produced by reacting the compound of formula (SM-α-1) with benzoyl chloride in the presence of a suitable base. In some embodiments, the base is an amine base. In some variations, the base serves as a reaction solvent. In some embodiments, the base is pyridine, piperidine, trimethylamine, diisopropylethylamine, ethylamine, ammonia, or aniline. In one variation, the base is pyridine.

[0068] In some embodiments, the compound of formula (A-α-1) is produced by reacting the compound of formula (SM-α-1) with benzoyl chloride at a temperature between 10° C. and 30° C., between 10° C. and 25° C., between 15° C. and 25° C., or between 20° C. and 25° C. In one variation, the reaction is carried out between 20° C. and 25° C.

[0069] In some embodiments, the compound of formula (A-α-1) is produced by reacting the compound of formula (SM-α-1) with benzoyl chloride for a reaction time between 30 minutes and 3 hours, 45 minutes and 3 hours, 1 hour and 3 hours, 1.5 hours and 3 hours, 2 hours and 3 hours, 2.5 hours and 3 hours, or 3 hours and 5 hours, or less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, or less than 4 hours. In one variation, the reaction time is 3 hours.

[0070] Referring again to Figure 1, in some embodiments, the compound of formula (E-α-1) is produced by reacting the compound of formula (D-α-1) with hydrogen in the presence of Pd / C to selectively deprotect the phosphate group. In some variations, the compound of formula (E-α-1) is [ka] It is.

[0071] In some embodiments, the compound of formula (E-α-1) is produced by reacting the compound of formula (D-α-1) with hydrogen and Pd / C, where the Pd / C used is 5 w / w%, 10 w / w%, 15 w / w%, or 20 w / w%. In one variation, the Pd / C used is 15 w / w%.

[0072] In some embodiments, the compound of formula (E-α-1) is produced by reacting the compound of formula (D-α-1) with hydrogen and Pd / C at a temperature between 15° C. and 30° C., between 20° C. and 30° C., or between 25° C. and 30° C. In one variation, the reaction is carried out between 20° C. and 30° C.

[0073] In some embodiments, the compound of formula (E-α-1) is produced by reacting the compound of formula (D-α-1) with hydrogen and Pd / C for a reaction time of between 4 hours and 13 hours, 6 hours and 13 hours, 7 hours and 13 hours, 8 hours and 12 hours, 9 hours and 12 hours, or 10 hours and 12 hours, or less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, or less than 1 hour. In one variation, the reaction time is between 10 and 12 hours.

[0074] Referring again to Figure 1, in some embodiments, the compound of formula (G-α-1), or a salt thereof, or a hydrate of any of the foregoing, is generated by deprotecting a compound of formula (E-α-1). In some variations, the compound of formula (G-α-1) is [ka] It is.

[0075] In some embodiments, the compound of formula (G-α-1) is produced by reacting the compound of formula (E-α-1) with a suitable base. In some embodiments, the base is an inorganic base. In some embodiments, the base is KOMe, NaOMe, LiOMe, t BuOk, t BuONa, t The base is BuOLi, NaOEt, KOEt, or LiOEt. In one variation, the base is KOMe. Those skilled in the art will understand that the choice of base used in this step determines the specific salt form of the compound of formula (G-α-1). For example, if NaOMe is used instead of KOMe, the sodium salt shown below is obtained. [ka]

[0076] In some embodiments, the compound of formula (G-α-1) is produced by reacting the compound of formula (E-α-1) at a temperature between 10° C. and 30° C., between 15° C. and 30° C., or between 20° C. and 30° C. In one variation, the reaction is carried out between 20° C. and 25° C.

[0077] In some embodiments, the compound of formula (G-α-1) is produced by reacting the compound of formula (E-α-1) for a reaction time of between 10 hours and 30 hours, between 15 hours and 30 hours, between 20 hours and 30 hours, between 20 hours and 25 hours, or between 20 hours and 22 hours, or less than 35 hours, less than 30 hours, less than 25 hours, or less than 22 hours. In one variation, the reaction time is between 20 and 22 hours. Compositions containing the alpha isomer of M1P

[0078] In some aspects, a composition is provided that is produced according to the method provided herein.In some embodiments, the composition comprises the alpha isomer of mannose-1-phosphate (M1P), or its salt, or any of the hydrates of the above, and the purity of the composition is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.In one variation, the purity of the composition is at least 96%.In one variation of the above, purity refers to the amount of the main component (in this case, the alpha isomer of mannose-1-phosphate (M1P), or its salt, or any of the hydrates of the above) in a sample of the composition.

[0079] In some embodiments, a composition comprising the alpha isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing, contains 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%, or less than 1% of the beta isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 2% of the beta isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing.

[0080] In some embodiments, a composition comprising the alpha isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing, 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%, or less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing.

[0081] In some embodiments, a composition comprising the alpha isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing, 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%, or less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing.

[0082] In some embodiments, a composition comprising the alpha isomer of M1P, or a salt thereof, or a hydrate of any of the foregoing, 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%, or less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% [ka] or a salt thereof, or a hydrate of any of the foregoing. Crystalline Form C and Methods for Producing It

[0083] In another embodiment, a crystalline form of a potassium hydrate salt of M1P is provided. Such a crystalline form is referred to herein as "Form C". Form C is a dipotassium salt. In some variations, Form C may be hydrated with between 2.5 and 3.5 water molecules per M1P molecule. In one variation, Form C specifically has the structure [ka] is the dipotassium trihydrate salt having the formula:

[0084] Overall, Form C was observed to be more stable than the other identified polymorphs, including Forms A and B. Form C exhibits good crystallinity, exhibits physical and chemical stability, is only slightly hygroscopic, exhibits no morphological change at 25° C. in 80% RH, and exhibits good solubility of greater than 160 mg / mL in Tris buffer at pH 7.0 at 37° C. over 24 hours.

[0085] In certain embodiments, form C of M1P can be produced from (i) a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) crystalline form A of a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) crystalline form B of a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii).

[0086] In some embodiments, a method of producing crystalline form C of M1P comprises combining a starting composition comprising (i) a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) crystalline form A of the substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) crystalline form B of the substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii), with a solvent comprising water and alcohol to produce a suspension, and agitating the suspension to produce a product composition comprising crystalline form C of mannose-1-phosphate. In some embodiments, the starting composition comprises an alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, and 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%, or less than 1% of a beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing. In some embodiments, the starting composition comprises a mixture of crystalline form A and crystalline form B of mannose-1-phosphate. In some embodiments, the starting composition comprises crystalline form A of mannose-1-phosphate. In some embodiments, the starting composition comprises crystalline form B of mannose-1-phosphate. In some variations, the solvent comprising water and alcohol comprises water and methanol in a ratio between 5:1 and 1:10 v / v. In other variations, the suspension is equilibrated for a time period between 0 and 1 week, between 1 and 2 weeks, or between 2 and 3 weeks. In some embodiments, the suspension is equilibrated at a temperature between 0°C and 5°C, between 5°C and 10°C, between 10°C and 20°C, between 20°C and 30°C, or between 30°C and 60°C.

[0087] In another embodiment, there is provided crystalline form C of M1P produced according to any of the methods described herein. In yet another embodiment, there is provided a composition comprising crystalline form C of M1P.

[0088] In some embodiments, crystalline form C of M1P is a substantially pure alpha isomer of the potassium trihydrate salt of mannose-1-phosphate. In some variations, form C 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%, or less than 1% of the corresponding beta isomer. In one variation, form C has less than 1% of the corresponding beta isomer.

[0089] In some embodiments, Form C is 11.6, 13.4, 14.0, 14.6, 15.0, 16.1, 17.8, 19.0, 22.0, 22.4, 23.1, 23.2, 25.2, 25.8, 27.0, 27.6, 27.7, 28.5, 29.5, 30.1, 30.7, 32.2, 32.9, 34.5, 34.7, 35.1, 35.9, 36.6, 37.0, 37.8, 38.3, 39.0, and 39.7±0.2 having an X-ray powder diffraction (XRPD) pattern that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or all peaks selected from the 2 theta degrees.

[0090] In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all peaks selected from 11.6, 14.6, 15.0, 16.1, 17.8, 22.0, 23.2, 25.2, 25.8, 26.9, 27.7, 29.5, 30.7, 32.2, and 35.1±0.2 degrees 2-theta.

[0091] In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern that includes at least 1, 2, 3, 4, 5, 6, 7, or all peaks selected from 11.6, 14.6, 17.8, 22.0, 23.2, 27.7, 29.5, and 30.7±0.2 2-theta degrees. In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern that includes at least 1, 2, 3, 4, 5, or all peaks selected from 11.6, 14.6, 22.0, 27.7, 29.5, and 30.7±0.2 2-theta degrees. In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern that includes at least 1, 2, 3, 4, 5, or all peaks selected from 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7±0.2 2-theta degrees. In one variation, Form C has an X-ray powder diffraction (XRPD) pattern that includes peaks selected from 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7±0.2 2-theta. In another variation, Form C has an X-ray powder diffraction (XRPD) pattern that includes peaks selected from 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7±0.1 2-theta.

[0092] In one variation, Form C has an XRPD substantially as shown in FIG.

[0093] In another variation, Form C exhibits a TGA thermogram substantially similar to Figure 6. In some embodiments, Form C exhibits a TGA thermogram with a weight loss of about 5%-8% over the temperature range of 101-150°C.

[0094] In other embodiments, the crystalline form C of M1P has an average particle size between 1 μm and 20 μm. In some embodiments, form C does not undergo or does not undergo substantial morphological change when compressed under 5 MPa and / or 10 MPa for 5 minutes. In some embodiments, form C does not undergo or does not undergo substantial morphological change when crushed, whether under dry or wet conditions. In some embodiments, form C does not undergo or does not undergo substantial morphological change when stored in an open container at about 40° C. and about 75% relative humidity for 1 week. In some embodiments, form C does not undergo or does not undergo substantial morphological change when stored in a closed container at about 60° C. for 1 week.

[0095] In some embodiments, M1P form C (including any of the compositions comprising M1P form C produced according to the methods herein) is stable under one or more of the following conditions: when compressed under 5 MPa and / or 10 MPa for 5 minutes; when crushed, whether under dry or wet conditions; when stored in an open container at about 40° C. and about 75% relative humidity for 1 week; and / or when stored in a closed container at about 60° C. for 1 week.

[0096] In other embodiments, form C of M1P (including any of the compositions comprising form C of M1P produced according to the methods herein) is stable for up to about 12 months when stored under one or more of the following conditions: at about -20°C; at about 25°C and about 60% relative humidity; at about 5°C; or at about 40°C and about 75% relative humidity.

[0097] In some variations of the above, Form C is stable if its purity remains substantially unchanged over storage time. In certain variations of the above, Form C is stable if no or substantially no morphological changes occur and / or any change in purity over storage time is less than 10%, less than 5%, or less than 1%, or undetectable. Any suitable method can be used to characterize stability and purity, including, for example, by XRPD and / or chromatography.

[0098] In some aspects, compositions are provided that comprise at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by weight of crystalline form C that may be produced according to any of the methods described herein. In one variation, the composition comprises at least 98% by weight of crystalline form C. In some embodiments, the composition comprises 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 the structure [ka] The corresponding beta isomer of the potassium salt of M1P has the formula:

[0099] In some embodiments, the composition has less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% M1P crystalline form A and / or crystalline form B. In one variation, less than 0.5% of the composition is mannose-1-phosphate form A and / or form B.

[0100] In some embodiments, Form A is 9.1, 12.7, 13.3, 14.8, 16.0, 16.4, 17.8, 18.2, 20.3, 20.6, 21.1, 21.5, 23.0, 23.3, 23.5, 24.4, 24.7, 25.2, 26.4, 27.1, 27.3, 28.1, 29.0, 29.7, 30.8, 31.3, 32.0, 33.0, 33.7, 34.4, 35.1, 36.3, 37.3, 37.7, 38.0, 38.6, 39.1, and 39.4 ±0.2 and ±0.2 having an X-ray powder diffraction (XRPD) pattern including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or all 38 peaks selected from the 2 theta degrees.

[0101] In some embodiments, Form A has an X-ray powder diffraction (XRPD) pattern comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 peaks selected from 9.1, 13.3, 16.0, 17.8, 20.3, 21.1, 21.5, 23.5, 25.2, 26.4, 27.1, 28.1, 29.0, 31.3, 32.0, and 33.0±0.2 degrees 2-theta.

[0102] In some embodiments, Form A has an X-ray powder diffraction (XRPD) pattern comprising at least 1, 2, 3, 4, 5, or all 6 peaks selected from 20.3, 21.1, 23.5, 27.1, 28.1, and 29.0±0.2 degrees 2-theta.

[0103] In some variations, crystalline form A has an XRPD substantially as shown in FIG.

[0104] In some embodiments, Form B is 4.2, 8.3, 12.2, 13.5, 14.2, 15.7, 16.1, 16.4, 16.7, 17.2, 17.6, 19.2, 19.8, 20.6, 21.4, 22.6, 23.2, 23.9, 24.5, 25.8, 26.2, 26.6, 27.5, 28.0, 28.6, 28.9, 29.3, 29.8, 30.6, 30.9, 31.3, 32.3, 32.9, 33.4, 34.0, 34.5, 34.8, 35.3, 36.7, 37.3, 37.6, 39.3, and 39.9±0.2. having an X-ray powder diffraction (XRPD) pattern including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or all 43 peaks selected from the 2 theta degrees.

[0105] In some embodiments, Form B has an X-ray powder diffraction (XRPD) pattern comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 peaks selected from 8.3, 14.2, 16.1, 16.4, 17.2, 19.8, 21.4, 23.2, 23.9, 24.5, 26.2, 26.6, 27.5, 28.6, 29.3, 29.8, 30.6, 32.3, 32.9, and 34.8±0.2 degrees 2-theta.

[0106] In some embodiments, Form B has an X-ray powder diffraction (XRPD) pattern comprising at least 1, 2, 3, 4, 5, or all 6 peaks selected from 8.3, 14.2, 19.8, 21.4, 23.9, and 27.5±0.2 degrees 2-theta.

[0107] In some variations, crystalline form B has an XRPD substantially as shown in FIG. Pharmaceutical Compositions

[0108] In some aspects, the composition described herein is provided, which further comprises at least one pharma- ceutically acceptable carrier, additive, or stabilizer.In some embodiments, the pharma-ceutically acceptable carrier, additive, or stabilizer is non-toxic to the cell or subject exposed thereto at the dosage and concentration used.In many cases, the physiologically acceptable carrier is a pH-buffered aqueous solution.

[0109] In one aspect, lipid particles are provided that contain any of the compositions described herein (e.g., including compositions that include the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a hydrate of any of the foregoing) encapsulated within the lipid particle. In some embodiments, lipid particles refer to particles formed by lipids in an aqueous solution. Suitable examples of lipid particles include, but are not limited to, liposomes, micelles, solid lipid nanoparticles, niosomes, lipospheres, emulsomes, and emulsions.

[0110] In some variations, encapsulation in lipid particles refers to lipid particles that provide active agents or therapeutic agents, such as the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a composition comprising any of the above-mentioned hydrates, by full encapsulation, partial encapsulation, or both.In some variations, at least a portion of the composition comprising the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a composition comprising any of the above-mentioned hydrates, can be encapsulated by lipid particles and be confined within the core of the lipid particles and / or within the inner surface (e.g., membrane) of the lipid particles.Alternatively, in other variations, the entire composition comprising the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a composition comprising any of the above-mentioned hydrates can be encapsulated by lipid particles and be confined within the core of the lipid particles and / or within the inner surface (e.g., membrane) of the lipid particles.

[0111] Any lipid particle known in the art that is suitable for delivering the encapsulated carbohydrate of the present disclosure to the interior of a cell may be used. Examples of suitable lipid particles include, but are not limited to, liposomes, micelles, solid lipid nanoparticles, and niosomes. Liposomes

[0112] In some embodiments, the lipid particles of the present disclosure can be liposomes. As used herein, liposomes refer to vesicles composed of lamellar phase lipid bilayers. Any suitable liposomes known in the art can be used. In some embodiments, liposomes have lamellar nanostructures. In some variations, lamellar nanostructures refer to nanostructures, such as lipid particles, that include amphiphilic parallel bilayers separated by cavities.

[0113] The liposome of the present disclosure can be prepared by any suitable method known in the art and disclosed herein.The examples of suitable methods for preparing liposome include, but are not limited to, disruption of biological membranes such as by ultrasonication, thin film hydration method, emulsion, French pressure cell, extrusion, and mechanical dispersion including reconstitution of dried vesicles; solvent dispersion including ethanol injection, ether injection, double emulsion, reverse phase, and evaporation; and detergent removal method.

[0114] In certain embodiments, the liposome is a stealth liposome, which may be immune-tolerant. In some variations, stealth liposome refers to a liposome that can avoid detection by the subject's immune system. Thus, stealth liposomes may be immune-tolerant.

[0115] In one aspect, provided herein is a composition comprising (i) a liposome comprising one or more phospholipids, and (ii) an alpha isomer of mannose-1-phosphate encapsulated in the liposome, or a salt thereof, or a hydrate of any of the foregoing. In one embodiment, provided herein is a composition comprising (i) a liposome comprising one or more phospholipids conjugated to polyethylene glycol (PEG), and (ii) an alpha isomer of mannose-1-phosphate encapsulated in the liposome, or a salt thereof, or a hydrate of any of the foregoing.

[0116] In some embodiments, a composition is provided that includes a liposome having a lipid membrane surrounding an intraliposomal compartment, the intraliposomal compartment encapsulating an alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing; an intraliposomal buffering agent; an extraliposomal buffering agent; and, optionally, a radical-scavenging antioxidant.

[0117] In some embodiments, provided herein are compositions comprising: liposomes having a lipid membrane surrounding an intraliposomal compartment, the liposomes encapsulating in the intraliposomal compartment an alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing; an intraliposomal buffering agent comprising a buffer salt and optionally an acid, wherein the buffer salt has a pKa between 6 and 8.5; an extraliposomal buffering agent comprising a buffer salt and a tonicity adjusting agent, wherein the buffer salt has a pKa between 6 and 8.5; and optionally a radical scavenging antioxidant.

[0118] In some variations, 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. In one variation, 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 conjugated to polyethylene glycol (PEG) having an ethanolamine head group and at least one saturated fatty acid tail.

[0119] In some variations of the composition comprising liposomes and the alpha isomer of M1P, the alpha isomer of M1P is produced according to any of the methods provided herein. In some embodiments, the composition contains 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 the beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing.

[0120] In some variations of the composition comprising liposomes and the alpha isomer of M1P, the alpha isomer of the potassium hydrate salt of mannose-1-phosphate is Form C. In other embodiments, less than 0.5% of the composition is Form A and / or Form B of mannose-1-phosphate. Micellar

[0121] In some embodiments, the lipid particles of the present disclosure may be micelles. In some variations, micelles refer to aggregates of surfactant molecules (e.g., soaps, detergents, fatty acids, lipids, phospholipids, etc.) dispersed in a liquid colloid. Micelles in aqueous solution may form aggregates in which the hydrophilic head regions are in contact with the surrounding solvent and sequester the hydrophobic tail regions in the interior of the micelle. Any suitable micelles known in the art may be used. In some embodiments, the micelles may be spherical. Micelles of the present disclosure may be prepared by any suitable method known in the art. Examples of suitable methods for preparing micelles include, but are not limited to, direct dissolution, and direct dialysis or microemulsion dialysis, which may include preparation by detergent or water-miscible solvent removal methods. Solid Lipid Nanoparticles

[0122] In some embodiments, the lipid particles of the present disclosure may be solid lipid nanoparticles. In some variations, solid lipid nanoparticles (SLNs) refer to lipid-in-water emulsions composed of lipids that are generally solid at a temperature of at least 50° C. and typically contain a solid lipid core matrix capable of solubilizing lipophilic molecules. In some variations, the solid lipid nanoparticles have diameters ranging from 10 to 1000 nanometers. Solid lipid nanoparticles can protect incorporated active compounds, such as the carbohydrates of the present disclosure, from chemical degradation and can also exhibit flexibility in modulating the release of such compounds. The lipid core of the solid lipid nanoparticles may be stabilized by surfactants (e.g., emulsifiers). The lipids may typically include triglycerides, diglycerides, monoglycerides, fatty acids, steroids, and / or waxes. Any suitable solid lipid nanoparticles known in the art may be used. The solid lipid nanoparticles of the present disclosure may be prepared by any suitable method known in the art. Examples of suitable methods for preparing solid lipid nanoparticles include, but are not limited to, microemulsification, high pressure homogenization, precipitation, and film ultrasonic dispersion. Niosomes

[0123] In some embodiments, the lipid particles of the present disclosure can be niosomes. In some variations, niosomes refer to vesicular structures that contain an aqueous core formed from a bilayer of nonionic surfactant molecules. Niosomes are structurally similar to liposomes in that they have a lamellar structure, but the materials used to prepare niosomes make them more stable against hydrolysis. Examples of suitable niosome preparation materials include, but are not limited to, sterols and one or more nonionic surfactants. Any suitable niosomes known in the art can be used. The niosomes of the present disclosure can be prepared by any suitable method known in the art. Examples of suitable methods for preparing micelles include, but are not limited to, ether injection, stirring, bubble method, reverse phase evaporation, sonication, multi-membrane extrusion, and microfluidigation. formulation

[0124] Examples of suitable formulations incorporating the compositions described herein include, but are not limited to, solutions, injections, inhalants, microspheres, aerosols, gels, ointments, creams, lotions, powders, dry vesicular powders, tablets, and capsules. The pharmaceutical composition may include a pharma- ceutically acceptable non-toxic diluent carrier, which is a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include, but are not limited to, distilled water, buffered water, saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical composition or formulation of the present disclosure may further include, but are not limited to, other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers and additives. The composition may also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents. The pharmaceutical compositions of the present disclosure may also include any of a variety of stabilizers, such as antioxidants.

[0125] In some variations, the pharmaceutical compositions described herein can be formulated for injection into the subject in need thereof, for example, the human being in need thereof.The preparation suitable for parenteral administration includes aqueous and non-aqueous isotonic sterile injection solution, which can contain antioxidant, buffer, bacteriostatic agent, and solute that makes the preparation isotonic with the blood of intended recipient, and aqueous and non-aqueous sterile suspension, which can contain suspending agent, solubilizer, thickener, stabilizer, and preservative.

[0126] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually 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, and in particular substantially free of any endotoxins that may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic, and made under GMP conditions.

[0127] In some variations, the compositions provided herein are formulated for intravenous administration.

[0128] In some variations of compositions comprising lipid particles (including, for example, liposomes) and the alpha isomer of M1P, after the composition is stored at controlled room temperature, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 94%, at least about 95%, at least about 96%, at least about 97%, or at least about 98%. In one variation, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 98%.

[0129] In some variations of compositions comprising lipid particles (including, for example, liposomes) and the alpha isomer of M1P, after the composition is stored at ambient temperature, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%. In one variation, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 95%.

[0130] Suitable phospholipids, buffering agents and radical scavenging antioxidants may be used in the pharmaceutical compositions, which are described in further detail below. Phospholipids

[0131] 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.

[0132] In one embodiment, 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 conjugated to polyethylene glycol (PEG) having an ethanolamine head group and at least one saturated fatty acid tail.

[0133] In certain embodiments of phospholipids having an ethanolamine head group at (a) of the lipid membrane, the unsaturated fatty acid tails independently comprise at least one C10-28 carbon chain. In certain embodiments, the unsaturated fatty acid tails are optionally substituted C10-28 alkenyl or optionally substituted C10-28 alkynyl. In some embodiments, each of the fatty acid chains is unsubstituted C10-28 alkenyl. In some embodiments, the alkenyl is straight chain or branched. In some embodiments, each of the fatty acid chains has one or more double bonds. In some embodiments, each double bond has a cis configuration. In some embodiments, each double bond has a trans configuration. In some embodiments, each of the fatty acid chains is a C10-28 alkenyl substituted by a substituent selected from 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 an oleoyl tail group.

[0134] In some embodiments, the 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. In one variation, the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is [ka] or a salt thereof.

[0135] In certain embodiments of the phospholipids having a choline group at (b) of the lipid membrane, the unsaturated fatty acid tail independently comprises at least one C10-28 carbon chain. In certain embodiments, the unsaturated fatty acid tail is an unsubstituted C10-28 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 a cis configuration. In some embodiments, each double bond has a trans configuration. In some embodiments, the unsaturated fatty acid tail is a C10-28 alkenyl substituted with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, and heterocyclyl. In some variations, the phospholipids having a choline group at (b) of the lipid membrane have an oleoyl tail group.

[0136] In some variations, the 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. In some variations, the phospholipid having a choline group and at least one unsaturated fatty acid tail is [ka] or a salt thereof.

[0137] In some embodiments of the phospholipids having an ethanolamine head group at (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 with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, and heterocyclyl. In some variations, the phospholipids having an ethanolamine head group at (c) of the lipid membrane have a stearoyl tail group.

[0138] In some variations, the 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. In some variations, the phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is [ka] or a salt thereof.

[0139] In certain variations, the phospholipid conjugated to PEG is DSPE or a salt thereof. In some embodiments, the PEGylated phospholipid is DSPE-PEG. In some embodiments, the PEGylated phospholipid is DSPE-PEG. 2000 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: [ka] or a salt thereof.

[0140] In one embodiment, the lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG. In one variation, the lipid membrane comprises DOPE:DOPC:DSPE-PEG2000 in a ratio between 58.2:38.8:3 and 30:67:3.

[0141] In certain variations, PEG is present in the composition at a concentration ranging from about 0.5 mole percent to about 20 mole percent. In some embodiments, PEG is present in the composition at a concentration of about 0.5 mole percent, about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, about 5 mole percent, about 6 mole percent, about 7 mole percent, about 8 mole percent, about 9 mole percent, about 10 mole percent, about 11 mole percent, about 12 mole percent, about 13 mole percent, about 14 mole percent, about 15 mole percent, about 16 mole percent, about 17 mole percent, about 18 mole percent, about 19 mole percent, or about 20 mole percent. In some embodiments, PEG is present in the composition at a concentration of at least about 0.5 mol percent, at least about 1 mol percent, at least about 2 mol percent, at least about 3 mol percent, at least about 4 mol percent, at least about 5 mol percent, at least about 6 mol percent, at least about 7 mol percent, at least about 8 mol percent, at least about 9 mol percent, at least about 10 mol percent, at least about 11 mol percent, at least about 12 mol percent, at least about 13 mol percent, at least about 14 mol percent, at least about 15 mol percent, at least about 16 mol percent, at least about 17 mol percent, at least about 18 mol percent, at least about 19 mol percent, or at least about 20 mol percent, or between 0.5 mol percent and 50 mol percent, between 0.5 mol percent and 40 mol percent, between 0.5 mol percent and 30 mol percent, or between 0.5 mol percent and 20 mol percent.

[0142] In certain variations, PEG is present in the composition at a molecular weight ranging from about 200 Da to about 40,000 Da. In some embodiments, PEG is present in the composition at a molecular weight ranging from about 200 Da to about 10,000 Da. In some embodiments, PEG is present in the composition at a molecular weight ranging from about 200 Da to about 10,000 Da. In some embodiments, PEG is present in the composition at a molecular weight ranging from 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 They exist with molecular weights of 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.

[0143] In one aspect, a pharmaceutical composition is provided that includes i) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a salt thereof, ii) one or more phospholipids having a polar head group comprising glycerol, choline, phosphate, or serine and two unsaturated fatty acid tails, each unsaturated fatty acid tail comprising a C10-28 alkenyl chain, and iii) a liposome comprising polyethylene glycol (PEG) conjugated to at least one phospholipid, as well as any of the compositions described herein (e.g., including a composition comprising the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a hydrate of any of the foregoing) encapsulated in the liposome. In some embodiments, the fatty acid comprising a polar head group is choline. In some embodiments, the one or more phospholipids with polar head groups are 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or a salt thereof, or any combination of the above. In a particular variation, the one or more phospholipids with polar head groups include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or a salt thereof. In a particular variation, the phospholipid conjugated to PEG is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In one variation, the component (iii) of the liposome is DSPE-PEG2000. In another variation, the liposome does not include cholesterol or cholesterol esters. In certain variations, DOPE is present in the composition at about 15 to about 30 mole percent. In another variation, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 35 to about 75 mole percent. In yet another variation, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 60 to about 65 mole percent. In certain embodiments, DOPE is present in the composition at about 25 to about 30 mole percent.In yet another embodiment, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 35 to about 75 mole percent. In another embodiment, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 60 to about 65 mole percent. In some variations, DOPE is present in the composition at about 30 to about 60 mole percent. In another variation, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 35 to about 75 mole percent. In yet another variation, the one or more phospholipids having a polar head group are DOPC, and DOPC is present in the composition at about 60 to about 65 mole percent. In one variation, the average particle size of the liposomes in the composition is about 0.1 microns. Buffer

[0144] In some embodiments, the intraliposomal buffer comprises a buffer salt and, optionally, an acid. In some variations, an acid can be added to the intraliposomal buffer to maintain a neutral pH.

[0145] In certain embodiments, the pKa of the buffer salt is between 6 and 8.5. In one embodiment, the intraliposomal buffer is in a physiological pH range. In one embodiment, the intraliposomal buffer is in a pH range between 7.35 and 6.45. In one variation, the buffer salt is tris(hydroxymethyl)aminomethane (Tris). In other variations, the intraliposomal buffer comprises bicarbonate, Tris, or HEPES, or any combination thereof.

[0146] In some embodiments, the extraliposomal buffer comprises a buffer salt and a tonicity adjusting agent. In some variations, the pKa of the buffer salt is between 6 and 8.5. In one embodiment, the extraliposomal buffer is in the physiological pH range. In one variation, the buffer salt is tris(hydroxymethyl)aminomethane (Tris). In other variations, the extraliposomal buffer comprises bicarbonate, Tris, or HEPES, or any combination thereof.

[0147] In some variations, the tonicity adjusting agent comprises a sugar or saline, or a combination thereof. Suitable sugars that can be used for tonicity include, for example, sucrose and dextrose.

[0148] In other variations, the tonicity adjusting agent is an ionic tonicity adjusting agent. In one variation, the tonicity adjusting agent comprises saline.

[0149] In some embodiments, M1P and Tris, and optionally an acid, are present in the composition in a ratio suitable to maintain a neutral pH.

[0150] In one variation, the intraliposomal buffer comprises about 50 mM Tris and the extraliposomal buffer comprises 15 mM Tris and at least 145 mM saline. Antioxidants that scavenge radicals

[0151] Any suitable radical scavenging antioxidant can be used in the liposome composition 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 some variations, any suitable combination of radical scavenging antioxidants can also be used. Characteristics of the pharmaceutical composition

[0152] The pharmaceutical compositions described herein are optimized for delivery of M1P inside cells to treat diseases and disorders, such as congenital disorders of glycosylation (CDG).

[0153] In some embodiments, the pharmaceutical composition has a drug to lipid (D / L) ratio of at least 0.1. In some embodiments, the composition minimizes both lipid degradation and liposome aggregation. 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%, or less than 1%.

[0154] In some embodiments, the pharmaceutical 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%, or less than 1% total lipid impurities.

[0155] In some embodiments, the pharmaceutical composition maintains a pH range between 6.5 and 7 or between 7.35 and 7.45. In certain embodiments, the pharmaceutical composition maintains a physiological pH range.

[0156] In some embodiments, the pharmaceutical composition has a Z-average of 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.

[0157] In some embodiments, the composition has a polydispersity index of less than 0.1.

[0158] In some embodiments, no free M1P is detectable in the composition.

[0159] In some embodiments, the pharmaceutical composition has an encapsulation efficiency of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0160] In some embodiments, the pharmaceutical composition has a mean osmolality between 290 mOsm / kg and 320 mOsm / kg.

[0161] The compositions provided herein can have any one or more of the characteristics described above.For example, in some variations, the compositions provided herein have all of the following characteristics: (i) less than 10% lipid degradation, (ii) Z-average between 80nm and 130nm, (iii) encapsulation efficiency of at least 80%, and (iv) pH range between 6.5 and 7.

[0162] The stability of the compositions provided herein, characterized for example based on lipid degradation, total lipid impurities, pH, Z-average, PDI, free M1P, encapsulation efficiency, and osmolality, can be measured over a period of time over a range of temperatures, such as 5° C., 25° C., and 40° C. In some variations, the period is 1-3 months. In other variations, the period is at least 6 months, at least 1 year, or at least 2 years. Pharmaceutical medication

[0163] The pharmaceutical compositions described herein may be used in accordance with known methods, such as oral administration, intravenous administration as a bolus, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes.

[0164] The dosage and desired concentration of the pharmaceutical composition of the present disclosure may vary depending on the specific use envisaged. The determination of suitable dosage or route of administration is well within the skill of a person skilled in the art. Animal experiments provide reliable guidance for determining the effective dose for human treatment. Interspecies scaling of effective dose can be carried out according to 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.

[0165] For in vivo administration of any of the compositions of the present disclosure, dosages can vary from 10 ng / kg to 100 mg / kg of subject body weight per day.

[0166] Administration of compositions of the present disclosure may be continuous or intermittent depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art.

[0167] It is within the scope of this disclosure that different formulations are effective for different treatments and different disorders, and that administration intended to treat a particular organ or tissue may require a different mode of delivery than delivery to another organ or tissue.Furthermore, dosage can be administered by one or more separate administrations, or by continuous infusion.For repeated administration over several days or longer depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs.However, other dosage regimes may also be useful.The progress of this treatment is easily monitored by conventional techniques and assays.

[0168] Thus, in some variations, the compositions provided herein can be administered chronically or intermittently to a subject (including, for example, a human) in need thereof. "Chronic" administration refers to administration of a pharmaceutical agent(s) in a continuous, rather than acute, manner to maintain an initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is cyclic in nature, rather than treatment that is given continuously without interruption. therapeutic use

[0169] The present disclosure provides compositions capable of delivering carbohydrates to the interior of a cell. These compositions are useful for delivering the phosphorylated carbohydrates of the present disclosure to a subject in need of such carbohydrates.

[0170] In some embodiments, the subject is a mammal. In one embodiment, the subject is a human. In some variations, the subject may be at risk. For example, in one variation, the subject is a human at risk. 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 disease symptoms, and may or may not show detectable disease or disease symptoms prior to the treatment methods described herein. In certain variations, an "at risk" individual is an individual who has risk factors known in the art that are measurable parameters that correlate with the development of a particular disease, disorder, or condition. A subject who has 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 who does not have one or more of these risk factors.

[0171] In some embodiments, congenital disorders of glycosylation (CDG) are a group of genetic disorders that result in metabolic abnormalities in which glycosylation of various tissue proteins and / or lipids is deficient or absent. Congenital disorders of glycosylation may also be known as CDG syndrome. CDG syndrome can often cause severe, sometimes fatal, dysfunction of several different organ systems, such as the nervous system, brain, muscles, and intestines, in affected infants. Signs of CDG syndrome may range from severe growth retardation and hypotension beginning in infancy to hypoglycemia and protein-losing enteropathy with normal development. Growth retardation may be a common early sign of CDG diagnosis. One of the most common subtypes of CDG syndrome is CDG-Ia type (also known as PMM2-CDG), whose genetic defect results in the loss of phosphomannomutase 2, the enzyme responsible for the conversion of mannose-6-phosphate to mannose-1-phosphate.

[0172] CDG syndrome can be classified into type I (CDG-I) and type II (CDG-II). Such classification can depend on the nature and location of biochemical defects in the metabolic pathway related to the action of oligosaccharyltransferase. Methods for screening CDG subtypes can include, for example, the analysis of transferrin glycosylation status by isoelectric focusing or ESI-MS. CDG type I includes, for example, Ia (PMM2-CDG), Ib (MPI-CDG), Ic (ALG6-CDG), Id (ALG3-CDG), Ie (DPM1-CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), Ii (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), 1L (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 includes, for example, IIa (MGAT2-CDG), IIb (GCS1-CDG), IIc (SLC335C1-CDG), IId (B4GALT1-CDG), IIe (COG7-CDG), IIf (SLC35A1-CDG), IIg (COG1-CDG), IIh (COG8-CDG), IIi (COG5-CDG), IIj (COG4-CDG), IIL (COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG.

[0173] Congenital disorders of glycosylation (CDGs) that may be treated with the compositions of the present disclosure include, for example, Ia (PMM2-CDG), Ib (MPI-CDG), Ic (ALG6-CDG), Id (ALG3-CDG), Ie (DPM1-CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), Ii (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), 1L (ALG9-CDG), Im (DOLK-CDG), In (RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG), Iq (SRD5A3-CDG), I These include r(DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS-CDG, I / IIx, IIa(MGAT2-CDG), IIb(GCS1-CDG), IIc(SLC335C1-CDG), IId(B4GALT1-CDG), IIe(COG7-CDG), IIf(SLC35A1-CDG), IIg(COG1-CDG), IIh(COG8-CDG), IIi(COG5-CDG), IIj(COG4-CDG), IIL(COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG.

[0174] In some embodiments, "treatment" or "treating" includes an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms resulting from a disease or condition and / or reducing the severity of the disease or condition), b) delaying or halting the onset of one or more clinical symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, preventing or delaying the worsening or progression of a disease or condition, and / or preventing or delaying the spread of a disease or condition), and / or c) alleviating the disease, i.e., causing a reduction in clinical symptoms (e.g., ameliorating the disease state, causing partial or complete remission of a disease or condition, enhancing the effect of another drug therapy, delaying the progression of a disease, increasing quality of life, and / or prolonging survival).

[0175] In some embodiments, "prevention" or "preventing" includes any treatment of a disease or condition that keeps the clinical symptoms of the disease or condition from occurring. The compounds, in some embodiments, may be administered to subjects (including humans) at risk or who have a family history of the disease or condition.

[0176] 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.

[0177] In some variations, a "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement in a particular disease, disorder, or condition, such as a congenital disorder of glycosylation. The therapeutically effective amount herein may vary according to factors such as the disease state, the age, sex, and weight of the subject, and the ability of the composition of the present disclosure to induce a desired response in the subject. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the composition of the present disclosure are outweighed by the therapeutically beneficial effects.

[0178] In one aspect, provided herein is a method for delivering a composition of the present disclosure to a subject in need thereof. In some embodiments, the method comprises administering to the subject any of the compositions described herein.

[0179] In another aspect, provided herein is a method for delivering the composition of the present disclosure to the inside of a cell of a subject in need thereof.In some embodiments, the method comprises administering to the subject any of the compositions described herein.In some embodiments, at least a portion of the administered composition crosses the cell plasma membrane and delivers carbohydrates to the inside of the cell.

[0180] In another aspect, provided herein is a method for treating congenital disorders of glycosylation (CDG) in a subject in need thereof by administering to the subject a composition (including a pharmaceutical composition) provided herein. In some embodiments, the method comprises administering to the subject any of the compositions described herein. In some embodiments, the congenital disorders of glycosylation (CDG) is a CDG-Ia disorder. In some embodiments, administration of the composition induces a 0.05-fold to at least 3-fold increase in the production of higher order lipid-linked oligosaccharides by cells in the human compared to the production of higher order lipid-linked oligosaccharides by cells in the human when the composition is not administered to the subject. Manufactured Products and Kits

[0181] The present disclosure also provides an article of manufacture and / or kit containing the composition of the present disclosure. The article of manufacture and / or kit of the present disclosure may include one or more containers containing the 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 article of manufacture and / or kit further includes instructions for use according to any of the methods of the present disclosure. In some embodiments, these instructions include instructions for administering the composition of the present disclosure according to any of the methods of the present disclosure to deliver carbohydrates to a subject in need of delivering carbohydrates, to deliver carbohydrates to the interior of a cell of a subject in need of delivering carbohydrates to the interior of a cell, or to treat congenital disorders of glycosylation (CDG) in a subject in need of treating congenital disorders of glycosylation (CDG). In some embodiments, the instructions include instructions for how to detect congenital disorders of glycosylation (CDG), for example, in a subject, tissue sample, or cell. The article of manufacture and / or kit may further include instructions for selecting suitable subjects for treatment based on whether the subject has the disease and the identification of the stage of the disease.

[0182] The instructions generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be single dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. The instructions provided 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 sheet of paper included in the articles of manufacture and / or kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0183] The label or package insert indicates that the composition is used to deliver carbohydrates and / or treat, for example, congenital disorders of glycosylation (CDG). Instructions for use can be provided for practicing any of the methods described herein.

[0184] The article of manufacture and / or kit of the present disclosure can be placed in suitable packaging. Suitable packaging includes, for example, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). Packages for use with certain devices, such as inhalers, nasal administration devices (e.g., atomizers) or injection devices, such as mini-pumps, are also contemplated. The article of manufacture and / or kit can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be penetrated by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be penetrated by a hypodermic needle). In some variations, the container can further include a second pharma- ceutically active agent.

[0185] The article of manufacture and / or kit may optionally provide additional components, such as buffers and interpretive information. Typically, the article of manufacture and / or kit includes a container and a label or package insert(s) on or associated with the container. EXAMPLES

[0186] The subject matter of the present disclosure will be better understood by reference to the following examples, which are provided as illustrations of the invention and are not intended to be limiting. Example 1 Synthesis of mannose-1-phosphate

[0187] This example demonstrates the synthesis of compound 130 on an 80+ kg scale. The synthetic steps of this example were carried out according to the scheme provided in Figure 1. Additionally, a continuous flow production system was used in steps 3 and 4, and these steps were carried out according to both Figures 1 and 2. Please refer to both figures for the procedures of steps 3 and 4. Step 1: Synthesis of (2R,3S,4S,5R,6R)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetrabenzoate (compound 106) [ka]

[0188] To a solution of compound 102 (1.0 equiv.) in pyridine (6V) was added compound 104 (benzoyl chloride, 5.5 equiv.) at a temperature between 10°C and 20°C. The solution was stirred for 3 h at a temperature between 20°C and 25°C. The reaction mixture was then diluted with toluene (10v) and water (10v). The organic components were separated and diluted with NaHCO 3 (2 × 4V) and H 2 It was washed with O (3 V) and concentrated to 5 V to 6 V. Then, the solvent was exchanged with toluene (4 × 5 V) and the mixture was used in the next step without further purification. Step 2: Synthesis of (2R,3R,4S,5S,6S)-2-((benzoyloxy)methyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 110) [ka]

[0189] A solution of compound 106 (1.0 equiv.) in toluene was diluted with THF (6 V). 2 DMAPA (2.0 equiv.) was added under protection. The reaction mixture was stirred at 60-65 °C for 12-14 h and diluted with EtOAc (5 V). The resulting mixture was then diluted with 5% H 2 SO 4It was quenched with aqueous solution and separated. The organic layer was decolorized by CUNO filtration, concentrated and crystallized in methylcyclohexane to give compound 110 as a white solid. Step 3: Synthesis of (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphaneyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 114) [ka]

[0190] Two solutions were prepared. The first solution contained compound 110 (1.0 equiv.) and DCI (1.77 equiv.) in acetonitrile (10 V) and was stored at −20 to −14° C. in tank 202. The second solution contained compound 112 ((BnO) 2 PNiPr 2 , 1.3 equiv.) and was stored at RT in tank 204. 2 SO 3 A quench solution containing (4.8 equiv.) was prepared separately and stored in tank 230. The first two solutions were loaded into pre-cooling tubes 206 and 208, respectively, and combined in flow reactor 214 at a temperature between 0° C. and 5° C. for a total residence time of 1 min. The reaction stream containing compound 114 is the product of this step and is subjected to the second flow chemistry step without isolation or purification. Step 4: Synthesis of (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 118) [ka]

[0191] The reaction stream containing compound 114 is fed into flow reactor 224 and treated with oxidant 218 (H) at a temperature between 20° C. and 25° C. for a residence time of 15 minutes.2 O 2 , 3 equiv.). The resulting reaction stream was collected in tank 228 where it was then mixed with NaCl at a temperature between 0° C. and 10° C. for a residence time between 3 and 10 minutes at 0-5° C. 2 SO 3 The resulting reaction mixture was diluted with toluene and placed in a centrifugal extractor for immediate separation. The reaction mixture was concentrated and then dissolved in EtOAc (3V-4V). The resulting reaction mixture was used in the next step without further purification. Step 5: Synthesis of (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 126) [ka]

[0192] To a solution of compound 118 (1.0 equiv.) in EtOAc was added MeOH (5V-7V) in an autoclave. To the reaction mixture was added wet Pd / C (15 w / w%, 0.15-0.17 equiv.). The autoclave was sequentially filled with N 2 Vacuum purge 3 times with H 2 The mixture was vacuum purged three times at 45-50 psi H at temperatures between 20°C and 30°C. 2 After stirring under atmospheric pressure for 18 hours, the reaction mixture was filtered and the solvent was switched to MeOH to give compound 126 as a yellow MeOH solution (97.7% yield), which was used in the next step without further purification. Step 6: Synthesis of potassium (2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl phosphate (potassium salt of mannose-1-phosphate, compound 130). [ka]

[0193] A solution of compound 126 (1.0 equiv.) in MeOH (10 V) was dried by solvent exchange with dry MeOH. To this solution was added a solution of KOMe (4.0 equiv.) in MeOH, and the resulting reaction mixture was heated at a temperature between 20° C. and 25° C. under N 2 The mixture was stirred under ambient atmosphere for 20-22 hours. The solid product was then isolated by filtration and redissolved in MeOH (3V) to form a slurry, which was then filtered again to give a crude white solid. This solid was then washed with H 2 Recrystallization in 0:MeOH afforded the hydrate of compound 130 (60-70% yield) as a white solid. Compound 130 can then be subjected to further crystallization procedures to produce Form C, as described in Example 6 below. Example 2 Instability of Compound 110

[0194] In the present invention, the use of flow chemistry techniques was essential to produce a final product that is substantially pure in the desired alpha isomer of M1P because, under the conditions required for the conversion of compound 110 to compound 118, a significant amount of isomerization of the reaction intermediate from the alpha form to the beta form occurs over time. The flow chemistry techniques described herein reduce the amount of isomerization in this conversion.

[0195] Table 1 demonstrates the instability of compound 110 over time in solution. If the reaction solution containing 110 and DCI is stored and not used immediately after preparation, the amount of the beta isomer of the product 118 increases. [Table 1-1] [Table 1-2] Example 3 Batch versus flow chemistry synthesis of compound 118

[0196] This example compares the production of intermediate D using batch synthesis as compared to flow chemistry synthesis following the procedure described in Example 1 above.

[0197] For the batch synthesis, compound 110 (1.0 equiv.) and DCI (1.77 equiv.) were dissolved in acetonitrile (10 V). To this solution was added (BnO) 2 PN i Pr 2 (1.5 eq.) was added and the resulting mixture was stirred at -20 to -15 °C to produce compound 114. t BuOOH (1.6-1.8 equiv.) was added to the reaction mixture, which was stirred at −10 to −5 °C to produce compound 118. The reaction mixture was diluted with 10% citric acid, followed by 7% NaHCO 3 The crude product was washed with 1,2-dichloromethane and then purified by silica gel chromatography to remove the undesired beta isomer of 118. At the 100 g scale, this procedure successfully yielded 118 in 95% purity with only 2-3% of the beta isomer of 118 present as an impurity. However, at the larger (25 kg) scale, the amount of beta isomer present increased to 15%. This highlights the importance of the flow chemistry techniques described herein, which allow for large-scale production of the substantially pure alpha isomer of 118, and therefore large-scale quantities of M1P in the substantially pure alpha isomer form. Example 4 Preparation and characterization of polymorphic form A (potassium hydrate salt) of M1P

[0198] This example demonstrates the synthesis and characterization of polymorphic Form A of M1P, a dipotassium dihydrate salt.

[0199] Form A of M1P was prepared using the following procedure: Approximately 500 mg of M1P was added to a vial. 3 mL of MeOH was added to the vial under stirring at 5° C. After stirring at 5° C. for approximately 5 days, the resulting suspension was filtered. The resulting solid was air-dried under ambient conditions for approximately 22 hours. Approximately 420 mg of Form A of M1P was obtained as a white solid in 84% yield. See Table 2 for characterization data of Form A. [Table 2] Example 5 Preparation and characterization of polymorphic form B (potassium hydrate salt) of M1P

[0200] This example demonstrates the synthesis and characterization of polymorphic Form B of M1P, a dipotassium trihydrate salt.

[0201] Form B of M1P was prepared using the following procedure: Approximately 500 mg of M1P was dissolved in 1.5 mL of water at ambient temperature. The solution was filtered through a syringe with a 0.45 μm membrane filter. The filtered 1.5 mL of solution was quickly added to 7.5 mL of MeOH in a 20 mL glass vial. The clear solution quickly became a suspension, and the suspension was stirred at 25° C. After stirring at 25° C. for approximately 6 days, the suspension was filtered at 4,000 rpm through a 0.45 μm nylon membrane filter. The resulting solid was air-dried under ambient conditions for approximately 22 hours. Approximately 360 mg of Form B of M1P was obtained as a white solid in 72% yield. See Table 3 for characterization data of Form B. [Table 3] Example 6A Preparation and characterization of polymorphic form C (potassium hydrate salt) of M1P

[0202] This example demonstrates the synthesis and characterization of polymorphic Form C of M1P, a dipotassium trihydrate salt.

[0203] Form C of M1P was prepared using the following procedure: Approximately 500 mg of M1P was added to an 8 mL glass vial. 3 mL of water:MeOH (1:10, v / v) was added to the vial under stirring at 25° C., thereby obtaining a suspension. Approximately 5 mg of form C seeds were added to the suspension. After stirring at 25° C. for approximately 6 days, the suspension was filtered at 4,000 rpm through a 0.45 μm nylon membrane filter. The resulting solid was air-dried under ambient conditions for approximately 22 hours. Approximately 389 mg of form C of M1P was obtained as a white solid in 77% yield.

[0204] Form C of M1P was also prepared using the following process. The potassium salt of M1P (0.97-1.03 equiv.) was dissolved in water (2.3-2.4 V) in a reactor. An additional amount of water (0.2-1.6 V) was added to the reactor through a playhead and the temperature of the reactor was adjusted between 20-30°C. The solution was stirred for 1-2 h until clear. The temperature was adjusted to 0-10°C and then MeOH (2.5-3.0 V) was added slowly. Seed crystals of form C of M1P (0.1%-2.0%×) were added and the mixture was stirred at 0-10°C for 14-20 h. Then, H 2 O:MeOH (volume ratio 1 / 3, 2.5-3.0x (target 2.7x)) was added slowly over 4 hours at 0-10°C and stirring was continued for 6-8 hours. Stirring was continued and additional MeOH was added until a sample taken for XRPD analysis demonstrated Form C according to the internal standard. Finally, the crystals were isolated by active filter drying (AFD). [Table 4] Example 6B Polymorphism Screening

[0205] This example describes a polymorph screening carried out using several different solvent systems and a variety of conditions. Overall, it was surprisingly observed that only certain solvent systems and certain conditions resulted in the production of Form C of M1P (as described in Example 6A above).

[0206] Equilibration with solvents at 5 °C for 2 weeks: Approximately 50-150 mg of M1P (mixture of Forms A and B) was equilibrated with stirring in 0.04-0.40 mL of the following solvents at 5 °C for 2 weeks: water, methanol (MeOH), ethanol (EtOH), isopropanol, acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, isopropyl acetate (IPAc), tetrahydrofuran (THF), dichloromethane (DCM), 2-methyl THF, and various ratios of water / methanol (10:1, 5:1, 3:2, 1:1, 2:3, 1:2, 1:5, and 1:10, v:v). Based on XRPD, Form C was only observed when 5:1, 3:2, 1:1, 2:3, 1:2, and 1:10, v:v of water / methanol was used. No XRPD was obtained for the water and water / MeOH (10:1, v:v) samples. XPRD for the remaining samples showed Form A, Form B, or a mixture of Form A and Form B.

[0207] Equilibration with solvents at 25 °C for 1 week: Approximately 50-150 mg of M1P (mixture of Form A and Form B) was equilibrated with stirring in 0.04-0.50 mL of the following solvents at 25 °C for 1 week: water, methanol (MeOH), ethanol (EtOH), isopropanol, acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, isopropyl acetate (IPAc), tetrahydrofuran (THF), dichloromethane, 2-methyl THF, and various ratios of water / methanol (10:1, 5:1, 3:2, 1:1, 2:3, 1:2, 1:5, and 1:10, v:v). Based on XRPD, Form C was only observed when 5:1, 3:2, 1:1, 2:3, 1:2, and 1:10, v:v of water / methanol was used. No XRPD was obtained for the water and water / MeOH (10:1, v:v) samples. XPRD for the remaining samples showed Form B, or a mixture of Forms A and B.

[0208] Equilibration under temperature cycling: Approximately 50–150 mg of M1P (a mixture of Forms A and B) was equilibrated under temperature cycling between 5 °C and 50 °C at a heating / cooling rate of 0.1 °C / min for 10 cycles in 0.04–0.40 mL of the following solvents, with the equilibration achieved under stirring: water, methanol (MeOH), ethanol (EtOH, isopropanol, acetone, methyl ethyl ketone, ethyl acetate, acetonitrile, isopropyl acetate (IPAc), tetrahydrofuran (THF), anisole, 2- methyl THF, and water / methanol in various ratios (10:1, 5:1, 3:2, 1:1, 2:3, 1:2, 1:5 and 1:10, v:v). Based on XRPD, Form C was only observed when 3:2, 1:1 and 1:2, v:v, water / methanol was used. XRPD was not obtained for the water and water / MeOH (10:1 and 5:1, v:v) samples because M1P was too soluble. XPRD for the remaining samples showed Form A or a mixture of Form A and Form B.

[0209] Crystallization from hot saturated solutions by slow cooling: Approximately 50 mg of M1P (mixture of Forms A and B) was dissolved at 50° C. in a minimum amount of the following solvents: water / MeOH (1:1, v:v), water / EtOH (1:1, v:v), water / IPA (8:2, v:v), water / ACN (8:2, v:v), water / acetone (8:2, v:v), and water / THF (8:2, v:v). The resulting solution was filtered and the clear solution was cooled to 5° C. at 0.1° C. / min. Samples that were free of precipitate at 5° C. were further cooled to −20° C. XRPD was not performed on the above samples as M1P was too soluble.

[0210] Crystallization from hot saturated solutions by rapid cooling: Approximately 50 mg of M1P (mixture of Forms A and B) was dissolved at 50° C. in a minimum amount of the following solvents: water / MeOH (1:1, v:v), water / EtOH (1:1, v:v), water / IPA (8:2, v:v), water / ACN (8:2, v:v), water / acetone (8:2, v:v), and water / THF (8:2, v:v). The resulting solution was filtered and the clear solution was placed in an ice bath at 0° C. and stirred. Samples that did not have any precipitate at 5° C. were further cooled to −20° C. XRPD was performed only for water / MeOH (1:1, v:v), which indicated Form C. For the rest of the above samples, XRPD was not obtained as M1P was too soluble.

[0211] Crystallization by addition of antisolvent: Approximately 1 g of M1P (a mixture of Form A and Form B) was dissolved in 2 mL of water at ambient temperature (approximately 20-22 °C). The resulting solution was filtered. Then, 0.1 mL of the clear solution was pipetted into a vial, and 3-5 times the antisolvent to be tested was slowly added to the clear solution. Samples that did not have a precipitate were further cooled to -20 °C. Antisolvents tested included methanol (MeOH) (0.3 mL), ethanol (EtOH) (0.3 mL), isopropanol (IPA) (0.3 mL), acetone (0.5 mL), methyl ethyl ketone (MEK) (0.5 mL), acetonitrile (ACN) (0.5 mL), tetrahydrofuran (THF) (0.5 mL), 2-methyl THF (0.5 mL), and 1,4-dioxane (0.5 mL). The precipitate was collected by centrifugal filtration. The solid portion (wet cake) was probed by XRPD. XRPD was obtained only for the water / methanol system, which indicated Form B. For the remainder of the above samples, XRPD was not obtained as M1P was too soluble.

[0212] Crystallization by inverse addition of antisolvent: Approximately 1 g of M1P (mixture of Form A and Form B) was dissolved in 2 mL of water at ambient temperature (approximately 20-22 °C). The resulting solution was filtered. Then, 0.1 mL of the clear solution was added to 5x the antisolvent to be tested in a vial. Samples that did not have precipitate were further cooled to -20 °C. Antisolvents tested include methanol (MeOH), ethanol (EtOH), isopropanol (IPA), acetone, methyl ethyl ketone (MEK), acetonitrile (ACN), tetrahydrofuran (THF), 2-methyl THF, and 1,4-dioxane. The precipitate was collected by centrifugal filtration. The solid portion (wet cake) was probed by XRPD. Samples with different XRPD patterns were analyzed by DSC, TGA, IC, KF, 1 Further analysis was performed including H-NMR and PLM. Based on XRPD, Form B was observed to be obtained in water / methanol, water / ethanol, water / isopropanol, water / acetone and water / 1,4-dioxane. For the rest of the above samples, XRPD was not obtained since M1P was too soluble.

[0213] Crystallization by vapor diffusion: Approximately 500 mg of M1P (a mixture of Form A and Form B) was dissolved in 0.5 mL of water at ambient temperature (approximately 20-22 °C). The resulting solution was filtered. The clear solution was then transferred to a small vial without a cap. The small vial without a cap was placed into a larger vial. The antisolvent to be tested was then added to the larger vial. Antisolvents to be tested include methanol (MeOH), ethanol (EtOH), acetone, methyl ethyl ketone (MEK), heptane, methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), 2-methylTHF, and acetonitrile (ACN). The larger vial was then tightly capped and placed at ambient conditions. The precipitate was collected by centrifugal filtration. The solid portion (wet cake) was probed by XRPD. XRPD of the water / methanol and water / acetone samples showed a mixture of Form A and Form B. XPRD in water / methanol showed Form A. XRPD was not obtained for the remainder of the samples above as M1P was too soluble. Example 7 Water activity experiment

[0214] This example demonstrates that form C of M1P is stable over a wide water activity range. In general, form C is the more stable form compared to forms A and B. Furthermore, forms A and B convert to form C under certain specific conditions. Water activity experiments were performed in several different water / EtOH systems at 25° C. to determine the relative stability of forms A, B, and C of M1P under these conditions.

[0215] Approximately 5 mg each of M1P forms A, B, and C were combined and added to 0.2 mL of a saturated solution in a water / EtOH system. The resulting suspensions were stirred at 5° C., 25° C., or 50° C. for 4 or 11 days. After 4 or 11 days, the solids (wet cakes) were isolated by centrifugal filtration and analyzed by XRPD to determine which form remained. The results are summarized in Table 5. [Table 5-1] [Table 5-2] Example 8 Moisture sorption and desorption experiments

[0216] The moisture sorption behavior of Form C of M1P was investigated using dynamic vapor sorption (DVS) at 25° C. The relative humidity (RH) was increased from 0% to 80% with a minimum equilibration time of 60 minutes and a maximum equilibration time of 360 minutes. As shown in Table 6, Form C absorbed 1.9% (w / w) H at 80% RH. 2 XRPD analysis was performed after the DVS experiment and no morphological changes were observed, demonstrating the low hygroscopicity and favorable stability of Form C. [Table 6] Example 9 Stability of Form C

[0217] This example shows the stability of crystalline form C in several stress tests. Form C was subjected to compression, dry milling simulation, and wet granulation simulation. The results are summarized in Tables 7-9. Compression Studies

[0218] Approximately 10 mg of M1P Form C was compressed under 5 MPa or 10 MPa for 5 minutes using a hydraulic press. Potential morphological changes and crystallinity were assessed by XRPD. Based on the XPRD results from the compression study, no morphological changes were observed from compression at 5 MPa and 10 MPa, and crystallinity decreased by exhibiting broad peaks. Dry grinding research

[0219] Approximately 10 mg of M1P Form C was manually ground with a mortar and pestle for 3 minutes. Potential morphological changes and crystallinity were assessed by XRPD. XPRD results from the dry grinding study showed no morphological changes after 3 minutes of grinding time, and reduced crystallinity by exhibiting broad peaks. Wet Granulation Simulation

[0220] Ethanol was added dropwise to approximately 10 mg of M1P Form C until the sample was fully wetted. The wet sample was gently ground with a mortar and pestle. After granulation, the sample was allowed to dry for 10 minutes under ambient conditions. Potential morphological changes and crystallinity were evaluated by XRPD. XRPD results from the wet granulation study did not indicate any morphological changes in ethanol as the granulation solvent. Example 10 Solubility of M1P form C

[0221] This example demonstrates the solubility of M1P form C. 242.1 mg of M1P form C (equivalent to 160 mg of M1P free base) was weighed into a 2 mL glass vial. 1 mL of dissolution medium (Tris buffer, 50 mM, pH 7.0) was added. The resulting solution was stirred at 37° C. and 400 rpm for 2 and 24 hours, and then centrifuged at 37° C. and 14,000 rpm for 5 minutes. At each of the 2 and 24 hour time points, the supernatant was analyzed by pH meter to determine the solubility. M1P form C demonstrated favorable solubility of more than 160 mg / mL. Example 11 Characterization of the polymorphic form C of M1P with 12-month stability

[0222] Form C of M1P was produced according to the above examples. XRPD was used on samples taken at 1, 3, 6, 9 and 12 months and tested under the following four conditions: -20℃±5℃, 25°C±2°C at 60% relative humidity (RH)±5%RH, 40°C ±2°C at 75% RH ±5% RH, and 5℃±3℃.

[0223] In each case, the XRPD of the samples corresponded to the reference standard spectrum for Form C of M1P, indicating that the samples remained as Form C at all time points tested. No change in polymorphic form was observed at any of the temperature conditions under study.

[0224] Additionally, no substantial changes were observed with respect to the purity of Form C, as determined based on chromatography.

Claims

1. A crystalline form C of mannose-1-phosphate, which is a substantially pure alpha isomer of the potassium trihydrate salt of mannose-1-phosphate.

2. The crystal morphology C according to claim 1, which exhibits an X-ray powder diffraction (XRPD) pattern including peaks at 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7 ± 0.2 2 theta degrees.

3. The crystal morphology C according to claim 1, which shows an XRPD substantially similar to that in Figure 1.

4. The crystal morphology C according to any one of claims 1 to 3, which exhibits a TGA thermogram with a weight loss of about 5% to 8% in a temperature range of 101 to 150°C.

5. The crystal morphology C according to any one of claims 1 to 3, showing a TGA thermogram substantially similar to that in Figure 6.

6. The crystal morphology C according to any one of claims 1 to 3, having an average particle size between 1 μm and 20 μm.

7. The crystalline form C according to any one of claims 1 to 3, which is stable when compressed at 5 MPa and / or 10 MPa for 5 minutes.

8. The crystalline form C according to any one of claims 1 to 3, which is stable when pulverized, regardless of whether it is under dry or wet conditions.

9. The crystalline form C according to any one of claims 1 to 3, which is stable when stored in an open container at 40°C and 75% relative humidity for one week, or at approximately 40°C and approximately 75% relative humidity for one week in an open container.

10. In a closed container at approximately 60°C for one week, Up to approximately 12 months at approximately -20°C. At approximately 25°C and 60% relative humidity, it can last up to approximately 12 months. Up to approximately 12 months at approximately 5°C, and / or Up to approximately 12 months at approximately 40°C and approximately 75% relative humidity. The crystalline form C according to any one of claims 1 to 3, which is stable when stored.

11. A composition comprising at least 98% by weight of the crystalline form C described in any one of claims 1 to 3.

12. Less than 1% of the composition is structure 【Chemistry 46】 The composition according to claim 11, which is a beta isomer of the potassium salt of mannose-1-phosphate having the following.

13. The composition according to claim 11, wherein less than 0.5% of the composition is form A and / or form B of mannose-1-phosphate.

14. Morphology A of mannose-1-phosphate exhibits an XRPD pattern including peaks at 20.3, 21.1, 23.5, 27.1, 28.1, and 29.0 ± 0.2 2 theta degrees, Morphology B of mannose-1-phosphate exhibits an XRPD pattern with peaks at 8.3, 14.2, 19.8, 21.4, 23.9, and 27.5 ± 0.2 degrees 2 theta. The composition according to claim 13.

15. A method for producing crystalline form C of mannose-1-phosphate, a) A starting composition comprising (i) a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) crystalline form A of a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) crystalline form B of a substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i) to (iii), is combined with a solvent containing water and alcohol to produce a suspension; b) The step of stirring the suspension to produce a product composition containing crystalline form C of mannose-1-phosphate. Methods that include...

16. The method according to claim 15, wherein the starting composition comprises an alpha isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing, and has less than 2% of a beta isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing.

17. The method according to claim 15, wherein the starting composition comprises a mixture of crystalline forms A and B of mannose-1-phosphate.

18. The method according to claim 15, wherein the starting composition comprises crystalline form A of mannose-1-phosphate.

19. The method according to claim 15, wherein the starting composition comprises crystalline form B of mannose-1-phosphate.

20. A crystalline form C of mannose-1-phosphate produced according to the method described in any one of claims 15 to 19.

21. A method for generating a continuous flow, a) A step of preparing a compound of formula B-α-1 and a nucleophilic catalyst at a temperature between -30°C and -10°C to obtain a pre-cooled solution, wherein the compound of formula B-α-1 is 【Transformation 34】 The steps and b) The pre-cooled solution and phosphorylating agent are continuously mixed in the first reactor at a temperature between -30°C and 5°C for a residence time between 30 seconds and 5 minutes to form the compound of formula (C-α-1). 【Chemistry 35】 A step of producing an intermediate composition containing, c) The step of transferring the intermediate composition from the first reactor to the second reactor, d) The intermediate composition is continuously mixed with an oxidant in the second reactor at a temperature between 0°C and 30°C for a residence time between 10 and 30 minutes to form the compound of formula (D-α-1). 【Transformation 36】 The steps include generating a reaction signal and Methods that include...

22. The aforementioned pre-cooled solution contains less than 1% of the compound of formula (B-β-1). 【Chemistry 37】 It has, and / or The reaction mixture containing the compound of formula (D-α-1) contains less than 2% of the compound of formula (D-β-1). 【Transformation 38】 The method according to claim 21, comprising:

23. The method according to claim 21 or 22, wherein the intermediate composition containing the compound of formula (C-α-1) contains less than 1% of the compound of formula (B-α-1) and less than 1% of the compound of formula (B-β-1).

24. The method according to claim 21 or 22, wherein the nucleophilic catalyst is 4,5-dicyanoimidazole.

25. The phosphorylating agent is (BnO) ２ PN ｉ Pr ２ The method according to claim 21 or 22.

26. The aforementioned oxidant is H ２ O ２ The method according to claim 21 or 22.

27. H between 1 and 3 molar equivalents ２ O ２ The method according to claim 26, wherein the above is used.

28. The method according to claim 21 or 22, wherein the organic solvent is used in the pre-cooled solution.

29. The method according to claim 21 or 22, wherein the phosphorylating agent is at least partially dissolved in the organic solvent.

30. The method according to claim 28, wherein the organic solvent includes a polar aprotic solvent.

31. The method according to claim 28, wherein the organic solvent is acetonitrile, THF, or 2-MeTHF.

32. The method according to claim 28, wherein the organic solvent includes acetonitrile.

33. The method according to claim 21 or 22, wherein the pre-cooled solution is provided at a flow rate between 0.5 mL / min and 1 L / min.

34. The method according to claim 21 or 22, wherein the phosphorylating agent is transferred at a flow rate between 0.5 mL / min and 1 L / min.

35. The method according to claim 21 or 22, wherein the oxidant is supplied at a flow rate between 0.5 mL / min and 1 L / min.

36. The method according to claim 21 or 22, further comprising the step of quenching the reaction mixture.

37. The method according to claim 36, wherein the reaction mixture is quenched with an aqueous solution containing a reducing agent.

38. where the reducing agent is Na ２ SO ３ The method according to claim 37, wherein the reducing agent is Na ２ SO ３ .

39. The method according to claim 36, wherein the reaction mixture is quenched at a temperature between 0°C and 25°C.

40. The method according to claim 36, further comprising the step of extracting the compound of formula (D-α-1) into an organic solvent.

41. The method according to claim 40, wherein the organic solvent includes toluene.

42. The aforementioned organic solvent is dissolved in water, followed by Na ２ SO ４ The method according to claim 40, further comprising the step of washing with a 10% solution of the following:

43. The method according to claim 40, further comprising the step of isolating the compound of formula (D-α-1) by evaporating at least a portion of the organic solvent.

44. The method according to claim 43, wherein the compound of formula (D-α-1) is not subjected to further purification.

45. The method according to claim 21 or 22, further comprising the step of deprotecting the compound of formula (D-α-1) to produce a composition comprising an alpha isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing, wherein less than 2% of the composition is a beta isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing.

46. Compound of formula (A-α-1) 【Chemistry 39】 The method according to claim 21 or 22, further comprising the step of reacting with DMAPA to selectively remove the Bz group of the oxygen bonded to the anomeric center to produce the compound of formula (B-α-1).

47. Compound of formula (SM-α-1) 【Chemistry 40】 The method according to claim 46, further comprising the step of reacting with benzoyl chloride to produce a compound of formula (A-α-1).

48. The phosphate group of the compound of formula (D-α-1) is selectively deprotected, and the structure 【Chemistry 41】 The method according to claim 21 or 22, further comprising the step of producing a compound of formula (E-α-1) having .

49. The compound of formula (E-α-1) is deprotected to obtain the compound of formula (G-α-1). 【Chemistry 42】 The method according to claim 48, further comprising the step of producing a salt thereof or a hydrate of any of the foregoing.

50. A composition produced according to the method of claim 21.

51. A composition comprising an alpha isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the above, wherein the purity of the composition is at least 96%.

52. The composition according to claim 51, wherein less than 2% of the composition is a beta isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the above.

53. Less than 1% of the above composition 【Chemistry 43】 The composition according to any one of claims 50 to 52.

54. Less than 1% of the above composition 【Chemistry 44】 The composition according to any one of claims 50 to 52.

55. Less than 1% of the above composition 【Chemistry 45】 The composition according to any one of claims 50 to 52.

56. It contains the dipotassium salt of α-D(+)mannose-1-phosphate, The composition according to any one of claims 50 to 52, further comprising, if necessary, at least one pharmaceutically acceptable carrier, additive, and / or stabilizer.

57. (i) liposomes, and (ii) An alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the above, encapsulated in the liposome. A composition containing the following:

58. The composition according to claim 57, wherein the liposome comprises one or more phospholipids conjugated to polyethylene glycol (PEG).

59. A liposome having a lipid membrane surrounding an internal compartment, The liposome encapsulates an alpha isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the aforementioned in its internal compartment. The aforementioned lipid membrane (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. Liposomes, An intraliposomal buffer comprising a buffer salt and optionally an acid, wherein the pKa of the buffer salt is between 6 and 8.5, An extraliposome buffer comprising a buffer salt and a tonicity modifier, wherein the pKa of the buffer salt is between 6 and 8.5; If necessary, antioxidants that scavenge radicals and A composition containing the following:

60. The aforementioned lipid membrane (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 conjugated to polyethylene glycol (PEG), having an ethanolamine head group and at least one saturated fatty acid tail. The composition according to claim 59, comprising:

61. The composition according to claim 59 or 60, wherein an alpha isomer of the mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing is produced according to the method of claim 21.

62. The composition according to any one of claims 57 to 60, wherein the alpha isomer of mannose-1-phosphate, a salt thereof, or any of the above hydrates is the dipotassium salt of α-D(+)mannose-1-phosphate.

63. The composition according to any one of claims 57 to 60, wherein less than 1% of the composition is a beta isomer of mannose-1-phosphate, a salt thereof, or a hydrate of any of the foregoing.

64. The composition according to any one of claims 57 to 60, wherein the alpha isomer of the potassium hydrate salt of mannose-1-phosphate is form C.

65. The composition according to claim 64, wherein less than 0.5% of the composition is form A and / or form B of mannose-1-phosphate.

66. A composition according to any one of claims 57 to 60, formulated for intravenous administration.

67. A composition according to any one of claims 57 to 60, formulated for injection.

68. The composition according to any one of claims 57 to 60, wherein, after the composition is stored at a controlled room temperature, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 98%.

69. The composition according to any one of claims 57 to 60, wherein, after the composition is stored at ambient temperature, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 95%.

70. The composition according to any one of claims 57 to 60, further comprising at least one pharmaceutically acceptable carrier, additive, and / or stabilizer.

71. The composition according to claim 11 for treating glycosylation-related congenital disorders (CDG) in subjects requiring treatment of such CDG.

72. The composition according to any one of claims 50, 51, 52, or 57-60 for treating glycosylation-related congenital disorders (CDGs) in subjects requiring treatment of such CDGs.

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