Continuous flow processes for production of mannose-1-phosphoric acid, polymorphs of mannose-1-phosphoric acid, and compositions and uses related thereto

Abstract

Provided herein are methods of synthesizing an alpha isomer of mannose-1-phosphoric acid (M1P) and compositions comprising an alpha isomer of mannose-1-phosphoric acid (M1P). For example, such methods employ flow chemistry techniques to obtain a substantially pure alpha-isomer of M1P. Also provided are compositions, including pharmaceutical compositions, comprising substantially pure alpha isomers of M1P. Also provided herein are polymorphic forms of M1P, including polymorphic forms that can be produced from a substantially pure alpha isomer of M1P.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 343,447, filed May 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to mannose-1-phosphate ("M1P"), and more particularly to continuous flow methods for producing M1P, including specific polymorphs of M1P, and compositions comprising M1P produced using such continuous flow methods. Background Art

[0004] Glycosylation, the enzymatic attachment of carbohydrates (glycans) to proteins and lipids, is a co- and post-translational modification (PTM) that is more common than any other PTM as it applies to the majority of proteins synthesized in the rough endoplasmic reticulum (ER). Glycosylation plays a key role in a variety of biological processes for membrane and secretory proteins. In the ER, glycosylation defines protein structure and folding and serves as a quality control mechanism to direct correctly folded proteins to be exported to the Golgi apparatus or to target misfolded proteins for degradation. Glycan moieties can also serve as ligands for cell surface receptors to mediate cell attachment or stimulate signal transduction pathways. Congenital disorders of glycosylation, also known as CDG syndromes, are a group of rare genetic diseases in which tissue proteins and / or lipids carry defective glycosylation and / or lack glycosylation. These diseases are associated with a number of enzyme defects and often cause severe, sometimes fatal, damage to the nervous system, muscle, intestine, and several other organ systems.

[0005] Common clinical symptoms in children with CDG include hypotonia in children with developmental delay, growth retardation, failure to thrive, liver dysfunction, coagulopathy, hypothyroidism, esotropia, abnormal fat patterns and nipple inversion, hypoglycemia, seizures, cerebellar hypoplasia, and stroke-like episodes. In older adolescence or adulthood, manifestations may include ataxia, cognitive impairment, absent puberty in females, small testicles in males, retinitis pigmentosa, scoliosis, joint contractures, and peripheral neuropathy.

[0006] CDG can be divided 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, namely the biosynthesis of polytrichol pyrophosphate-linked oligosaccharides (DLO) that occurs in the ER, or the transfer of DLO to asparagine residues of nascent polypeptides. CDG type II involves defects in the further processing (synthesis or hydrolysis) of glycans bound to proteins. Currently, 22 CDG type I and 14 type II variants have been identified. One of the most common subtypes of CDG is CDG-Ia (about 70% of all CDG cases), which is characterized by the loss or reduction of phosphomannomutase 2 (PMM) activity, resulting in a lack or deficiency 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").

[0007] Patients with reduced PMM activity have reduced M1P production, associated with symptoms of multi-visceral damage. To overcome defects in PMM production, it is important to provide the required substrate (i.e., M1P) to downstream enzymes. However, delivery and maintenance of such a systemic supply of M1P is problematic because extracellular enzymes within body fluids degrade M1P when delivered exogenously by oral or intravenous administration. Another problem associated with exogenously delivered M1P is that its high polarity prevents M1P from penetrating into the interior of cells (i.e., the cytosol) and thereby treating defects in PMM production.

[0008] Potential solutions include the use of delivery vehicles, such as lipid particles, to encapsulate and deliver M1P to the interior of cells (see WO 2015 / 053910 and WO 2020 / 205530). However, in order to make such formulations viable on a commercial scale, commercially viable methods are needed to produce M1P on a large scale. Current methods for producing M1P are generally limited by the difficulty in obtaining the desired M1P isomer in pure form and on a commercially viable and economical scale. During the conventional production process of M1P, synthetic intermediates are degraded into the β-anomer form, which results in a mixture of α and β-anomers in the final product composition. Isolating the pure α isomer of M1P requires expensive and time-consuming purification methods.

[0009] Therefore, there is a need in the art for a commercially viable method for producing M1P on a large scale. Summary of the invention

[0010] In a first aspect, a continuous flow production method is provided, comprising:

[0011] a) providing a compound of formula B-α-1 and a nucleophilic catalyst at a temperature of -30°C to -10°C to produce a pre-cooled solution, wherein the compound of formula B-α-1 is:

[0012]

[0013] wherein the pre-cooled solution has less than 1% of the compound of formula (B-β-1):

[0014]

[0015] b) continuously combining the pre-cooled solution and the phosphorylating agent in a first reactor at a temperature of -30°C to 5°C and a residence time of 30 seconds to 5 minutes to obtain an intermediate composition comprising a compound of formula (C-α-1):

[0016]

[0017] c) transferring the intermediate composition from the first reactor to a second reactor; and

[0018] d) continuously combining the intermediate composition and an oxidant in the second reactor at a temperature of 0° C. to 30° C. and with a residence time of 10 minutes to 30 minutes to produce a reaction mixture comprising a compound of formula (D-α-1):

[0019]

[0020] In some embodiments, the method further comprises deprotecting the compound of formula (D-α-1) to produce a composition comprising the α 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 β isomer of mannose-1-phosphate or a salt thereof or a hydrate of any of the foregoing.

[0021] In another aspect, a composition produced according to the above method is provided. In some embodiments, a composition comprising the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing is provided, wherein 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 a salt thereof, or a hydrate of any of the foregoing.

[0022] In another aspect, a method for producing Form C of mannose 1-phosphate is provided, comprising: combining a starting composition comprising (i) substantially pure α-isomer of mannose 1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) substantially pure Form A of the α-isomer of mannose 1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) substantially pure Form B of the α-isomer of mannose 1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii); and stirring the suspension to produce a product composition comprising Form C of mannose 1-phosphate.

[0023] In another aspect, provided is Form C of mannose-1-phosphate produced according to the method described in the previous paragraph. In some embodiments, provided is Form C of mannose-1-phosphate, which is a substantially pure alpha isomer of the potassium salt of mannose-1 phosphate trihydrate.

[0024] In another aspect, there is provided a composition comprising at least 98% by weight of Form C according to the above description.

[0025] In another aspect, a composition is provided comprising: (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.

[0026] In one embodiment, a composition is provided, comprising: a liposome having a lipid membrane encapsulating an intraliposomal compartment, wherein the liposome encapsulates the alpha isomer of mannose-1 phosphate, or a salt thereof, or a hydrate of any of the foregoing, in the intraliposomal compartment, and wherein the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail conjugated to polyethylene glycol (PEG); and 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 extraliposomal buffer comprising a buffer salt and a tonicity modifier, wherein the pKa of the buffer salt is between 6 and 8.5; and an optional free radical scavenging antioxidant.

[0027] In yet another aspect, provided is a method of 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present application may be understood by referring to the following description in conjunction with the accompanying drawings.

[0029] Figure 1 An exemplary scheme for preparing the M1P salt or its hydrate from (2S,3S,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrol is described.

[0030] Figure 2 Exemplary methods and systems for producing (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-((bis(benzyloxy)phosphoryl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate, or a salt or hydrate thereof, which can be deprotected to produce M1P, or a salt or hydrate thereof, are described.

[0031] Figure 3 An exemplary XRPD of Form A of the dipotassium salt of M1P dihydrate is depicted.

[0032] Figure 4 An exemplary XRPD of Form B of the dipotassium salt trihydrate of M1P is depicted.

[0033] Figure 5 An exemplary XRPD of Form C of the dipotassium salt trihydrate of M1P is depicted.

[0034] Figure 6 An exemplary TGA spectrum of Form C is depicted. Specific implementation plan

[0035] The following description sets forth 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 is provided as a description of exemplary embodiments.

[0036] In some aspects, provided herein is a method for producing pure or substantially pure alpha isomers of mannose-1-phosphate (including salts and hydrates thereof) involving continuous flow chemistry. In some variants, the composition produced has less than 2% of the beta isomers of mannose-1-phosphate (including salts and hydrates thereof). In other aspects, provided herein is a composition comprising pure or substantially pure alpha isomers of mannose-1-phosphate (including salts and hydrates thereof), and liposome preparations incorporating such pure or substantially pure alpha isomers of mannose-1-phosphate (including salts and hydrates thereof), and the purposes of such preparations. In some embodiments, the purity of the target compound is the amount of the compound (including salts and hydrates thereof) compared to all other components in the sample, expressed as a percentage. In some embodiments, the sample purity is measured by HPLC, including, for example, using an HPLC separation method to separate each component into a separate peak on a chromatogram. The peak area of ​​the target compound is compared to the peak area of ​​all other components, and purity is expressed as a percentage value.

[0037] Continuous flow production method

[0038] refer to Figure 1 , method 100 is an exemplary scheme for producing Form C of M1P. Form C can be produced from (2S, 3S, 4S, 5S, 6R) -6- (hydroxymethyl) tetrahydro -2H- pyran -2,3,4,5- tetraol (compound 102). In the first step of method 100, the alcohol group of compound 102 is protected using a suitable protecting agent such as benzoyl chloride (reagent 104) to produce (2R, 3S, 4S, 5R, 6R) -6- ((benzoyloxy) methyl) tetrahydro -2H- pyran -2,3,4,5- tetrabenzoate (compound 106) under suitable reaction conditions. For example, the reaction can be carried out at a temperature between about 10 ° C and 30 ° C in the presence of a suitable base such as pyridine. A suitable reaction time may be about 3 hours. In other embodiments, other suitable protecting agents, solvents and bases may be used. For example, a sufficient amount of a base such as diisopropylethylamine may be used in acetonitrile for the reaction.

[0039] Again, referring to method 100 ( Figure 1 ), compound 106 can be reacted with a suitable base such as DMAPA (reagent 108) in a suitable solvent such as THF to selectively remove the benzoyl group attached to the oxygen of the anomeric center to obtain (2R, 3R, 4S, 5S, 6S) -2- ((benzoyloxy) methyl) -6-hydroxytetrahydro-2H-pyran-3,4,5-triyl tribenzoate (compound 110) under suitable reaction conditions. For example, the reaction can be carried out at a temperature of about 60 ° C to 65 ° C for a sufficient amount of time, such as 12 to 14 hours. In other embodiments, other bases and solvents can be used to achieve selective deprotection.

[0040] Again, referring to method 100 ( Figure 1 ), compound 110 can be phosphorylated under suitable flow chemistry conditions to produce (2R, 3R, 4S, 5S, 6R) -2- ((benzoyloxy) methyl) -6- ((bis (benzyloxy) phosphinoyl) 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 of about -30 ° C to -10 ° C to produce a first pre-cooled solution. The pre-cooled solution can then be mixed with a suitable phosphorylation agent such as (BnO) 2PN at a temperature of about 0 ° C to 5 ° C. i The second solution of Pr2 (reagent 112) is continuously combined for a sufficient time to produce an intermediate composition comprising compound 114. The two solutions may be continuously combined for a total time of about 30 seconds to 5 minutes.

[0041] Again, referring to method 100 ( Figure 1 ), the intermediate composition comprising 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-triylbenzoate (Compound 118). For example, the intermediate composition comprising compound 114 can be continuously combined with a suitable oxidant such as H2O2 at a temperature of about 0°C to 30°C to produce compound 118. The intermediate compound comprising compound 114 can be continuously combined with the oxidant for a total time of about 10 minutes to 30 minutes.

[0042] Again, referring to method 100 ( Figure 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- (phosphoryloxy) tetrahydro -2H- pyran-3,4,5-triylbenzoate (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). The solution can then be reacted with gaseous H2 (reagent 122) at a temperature of about 20 ° C to 30 ° C for a sufficient time, such as 10 to 12 hours, to produce compound 126.

[0043] Again, referring to method 100 ( Figure 1), compound 126 can be deprotected under appropriate 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 of about 20° C. to 25° C. for a sufficient time, such as 20 to 22 hours, to produce compound 130 or a hydrate thereof.

[0044] In method 100, KOMe is used as a base to convert compound 126 into compound 130, which is a potassium salt. In other variations, other suitable salts may be used. It should be understood that the base used in this step determines the specific salt obtained from compound 126. For example, if NaOMe is used as a base, compound 126 will be converted into the corresponding sodium salt.

[0045] It is desirable to prepare substantially pure α-isomer forms of M1P or salts or hydrates thereof. Cyclic carbohydrates exist in "α" or "β" stereochemical forms or isomers, depending on the position of the substituents attached to the anomeric center. Because they are isomers of the anomeric center, this form is sometimes referred to as "anomeric matter." In the α anomer, the substituent attached to the anomeric carbon is on the opposite side of the substituent attached to another carbon adjacent to the epoxy group (i.e., trans). In the β anomer, the situation is reversed, and the substituent attached to the anomeric carbon is on the same side of the substituent attached to another carbon adjacent to the epoxy group (i.e., cis). In the "chair" and "planar" views, the α and β forms of M1P are as follows:

[0046]

[0047] 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.

[0048] To produce a substantially pure M1P alpha isomer, in some embodiments, flow chemistry is used to convert compound 110 to compound 118, which can then be deprotected to produce the M1P salt or hydrate as described above. Figure 2, describes an exemplary system 200 for producing compound 118 from compound 110 in two steps using flow chemistry.

[0049] In the first flow chemistry step, compound 110 is converted to compound 114. Tank 202 contains compound 110 and a nucleophilic catalyst such as DCI in a suitable solvent such as acetonitrile at a temperature of about -30°C to -10°C. Tank 204 contains reagent 112 in the same solvent. Tanks 202 and 204 are each continuously fed to pre-cooling tubes 206 and 208, respectively, by pumps 203 and 205, and then combined in mixer 210 and passed through reactor tube 212 at a temperature of about 0°C to 5°C, which is maintained by bath 216. The total residence time in mixer 210 and reactor tube 212, which together constitute flow reactor 214, is about 30 seconds to 5 minutes. The composition containing compound 114 is the product of this step and is carried forward in the second flow chemistry step without separation or purification.

[0050] In the second flow chemistry step, compound 114 is converted to compound 118. A composition comprising compound 114 and a suitable oxidant 218 is continuously combined in mixer 220 and passed through reactor tube 222 at a temperature of about 0°C to 30°C, which is maintained by bath 226. For example, the oxidant may be H2O2 ( Figure 1 The total residence time in the mixer 220 and the reactor tube 222, which together form the flow reactor 224, is about 10 minutes to 30 minutes. The reaction mixture containing compound 118 is the product of this step and is collected in tank 228.

[0051] After the second flow chemistry step, the reaction mixture containing compound 118 is quenched with an aqueous solution containing a reducing agent. Tank 230 contains an aqueous solution of Na2SO3 or another suitable reducing agent, which is transferred by pump 232 to tank 228 containing the reaction mixture containing compound 118. The quenching is performed at a temperature of about 0°C to 25°C for 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 in the extraction is toluene.

[0052] Therefore, in some aspects, a continuous flow production method is provided, which includes: a) providing a compound of formula (B-α-1) and a nucleophilic catalyst at a temperature of -30°C to -14°C to produce a pre-cooled solution; b) continuously combining the pre-cooled solution and a phosphorylating agent in a first reactor at a temperature of 0°C to 5°C and with a residence time of 30 seconds to 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 and an oxidant in the second reactor at a temperature of 20°C to 25°C and with a residence time of 10 minutes to 20 minutes to produce a reaction mixture comprising a compound of formula (D-α-1).

[0053] In some variations, the compound of formula (B-α-1) is:

[0054]

[0055] In some variations, the compound of formula (C-α-1) is:

[0056]

[0057] In some variations, the compound of formula (D-α-1) is:

[0058]

[0059] In some embodiments, in step a) of the above-mentioned continuous preparation method, suitable nucleophilic catalysts may include, for example, 1H-tetrazole, 5-(p-nitrophenyl)-1H-tetrazole, 5-ethylthio-1H-tetrazole, benzimidazole trifluoromethanesulfonate, a mixture of dimethylaminopyridine and 5-(p-nitrophenyl)-1H-tetrazole, a mixture of N-methylimidazole and 1H-tetrazole. In one variant, the nucleophilic catalyst is 4,5-dicyanoimidazole.

[0060] In some variants, an organic solvent is used in the pre-cooled solution of step a). In one embodiment, formula (B-α-1) compound and nucleophilic catalyst are provided in a suitable solvent such as an organic solvent. In some embodiments, the organic solvent is a polar aprotic solvent. In some embodiments, the polar aprotic solvent is acetone, methylene chloride, dimethylformamide, dimethyl propylene urea, dimethyl sulfoxide, ethyl acetate, hexamethylphosphoric acid triamide, 2-MeTHF or tetrahydrofuran. In one embodiment, wherein the organic solvent is acetonitrile, THF or 2-MeTHF. In a variant, the solvent comprises acetonitrile.

[0061] In some embodiments, in step a) of the continuous preparation method, the cooled solution containing the compound of formula (B-α-1) and the nucleophilic catalyst is maintained at a temperature of -30°C to -10°C, -30°C to -15°C, -30°C to -20°C, -20°C to -10°C, -20°C to -15°C or -30°C to -25°C.

[0062] In some embodiments, in step b) of the above continuous preparation method, suitable phosphorylating agents may include, for example, 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphosphorane, diethyldichlorophosphoramide, tris(1-pyrrolidinyl)phosphine, 2-chloro-1,3,2-benzodioxaphosphorane-4-one, dibenzyl N,N-diethylphosphoramide, diallyl N,N-diisopropylphosphoramide, bis(2-cyanoethyl)-N,N-diisopropylphosphoramide, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphoramide, or 2-cyanoethyl N,N-diisopropylchlorophosphoramide. In one variant, the phosphorylating agent is dibenzyl N,N-diisopropylphosphoramide, also known as (BnO)2PN i Pr2. 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 variant, the organic solvent is acetonitrile.

[0063] 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 of -30°C to 5°C, -30°C to 0°C, -30°C to -5°C, -20°C to 0°C, -20°C to -5°C, or -20°C to -10°C.

[0064] In some embodiments, in step b) of the continuous production process, the pre-cooled solution and the phosphorylating agent are continuously combined in the reactor for between 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, or 30 seconds to 1 minute; or a residence time of less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0065] 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 of 0.5 mL / min to 1 L / min. In some embodiments, in step b) of the continuous production method, the pre-cooled solution is provided at a flow rate of 0.5 mL / min to 1 L / min. In some embodiments, the phosphorylating agent is transferred at a flow rate of 0.5 mL / min to 1 L / min.

[0066] 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 variants, the compound of formula (B-α-1) is undetectable 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):

[0067]

[0068] In some variants, the compound of formula (B-α-1) is undetectable in the intermediate composition. In one variant, 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).

[0069] In some embodiments, in step d) of the above-mentioned continuous production method, 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 can be used. In one variant, H2O2 is used. In some embodiments, 1 to 3 molar equivalents of H2O2 are used. In some embodiments, the oxidant can be dissolved in a suitable organic solvent. In other embodiments, the oxidant is in an aqueous solution.

[0070] In some embodiments, in step d) of the continuous production method, the oxidant and the intermediate composition comprising the compound of formula (C-α-1) are continuously combined in a reactor at a temperature of 0°C to 30°C, 5°C to 30°C, 10°C to 30°C, 15°C to 30°C, 20°C to 30°C or 25°C to 30°C.

[0071] In some embodiments, in step d) of the continuous production process, the oxidant and the intermediate composition comprising the compound of formula (C-α-1) are continuously combined in the reactor for 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 a residence time of less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, or less than 12 minutes.

[0072] In some embodiments, in step d) of the continuous production method, the oxidant is provided at a flow rate of 0.5 mL / min to 1 L / min.

[0073] In some embodiments, the reaction mixture comprising a 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):

[0074]

[0075] In one variation, the reaction mixture comprising 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.

[0076] In some embodiments, the reaction mixture comprising a compound of formula (D-α-1) is quenched. In some embodiments, the reaction mixture is quenched with a solution comprising a reducing agent. In some variants, the solution is an aqueous solution. In some embodiments, the reducing agent is a disulfate. In other embodiments, the reducing agent is a thiosulfate. In one embodiment, the reducing agent is Na2S2O3. In one variant, the reducing agent is Na2SO3. In some embodiments, the reducing agent is a metal catalyst. In one variant, the reducing agent is manganese dioxide. In another variant, the reducing agent includes Fe 2+ ions. In some embodiments, the reducing agent is an iodide. In one variant, the reducing agent is potassium iodide. In another variant, the reducing agent is activated carbon. In some embodiments, the reducing agent is ascorbic acid. In some embodiments, the reaction mixture is quenched at a temperature between 0°C and 5°C, 0°C and 20°C, 0°C and 15°C, 0°C and 10°C, 0°C and 5°C, or 10°C and 200°C. In one variant, the reaction mixture is quenched at a temperature between 0°C and 25°C.

[0077] In another aspect of the present 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 is washed with water and then with 10% Na2SO4 solution. In some embodiments, the compound of formula (D-α-1) is separated by evaporating at least a portion of the organic solvent. In some embodiments, the compound of formula (D-α-1) is not further purified.

[0078] In one aspect of the invention, the compound of formula (D-α-1) is deprotected to produce a composition comprising an α 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 a β 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 a β isomer of mannose-1-phosphate or a salt thereof or a hydrate of any of the foregoing.

[0079] Reference again Figure 1 In some embodiments, the compound of formula (B-α-1) is prepared by reacting the compound of formula (A-α-1) with DMAPA to selectively remove the benzoyl (Bz) group attached to the oxygen of the anomeric center. In some variations, the compound of formula (A-α-1) is:

[0080]

[0081] In some embodiments, the compound of formula (B-α-1) is prepared by reacting the compound of formula (A-α-1) with an amine base to selectively remove the benzoyl (Bz) group attached to the oxygen of the anomeric center. In some embodiments, the amine base is DMAPA, N,N′-dimethyl-1,3-propylenediamine, N,N-dimethyldipropyltriamine, N 1 -isopropyldiethylenetriamine, 3-(methylamino)propylamine, N,N,N',N'-tetraethyldiethylenetriamine, 3,3'-imino(N,N-dimethylpropylamine) or diethylenetriamine. In one variant, the amine base is DMAPA.

[0082] In some embodiments, the compound of formula (B-α-1) is prepared by reacting a compound of formula (A-α-1) with an amine base at a temperature of 40° C. to 70° C., 50° C. to 75° C., 60° C. to 75° C., or 60° C. to 65° C. In one variation, the reaction is carried out at 60° C. to 65° C.

[0083] In some embodiments, the compound of formula (B-α-1) is prepared as follows: the compound of formula (A-α-1) is reacted with an amine base for 6 to 15 hours, 8 to 15 hours, 9 to 15 hours, 10 to 14 hours, 11 to 14 hours, or 12 to 14 hours; or for a reaction time of less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, or less than 13 hours. In one variant, the reaction time is 12 to 14 hours.

[0084] Reference again Figure 1 In some embodiments, the compound of formula (A-α-1) is prepared by reacting a 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:

[0085]

[0086] In some embodiments, the compound of formula (A-α-1) is prepared 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 variants, the base is used as a reaction solvent. In some embodiments, the base is pyridine, piperidine, trimethylamine, diisopropylethylamine, ethylamine, ammonia or aniline. In one variant, the base is pyridine.

[0087] In some embodiments, the compound of formula (A-α-1) is prepared by reacting a compound of formula (SM-α-1) with benzoyl chloride at a temperature of 10° C. to 30° C., 10° C. to 25° C., 15° C. to 25° C., or 20° C. to 25° C. In one variation, the reaction is carried out at 20° C. to 25° C.

[0088] In some embodiments, the compound of formula (A-α-1) is prepared by reacting the compound of formula (SM-α-1) with benzoyl chloride for 30 minutes to 3 hours, 45 minutes to 3 hours, 1 hour to 3 hours, 1.5 hours to 3 hours, 2 hours to 3 hours, 2.5 hours to 3 hours, or 3 hours to 5 hours; or for a reaction time of less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, or less than 4 hours. In one variant, the reaction time is 3 hours.

[0089] Reference again 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:

[0090]

[0091] In some embodiments, the compound of formula (E-α-1) is prepared by reacting the compound of formula (D-α-1) with hydrogen and Pd / C, wherein 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.

[0092] In some embodiments, the compound of formula (E-α-1) is produced by reacting a compound of formula (D-α-1) with hydrogen and Pd / C at a temperature of 15° C. to 30° C., 20° C. to 30° C., or 25° C. to 30° C. In one variation, the reaction is carried out at 20° C. to 30° C.

[0093] In some embodiments, the compound of formula (E-α-1) is prepared as follows: the compound of formula (D-α-1) is reacted with hydrogen and Pd / C for 4 to 13 hours, 6 to 13 hours, 7 to 13 hours, 8 to 12 hours, 9 to 12 hours or 10 to 12 hours; or for a reaction time of less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours or less than 1 hour. In one variant, the reaction time is 10 to 12 hours.

[0094] Reference again Figure 1 In some embodiments, the compound of formula (G-α-1) or a salt thereof or a hydrate of any of the foregoing compounds is prepared by deprotecting the compound of formula (E-α-1). In some variations, the compound of formula (G-α-1) is:

[0095]

[0096] In some embodiments, the compound of formula (G-α-1) is prepared by reacting a 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 BuOLi, NaOEt, KOEt or LiOEt. In one variation, the base is KOMe. Those skilled in the art will appreciate 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, a sodium salt as described below will be obtained:

[0097]

[0098] In some embodiments, the compound of formula (G-α-1) is prepared by reacting the compound of formula (E-α-1) at a temperature of 10° C. to 30° C., 15° C. to 30° C., or 20° C. to 30° C. In one variation, the reaction is carried out at 20° C. to 25° C.

[0099] In some embodiments, the compound of formula (G-α-1) is prepared as follows: the compound of formula (E-α-1) is reacted for 10 hours to 30 hours, 15 hours to 30 hours, 20 hours to 30 hours, 20 hours to 25 hours or 20 hours to 22 hours; or for a reaction time of less than 35 hours, less than 30 hours, less than 25 hours or less than 22 hours. In one variant, the reaction time is between 20 and 22 hours.

[0100] Compositions comprising the alpha isomer of M1P

[0101] In some aspects, a composition produced according to the method provided herein is provided. In some embodiments, the composition comprises an alpha isomer of mannose-1-phosphate (M1P) or a salt thereof, or a hydrate of any of the foregoing, 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 variant, the purity of the composition is at least 96%. In one variant of the foregoing, purity refers to the amount of the main component (in this case, the alpha isomer of mannose-1-phosphate (M1P), or a salt thereof, or a hydrate of any of the foregoing) in the composition sample.

[0102] In some embodiments, the 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.

[0103] In some embodiments, the 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 or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% or a salt thereof, or a hydrate of any of the foregoing.

[0104] In some embodiments, the 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 or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% or a salt thereof, or a hydrate of any of the foregoing.

[0105] In some embodiments, the 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 or a salt thereof, or a hydrate of any of the foregoing. In one variation, the composition contains less than 1% or a salt thereof, or a hydrate of any of the foregoing.

[0106] Crystal form C and preparation method thereof

[0107] In other aspects, a crystalline form of a potassium salt hydrate of M1P is provided. This crystalline form is referred to herein as "Form C". Form C is a dipotassium salt. In some variations, Form C can be hydrated with 2.5 to 3.5 water molecules per molecule of M1P. In one variation, Form C is specifically a dipotassium salt trihydrate having the following structure:

[0108]

[0109] Overall, Form C was observed to be more stable than other identified polymorphs, including Form A and Form B. Form C showed good crystallinity; showed physical and chemical stability and was only slightly hygroscopic; had no crystalline form changes at 25°C and 80% RH; and showed good solubility of >160 mg / mL in pH 7.0 Tris buffer at 37°C over 24 hours.

[0110] In certain aspects, Form C of M1P can be produced from (i) substantially pure α isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) substantially pure Form A of the α isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing. Or (iii) substantially pure Form B of the α isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii).

[0111] In some embodiments, the method for preparing Form C of M1P comprises: combining a starting composition comprising (i) substantially pure α-isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) substantially pure Form A of the α-isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) substantially pure Form B of the α-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 an alcohol to produce a suspension, and stirring the suspension to produce a product composition comprising Form C of mannose-1-phosphate. In some embodiments, the starting composition comprises the 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 the 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 variants, the solvent comprising water and alcohol comprises water and methanol in a ratio of 5:1 to 1:10 v / v. In other variants, the suspension is allowed to equilibrate for a period of 0 to 1 week, 1 to 2 weeks, or 2 to 3 weeks. In some embodiments, the suspension is equilibrated at a temperature of 0°C to 5°C, 5°C to 10°C, 10°C to 20°C, 20°C to 30°C, or 30°C to 60°C.

[0112] In another aspect, provided is Form C of M1P produced according to any of the methods described herein. In yet another aspect, provided is a composition comprising Form C of M1P.

[0113] In some embodiments, Form C of M1P is a substantially pure potassium salt trihydrate of the alpha isomer 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.

[0114] In some embodiments, Form C has a crystalline structure selected from the group consisting of 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. 29, 30, 31, 32 or all of the peaks at 39.9, 36.6, 37.0, 37.8, 38.3, 39.0, and 39.7 ± 0.2 ° 2θ.

[0115] In some embodiments, Form C 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 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 ° 2θ.

[0116] In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern comprising 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 0.7 ± 0.2 ° 2θ. In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern comprising 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 ° 2θ. In some embodiments, Form C has an X-ray powder diffraction (XRPD) pattern comprising 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 ° 2θ. In one variation, Form C has an X-ray powder diffraction (XRPD) pattern comprising peaks selected from 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7 ± 0.2 ° 2θ. In another variation, Form C has an X-ray powder diffraction (XRPD) pattern comprising peaks selected from 11.6, 14.6, 17.8, 23.2, 27.7, and 30.7 ± 0.1 ° 2θ.

[0117] In one variation, Form C has substantially Figure 5 XRPD shown.

[0118] In another variation, Form C exhibits substantially similar Figure 6 In some embodiments, Form C exhibits a TGA thermogram of about 5% to 8% weight loss in the temperature range of 101-150°C.

[0119] In other embodiments, Form C of M1P has an average particle size of 1 μm to 20 μm. In some embodiments, Form C does not undergo a crystalline form change, or does not undergo a significant crystalline form change, when compressed at 5 MPa and / or 10 MPa for 5 minutes. In some embodiments, Form C does not undergo a crystalline form change, or does not undergo a significant crystalline form change, when ground, whether under dry conditions or under wet conditions. In some embodiments, Form C does not undergo a crystalline form change, or does not undergo a significant crystalline form change, when stored in an open container at about 40°C and about 75% relative humidity for one week. In some embodiments, Form C does not undergo a crystalline form change, or does not undergo a significant crystalline form change, when stored in a closed container at about 60°C for one week.

[0120] In some embodiments, Form C of M1P (including any composition comprising Form C of M1P prepared according to the methods described herein) is stable under one or more of the following conditions: when compressed at 5 MPa and / or 10 MPa for 5 minutes; when ground, whether under dry or wet conditions; when stored in an open container for one week at about 40° C. and about 75% relative humidity; and / or when stored in a closed container at about 60° C. for one week.

[0121] In other embodiments, Form C of M1P (including any composition comprising Form C of M1P prepared according to the methods described herein) is stable when stored for up to about 12 months 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.

[0122] In some variations of the foregoing, Form C is stable when its purity remains substantially unchanged during storage. In certain variations of the foregoing, Form C is stable when Form C undergoes no or substantially no crystalline change; and / or when any change in purity during storage is less than 10%, less than 5%, or less than 1% or is undetectable. Stability and purity may be characterized using any suitable method, including, for example, by XRPD and / or chromatography.

[0123] In some aspects, provided are compositions comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by weight of Form C, which can be prepared according to any of the methods described herein. In one variation, the composition comprises at least 98% by weight of Form C. 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% of the corresponding beta isomer of the potassium salt of M1P having the following structure:

[0124]

[0125] 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% of Form A and / or Form B of M1P. In one variation, less than 0.5% of the composition is Form A and / or Form B of mannose-1-phosphate.

[0126] In some embodiments, Form A has a crystalline structure selected from the group consisting of 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. 37, 38, 39, 30, 31, 32, 33, 34, 35, 36, 37, or all 38 peaks at ±0.2° 2θ of 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 at ±0.2° 2θ of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15

[0127] 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° 2θ.

[0128] 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° 2θ.

[0129] In some variations, Form A has substantially Figure 3 XRPD shown.

[0130] In some embodiments, Form B has a crystalline structure selected from the group consisting of 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 34,35,36,37,38,39,40,41,42 or all 43 peaks at 34.5, 34.8, 35.3, 36.7, 37.3, 37.6, 39.3, and 39.9 ± 0.2 ° 2θ.

[0131] 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 at ±0.2 degrees 2θ 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.

[0132] In some embodiments, Form B has an X-ray powder diffraction (XRPD) pattern comprising at least 1, 2, 3, 4, 5, or all 20 peaks selected from 8.3, 14.2, 19.8, 21.4, 23.9, and 27.5 ± 0.2 degrees 2Θ.

[0133] In some variations, Form B has substantially Figure 4 XRPD shown.

[0134] Pharmaceutical composition

[0135] In some aspects, a composition as described herein is provided, further comprising at least one pharmaceutically acceptable carrier, excipient or stabilizer. In some embodiments, the pharmaceutically acceptable carrier, excipient or stabilizer is non-toxic to cells or subjects exposed thereto at the dose and concentration employed. Physiologically acceptable carriers are typically pH buffered aqueous solutions.

[0136] In one aspect, a lipid particle containing any composition described herein (including, for example, a composition comprising an α isomer of mannose-1-phosphate (M1P) or a salt thereof or a hydrate of any of the foregoing) encapsulated in a lipid particle is provided. In some embodiments, a lipid particle refers to a particle formed by lipids in an aqueous solution. Suitable examples of lipid particles include, but are not limited to, liposomes, micelles, solid lipid nanoparticles, vesicles (niosomes), lipid globules, emulsions, and emulsions.

[0137] In some variants, encapsulation in lipid particles refers to the lipid particles providing active or therapeutic agents, such as the lipid particles of any composition of the α isomer or its salt comprising mannose-1-phosphate (M1P), or any of the hydrates of the foregoing, with complete encapsulation, partial encapsulation or both. In some variants, at least a portion of any of the compositions comprising the α isomer or its salt or any of the hydrates of mannose-1-phosphate (M1P) or any of the foregoing can be encapsulated by lipid particles and located in the core of lipid particles and / or the inner surface (e.g., membrane) of lipid particles. Alternatively, in other variants, the entire composition comprising the α isomer or its salt or any of the hydrates of mannose-1-phosphate (M1P) can be encapsulated by lipid particles and positioned in the core of lipid particles and / or the inner surface (e.g., membrane) of lipid particles.

[0138] Any lipid particle known in the art suitable for delivering the encapsulated carbohydrates 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 vesicles.

[0139] Liposomes

[0140] 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 liposome known in the art can be used. In some embodiments, the liposomes have a lamellar nanostructure. In some variants, lamellar nanostructure refers to a nanostructure, such as a lipid particle comprising parallel amphiphilic bilayers separated by an inner cavity.

[0141] The liposomes of the present disclosure can be prepared by any suitable method known in the art and disclosed herein. Examples of suitable methods for preparing liposomes include, but are not limited to, disruption of biological membranes, such as by mechanical dispersion, including ultrasonic treatment, thin film hydration, emulsion, French pressure cell, extrusion and dry vesicle reconstruction); solvent dispersion, including ethanol injection, ether injection, double emulsion, inversion and evaporation; and detergent removal methods.

[0142] In certain embodiments, the liposome is a stealth liposome that can be immune-tolerant. In some variants, a stealth liposome refers to a liposome that is able to avoid detection by the subject's immune system. Thus, the stealth liposome can be immune-tolerant.

[0143] In one aspect, a composition is provided herein, comprising: (i) a liposome comprising one or more phospholipids; 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. In one embodiment, a composition is provided herein, comprising: (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.

[0144] In some embodiments, a composition is provided, comprising: a liposome having a lipid membrane surrounding an intraliposomal compartment, wherein the liposome encapsulates the alpha isomer of mannose-1-phosphate or a salt thereof, or a hydrate of any of the foregoing, in the intraliposomal compartment; an intraliposomal buffer; an extraliposomal buffer; and optionally a free radical scavenging antioxidant.

[0145] In some embodiments, provided herein is a composition comprising: a liposome having a lipid membrane surrounding an intraliposomal compartment, wherein the liposome encapsulates the alpha isomer of mannose-1-phosphate or a salt thereof, or a hydrate of any of the foregoing, in the intraliposomal compartment; 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 extraliposomal buffer comprising a buffer salt and a tonicity adjuster, wherein the pKa of the buffer salt is between 6 and 8.5; and optionally a free radical scavenging antioxidant.

[0146] In some variants, 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 variant, 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, which is conjugated to polyethylene glycol (PEG).

[0147] In some variations of the composition comprising liposomes and an 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.

[0148] In some variations of the composition comprising liposomes and the alpha isomer of M1P, the alpha isomer of potassium salt hydrate 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.

[0149] Micelles

[0150] In some embodiments, lipid particles of the present disclosure can be micelles. In some variants, micelles refer to aggregates of surfactant molecules (e.g., soaps, detergents, fatty acids, lipids, phospholipids, etc.) dispersed in liquid colloids. Micelles in aqueous solution can form aggregates with hydrophilic head regions in contact with surrounding solvents, thereby isolating hydrophobic tail regions inside micelles. Any suitable micelle known in the art can be used. In some embodiments, micelles can be spherical. Micelles 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, direct dissolution and direct or microemulsification dialysis, which can be prepared by detergent or water miscible solvent removal methods.

[0151] Solid lipid nanoparticles

[0152] In some embodiments, the lipid particles of the present disclosure can be solid lipid nanoparticles. In some variants, solid lipid nanoparticles (SLN) refer to lipids in an aqueous emulsion composed of lipids that are generally solid at a temperature of at least 50°C, and generally contain a solid lipid core matrix that can dissolve lipophilic molecules. In some variants, solid lipid nanoparticles have a diameter in the range of 10 to 1000 nanometers. Solid lipid nanoparticles can protect incorporated active compounds such as carbohydrates of the present disclosure from chemical degradation, and can also show the flexibility of regulating the release of such compounds. The lipid core of solid lipid nanoparticles can be stabilized by a surfactant (e.g., an emulsifier). Lipids can generally include triglycerides, diglycerides, monoglycerides, fatty acids, steroids and / or waxes. Any suitable solid lipid nanoparticles known in the art can be used. Solid lipid nanoparticles of the present disclosure can 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 membrane ultrasonic dispersion.

[0153] Niosome

[0154] In some embodiments, lipid particles of the present disclosure may be vesicle bodies. In some variants, vesicle bodies refer to vesicle structures formed by double layers of nonionic surfactant molecules and containing aqueous cores. Vesicle bodies are structurally similar to liposomes with lamellar structures, however, the materials used to prepare vesicle bodies make them more stable to hydrolytic degradation. Examples of suitable vesicle body preparation materials include but are not limited to sterols and one or more nonionic surfactants. Any suitable vesicle bodies known in the art can be used. Vesicle bodies 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, bubbling, reverse phase evaporation, ultrasonic treatment, multi-film extrusion and microfluidization.

[0155] preparation

[0156] Examples of suitable formulations for incorporating the compositions described herein may include, but are not limited to, solutions, injections, inhalants, microspheres, aerosols, gels, ointments, creams, lotions, powders, dry foam powders, tablets and capsules. Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable non-toxic diluent carrier, which is a vehicle commonly used to prepare a pharmaceutical composition for animal or human administration. 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 excipients. The composition may also include other substances close to physiological conditions, such as pH regulators and buffers, toxicity regulators, wetting agents and detergents. The pharmaceutical composition of the present disclosure may also include any of a variety of stabilizers, such as antioxidants.

[0157] In some variations, the pharmaceutical compositions described herein can be formulated for injection into a subject in need, such as a human in need thereof. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0158] The components used to prepare the pharmaceutical composition are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, and more usually at least pharmaceutical grade). In addition, compositions for in vivo use are generally sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is generally substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0159] In some variations, the compositions provided herein are formulated for intravenous administration.

[0160] In some variations of the composition comprising lipid particles (including, for example, liposomes) and the alpha isomer of M1P, 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% after the composition is stored at controlled room temperature. In one variation, the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 98%.

[0161] In some variations of the composition comprising lipid particles (including, for example, liposomes) and the alpha isomer of M1P, after storage of the composition 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%.

[0162] Suitable phospholipids, buffers, and free radical scavenging antioxidants may be used in the pharmaceutical compositions and are described in further detail below.

[0163] Phospholipids

[0164] 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.

[0165] 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 having an ethanolamine head group and at least one saturated fatty acid tail, which is conjugated to polyethylene glycol (PEG).

[0166] In certain embodiments of the phospholipids having an ethanolamine head group in (a) of the lipid membrane, the unsaturated fatty acid tails independently comprise at least one C 10-28 In certain embodiments, the unsaturated fatty acid tail is an optionally substituted C 10-28 Alkenyl or optionally substituted C 10-28 In some embodiments, each fatty acid chain is an unsubstituted C 10-28 In some embodiments, the alkenyl group is straight or branched. In some embodiments, each fatty acid chain 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 fatty acid chain is C substituted with a substituent selected from the following: 10-28 Alkenyl: acyl, hydroxy, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halogen, thiol, thioalkyl, alkyl, alkenyl, alkynyl, and heterocyclic. In some variations, the phospholipids having an ethanolamine head group in (a) of the lipid membrane have an oleoyl tail group.

[0167] 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 variant, the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is:

[0168]

[0169] or a salt thereof.

[0170] In certain embodiments of the phospholipids having a choline group in (b) of the lipid membrane, the unsaturated fatty acid tails independently comprise at least one C 10-28 In certain embodiments, the unsaturated fatty acid tail is an unsubstituted C 10-28 In some embodiments, the alkenyl group is straight 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 C substituted with a substituent selected from the following: 10-28 Alkenyl: acyl, hydroxy, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halogen, thiol, thioalkyl, alkyl, alkenyl, alkynyl, and heterocyclic. In some variations, the phospholipids having a choline group in (b) of the lipid membrane have an oleoyl tail group.

[0171] 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:

[0172] or a salt thereof.

[0173] In some embodiments of the phospholipids with an ethanolamine head group in (c) of the lipid membrane, the saturated fatty acid tails independently comprise at least one C 4-28 In some embodiments, the saturated fatty acid tail is an optionally substituted alkyl. In some embodiments, the saturated fatty acid tail is an unsubstituted C 4-28 In some embodiments, the saturated fatty acid tail is a C substituted with a substituent selected from the following 4-28 Alkyl: acyl, hydroxy, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halogen, thiol, thioalkyl, alkyl, alkenyl, alkynyl, and heterocyclic. In some variations, the phospholipids having an ethanolamine head group in (c) of the lipid membrane have a stearoyl tail group.

[0174] 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:

[0175]

[0176] or a salt thereof.

[0177] 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, DSPE-PEG is further conjugated to a carbohydrate. In some embodiments, DSPE-PEG is further conjugated to a monosaccharide. In some embodiments, DSPE-PEG is further conjugated to a galactose moiety. In some embodiments, DSPE-PEG-galactose has the following structure:

[0178]

[0179] or a salt thereof.

[0180] In one embodiment, the lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG. In one variant, the lipid membrane comprises DOPE:DOPC:DSPE-PEG in a ratio between 58.2:38.8:3 and 30:67:3 2000 .

[0181] In some variants, 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 ranging from 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 mole percent, at least about 1 mole percent, at least about 2 mole percent, at least about 3 mole percent, at least about 4 mole percent, at least about 5 mole percent, at least about 6 mole percent, at least about 7 mole percent, at least about 8 mole percent, at least about 9 mole percent, at least about 10 mole percent, at least about 11 mole percent, at least about 12 mole percent, at least about 13 mole percent, at least about 14 mole percent, at least about 15 mole percent, at least about 16 mole percent, at least about 17 mole percent, at least about 18 mole percent, at least about 19 mole percent, or at least about 20 mole percent; or within the range of between 0.5 mole percent and 50 mole percent, between 0.5 mole percent and 40 mole percent, between 0.5 mole percent and 30 mole percent, or between 0.5 mole percent and 20 mole percent.

[0182] 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 of about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 6,500 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 8,500 Da, about 9,000 Da, about 9,500 Da, or about 10,000 Da; or in the range of about 200 Da to about 10,000 Da.

[0183] In one aspect, a pharmaceutical composition is provided comprising: a liposome comprising: 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, wherein each unsaturated fatty acid tail comprises C 10-28 alkenyl chain; and iii) polyethylene glycol (PEG) conjugated to at least one phospholipid; and any composition described herein encapsulated in a liposome (including, for example, a composition comprising the alpha isomer of mannose-1-phosphate (M1P) or a salt thereof, or a hydrate of any of the foregoing). In some embodiments, the fatty acid comprising a polar head group is choline. In some embodiments, one or more phospholipids having a polar head group 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 foregoing. In certain variants, one or more phospholipids having a polar head group include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or a salt thereof. In certain variations, the phospholipid conjugated to PEG is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In one variation, component (iii) of the liposome is DSPE-PEG 2000. In another variant, the liposomes do not contain cholesterol or cholesterol esters. In certain variants, DOPE is present in the composition in the range of about 15 to about 30 mole percent. In another variant, one or more phospholipids with polar head groups are DOPC, wherein DOPC is present in the composition in the range of about 35 to about 75 mole percent. In yet another variant, one or more phospholipids with polar head groups are DOPC, wherein DOPC is present in the composition in the range of about 60 to about 65 mole percent. In certain embodiments, DOPE is present in the composition in the range of about 25 to about 30 mole percent. In yet another embodiment, one or more phospholipids with polar head groups are DOPC, wherein DOPC is present in the composition in the range of about 35 to about 75 mole percent. In other embodiments, one or more phospholipids with polar head groups are DOPC, wherein DOPC is present in the composition in the range of about 60 to about 65 mole percent. In some variants, DOPE is present in the composition in the range of about 30 to about 60 mole percent. In other variations, the one or more phospholipids having a polar head group is DOPC, wherein DOPC is present in the composition in the range of about 35 to about 75 mole percent. In yet another variation, the one or more phospholipids having a polar head group is DOPC, wherein DOPC is present in the composition in the range of 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.

[0184] Buffer

[0185] In some embodiments, the intraliposomal buffer comprises a buffer salt and optionally an acid. In some variations, an acid may be added to the intraliposomal buffer to maintain a neutral pH.

[0186] In certain embodiments, the pKa of the buffer salt is between 6 and 8.5. In one embodiment, the intraliposomal buffer is within the physiological pH range. In one embodiment, the pH range of the intraliposomal buffer is between 7.35 and 6.45. In one variant, the buffer salt is tris (hydroxymethyl) aminomethane (Tris). In other variants, the intraliposomal buffer includes bicarbonate, Tris or HEPES or any combination thereof.

[0187] In some embodiments, the extralipidic buffer comprises a buffer salt and a tension modifier. In some variants, the buffer salt has a pKa between 6 and 8.5. In one embodiment, the extralipidic buffer is within the physiological pH range. In one variant, the buffer salt is tris (hydroxymethyl) aminomethane (Tris). In other variants, the extralipidic buffer comprises bicarbonate, Tris or HEPES or any combination thereof.

[0188] In some variations, the tonicity adjusting agent comprises sugar or saline or a combination thereof. Suitable sugars that can be used for tonicity include, for example, sucrose and dextrose.

[0189] In other variations, the tonicity adjuster is an ionic tonicity adjuster. In one variation, the tonicity adjuster comprises saline.

[0190] In some embodiments, M1P and Tris, and optionally an acid, are present in the composition in a ratio suitable to maintain a neutral pH.

[0191] 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.

[0192] Free Radical Scavenging Antioxidants

[0193] Any suitable free radical scavenging antioxidant can be used in the liposome compositions provided herein. In some embodiments, the free radical scavenging antioxidant is butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or alpha tocopherol. In one variant, the free radical scavenging antioxidant is BHT. In some variants, any suitable combination of free radical scavenging antioxidants can also be used.

[0194] Properties of the pharmaceutical composition

[0195] The pharmaceutical compositions described herein are optimized for delivering M1P to the interior of cells to treat diseases and disorders such as congenital disorders of glycosylation (CDG).

[0196] 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 lipid degradation and liposome aggregation. In some variants, 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%.

[0197] 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.

[0198] In some embodiments, the pharmaceutical composition maintains a pH range between 6.5 to 7 or 7.35 to 7.45. In certain embodiments, the pharmaceutical composition maintains a physiological pH range.

[0199] In some embodiments, the pharmaceutical composition has a Z-average value between 80 nm and 130 nm, 80 nm and 120 nm, 80 nm and 110 nm, 80 nm and 100 nm, 90 nm and 130 nm, 90 nm and 120 nm, 90 nm and 110 nm, or 90 nm and 100 nm.

[0200] In some embodiments, the composition has a polydispersity index of less than 0.1.

[0201] In some embodiments, free M1P is not detected in the composition.

[0202] 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%.

[0203] In some embodiments, the pharmaceutical composition has an average osmolality of 290 mOsm / kg to 320 mOsm / kg.

[0204] The compositions provided herein may have any one or more of the above properties. For example, in some variants, the compositions provided herein have all of the following properties: (i) less than 10% lipid degradation; (ii) a Z-average value between 80 nm and 130 nm; (iii) an encapsulation efficiency of at least 80%; and (iv) a pH range between 6.5 and 7.

[0205] The stability of the compositions provided herein, characterized by, for example, lipid degradation, total lipid impurities, pH, Z-average, PDI, free M1P, encapsulation efficiency, and weight-gram molecular osmotic concentration, can be measured over a period of time at a range of temperatures, such as 5° C., 25° C., and 40° C. In some variations, the period of time is 1-3 months. In other variations, the period of time is at least 6 months, at least 1 year, or at least 2 years.

[0206] Drug dosage

[0207] The pharmaceutical compositions described herein can be used according to 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, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

[0208] The dosage and desired concentration of the pharmaceutical composition of the present disclosure may vary depending on the specific use envisioned. The determination of an appropriate dosage or route of administration is well within the skill of the ordinary technician. Animal experiments provide reliable guidance for determining effective doses for human treatment. Interspecies scaling can be performed 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., Pergamon Press, New York 1989, pp. 42-46.

[0209] For in vivo administration comprising any of the compositions of the present disclosure, the dosage may vary from 10 ng / kg to 100 mg / kg of subject body weight per day.

[0210] Administration of the compositions of the present disclosure can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled artisan.

[0211] Different formulations are effective for different treatments and different disorders, and administration intended to treat a specific organ or tissue may need to be delivered in a manner different from that for delivery to another organ or tissue, which is within the scope of the present disclosure. In addition, the dose can be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily detected by conventional techniques and assays.

[0212] 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 drug in a continuous mode, as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is performed continuously without interruption, but is in fact periodic.

[0213] Therapeutic Uses

[0214] The present disclosure provides compositions capable of delivering carbohydrates to the interior of cells. These compositions can be used to deliver the phosphorylated carbohydrates of the present disclosure to subjects in need of such carbohydrates.

[0215] In some embodiments, the subject is a mammal. In one embodiment, the subject is a person. In some variants, the subject may be at risk. For example, in one variant, the subject is a person at risk. A subject at risk of developing a specific disease, disorder or condition (such as congenital glycosylation disorder) may or may not have a detectable disease or symptom of the disease, and may or may not show a detectable disease or disease symptom before the treatment methods described herein. In some variants, an individual "at risk" is an individual with a risk factor, which is a measurable parameter associated with the development of a specific disease, disorder or condition as known in the art. Compared with a subject without one or more of these risk factors, a subject with one or more of these risk factors has a higher probability of developing a specific disease, disorder or condition such as congenital glycosylation disorder.

[0216] In some embodiments, congenital disorders of glycosylation (CDG) are a group of genetic disorders that lead to metabolic errors, in which the glycosylation of multiple tissue proteins and / or lipids is defective or defective. Congenital disorders of glycosylation may also be referred to as CDG syndrome. CDG syndrome can usually cause severe, occasionally fatal dysfunction of several different organ systems such as the nervous system, brain, muscle, and intestine in affected infants. The manifestations of CDG syndrome can range from severe developmental delay and hypotonia starting in infancy to hypoglycemia and protein-losing enteropathy with normal development. Developmental delay may be a common initial indication for CDG diagnosis. One of the most common subtypes of CDG syndrome is CDG-Ia (also known as PMM2-CDG), in which a genetic defect leads to the loss of phosphomannose mutase 2, an enzyme responsible for converting mannose-6-phosphate to mannose-1-phosphate.

[0217] CDG syndrome can be divided into type I (CDG-I) and type II (CDG-II). This classification may depend on the nature and location of the biochemical defect in the metabolic pathway relative to the action of the oligosaccharide transferase. Methods for screening CDG subtypes may include analyzing the transferrin glycosylation state by, for example, 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.

[0218] Congenital disorders of glycosylation (CDG) that can 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), Ir (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.

[0219] In some embodiments, "treatment" or "treating" includes methods for obtaining 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 caused by the disease or condition, and / or reducing the extent of the disease or condition); b) slowing or preventing the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the deterioration or progression of the disease or condition, and / or preventing or delaying the spread of the disease or condition); and / or c) alleviating the disease, i.e., causing regression of clinical symptoms (e.g., improving the disease state, providing partial or complete remission of the disease or condition, enhancing the effect of another drug, delaying the progression of the disease, improving the quality of life, and / or prolonging survival).

[0220] In some embodiments, "prevention" or "preventing" includes any treatment of a disease or condition that causes clinical symptoms of the disease or condition not to develop. In some embodiments, the compound can be administered to a subject (including a human) who is at risk of a disease or condition or has a family history of the disease or condition.

[0221] In some variations, an "effective amount" is an amount effective to achieve at least the desired therapeutic or preventive result, at dosages and for periods of time necessary. An effective amount can be provided in one or more administrations.

[0222] In some variations, a "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a particular disease, disorder, or condition (e.g., congenital glycosylation disorders). Therapeutically effective amounts herein may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the disclosed compositions to elicit a desired response in the subject. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the disclosed compositions.

[0223] In one aspect, provided herein are methods for delivering a composition of the present disclosure to a subject in need thereof. In some embodiments, the method comprises administering to a subject any composition described herein.

[0224] On the other hand, 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 any composition described herein to the subject. In some embodiments, at least a portion of the composition administered passes through the cytoplasmic membrane to deliver carbohydrates to the interior of the cell.

[0225] On the other hand, provided herein is a method for treating a congenital glycosylation disorder (CDG) in a subject in need thereof by administering a composition (including a pharmaceutical composition) provided herein to the subject. In some embodiments, the method comprises administering any composition described herein to the subject. In some embodiments, congenital glycosylation disorder (CDG) is a CDG-Ia disorder. In some embodiments, administration of the composition induces cellular production of higher order lipid-linked oligosaccharides in a subject to increase by 0.05 times to at least 3 times compared to cellular production of higher order lipid-linked oligosaccharides in a subject without administering the composition to the subject.

[0226] Products and kits

[0227] The present disclosure also provides products and / or kits containing the compositions of the present disclosure. The products and / or kits of the present disclosure may include one or more containers containing the compositions of the present disclosure. Suitable containers may include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed of various materials such as glass or plastic. In some embodiments, the products and / or kits also include instructions for use according to any method of the present disclosure. In some embodiments, these instructions include administering the compositions of the present disclosure according to any method of the present disclosure to deliver carbohydrates to a subject in need thereof, to deliver carbohydrates to the interior of a cell of a subject in need thereof, or to treat a description of a congenital glycosylation disorder (CDG) of a subject in need thereof. In some embodiments, the instructions include a description of how to detect, for example, a congenital glycosylation disorder (CDG) in a subject, a tissue sample, or a cell. The products and / or kits may further include instructions for selecting a subject suitable for treatment based on identifying whether the subject suffers from a disease and the stage of the disease.

[0228] The instructions typically include information about the dosage, administration regimen, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the articles 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 and / or kits), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0229] The label or package insert indicates that the composition is used for delivering carbohydrates and / or treating, for example, congenital disorders of glycosylation (CDG).Instructions for practicing any of the methods described herein may be provided.

[0230] The articles and / or kits of the present disclosure may be in suitable packaging. Suitable packaging includes, for example, vials, bottles, cans, and flexible packaging (e.g., sealed mylar or plastic bags). Packaging used in combination with specific devices such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices such as miniature pumps is also contemplated. Articles and / or kits may have sterile access ports (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). In some variants, the container may further include a second pharmaceutically active agent.

[0231] The article of manufacture and / or kit may optionally provide additional components, such as buffers and interpretative information. Typically, the article of manufacture and / or kit comprises a container and a label or package insert on or associated with the container.

[0232] Example

[0233] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the invention and not by way of limitation.

[0234] Example 1: Synthesis of Mannose-1-Phosphate

[0235] This example illustrates the synthesis of compound 130 on an 80+kg scale. The synthesis steps in this example are based on Figure 1 In addition, a continuous flow production system was used in steps 3 and 4, and these steps were performed according to Figure 1 and Figure 2 Proceed; the process of steps 3 and 4 refers to the two figures.

[0236] Step 1: Synthesis of (2R,3S,4S,5R,6R)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetrabenzoate (Compound 106)

[0237]

[0238] Compound 104 (benzoyl chloride, 5.5 eq.) was added to a solution of compound 102 (1.0 eq.) in pyridine (6 V) 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 (10 v) and water (10 v). The organic component was separated and washed with 7% NaHCO3 solution (2 x 4 V) and H2O (3 V), and concentrated to 5 V-6 V. The solvent was then exchanged with toluene (4 x 5 V), and the mixture was continued for the next step without further purification.

[0239] Step 2: Synthesis of (2R,3R,4S,5S,6S)-2-((Benzoyloxy)methyl)-6-hydroxytetrahydro-2H-pyran-3,4,5-triyl tribenzoate (Compound 110)

[0240]

[0241] A toluene solution of compound 106 (1.0 equiv.) was diluted with THF (6 V). DMAPA (2.0 equiv.) was added to the reaction mixture under N2 protection. The reaction mixture was stirred at 60-65 °C for 12-14 h and diluted with EtOAc (5 V). Thereafter, the resulting mixture was quenched with 5% H2SO4 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)phosphinoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (Compound 114)

[0242]

[0243] Two solutions were prepared. The first solution contained compound 110 (1.0 eq.) and DCI (1.77 eq.) in acetonitrile (10 V) at -20°C to -14°C and was stored in tank 202. The second solution contained compound 112 ((BnO)2PN i Pr2, 1.3 eq.) in acetonitrile (10 V) and stored in tank 204. A quenching solution containing Na2SO3 (4.8 eq.) in water (10 V) was prepared separately and stored in tank 230. The first two solutions were charged to pre-cooling tubes 206 and 208 and combined flow reactor 214, respectively, with a total residence time of 1 min and a temperature between 0° C. and 5° C. The reaction stream containing compound 114 is the product of this step and is forwarded in the second flow chemistry step without separation or purification.

[0244] 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)

[0245]

[0246] The reaction stream containing compound 114 enters the flow reactor 224 and is continuously combined with the oxidant 218 (H2O2, 3 equivalents) at a temperature between 20°C and 25°C for a residence time of 15 minutes. The resulting reaction stream is collected in a tank 228, and then combined with a Na2SO3 solution at a temperature between 0°C and 10°C for a residence time of 3 to 10 minutes at 0-5°C. The resulting reaction mixture is diluted with toluene and entered into a centrifugal extractor for immediate separation. The reaction mixture is concentrated and then dissolved in EtOAc (3V to 4V). The resulting reaction mixture is used in the next step without further purification.

[0247] Step 5: Synthesis of (2R,3R,4S,5S,6R)-2-((benzoyloxy)methyl)-6-(phosphoryloxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (Compound 126)

[0248]

[0249] In an autoclave, MeOH (5 V to 7 V) was added to a solution of compound 118 (1.0 eq.) in EtOAc. Wet Pd / C (15% w / w, 0.15-0.17 eq.) was added to the reaction mixture. The autoclave was vacuum purged three times with N2, followed by three times with H2. After stirring at 20°C to 30°C for 18 hours under an atmosphere of H2 at 45-50 psi, the reaction mixture was filtered and the solvent was switched to MeOH. Compound 126 (yield 97.7%) was obtained as a yellow MeOH solution and used in the next step without further purification.

[0250] Step 6: Synthesis of (2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl phosphate potassium (mannose-1-phosphate potassium salt, compound 130)

[0251]

[0252] A solution of compound 126 (1.0 equivalent) in methanol (10V) was dried over anhydrous methanol by solvent exchange. A methanol solution of KOMe (4.0 equivalent) was added to this solution, and the resulting reaction mixture was stirred for 20-22 hours under a N2 atmosphere at a temperature between 20°C and 25°C. The solid product was then separated by filtration, absorbed again in methanol (3V) to produce a slurry, and then filtered again to produce a crude white solid. The solid was recrystallized in H2O:MeOH to obtain a hydrate of compound 130 (60-70% yield) as a white solid. Compound 130 can then be subjected to a further crystallization procedure to produce Form C, as described in Example 6 below.

[0253] Example 2: Instability of Compound 110

[0254] In the present invention, the use of flow chemistry techniques is essential in producing a substantially pure final product of the desired α isomer of M1P. This is because a significant amount of isomerization of the reaction intermediate from the α form to the β form occurs over time under the conditions required to convert compound 110 to compound 118. The flow chemistry techniques described herein reduce the amount of isomerization in this conversion.

[0255] Table 1 shows the instability of compound 110 in solution over time. When the reaction solution containing 110 and DCI was stored and not used immediately after its preparation, the amount of the β isomer of product 118 increased.

[0256] Table 1: Instability of Compound 110 in Reaction Solution

[0257]

[0258] Example 3: Batch Synthesis and Flow Chemistry Synthesis of Compound 118

[0259] This example compares the production of Intermediate D using a batch synthesis with a flow chemistry synthesis according to the procedure described in Example 1 above.

[0260] For batch synthesis, compound 110 (1.0 eq.) and DCI (1.77 eq.) were dissolved in acetonitrile (10 V). To this solution was added (BnO)2PN i Pr2 (1.5 eq.) was added and the resulting mixture was stirred at -20 to -15 °C to produce compound 114. After the reaction was completed, 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 washed with 10% citric acid, followed by 7% NaHCO3, and then purified by silica gel chromatography to remove the unwanted β-isomer of 118. On a 100 g scale, this method successfully afforded 118 with 95% purity, with only 2-3% of the β-isomer of 118 present as an impurity. However, on a larger (25 kg) scale, the amount of β-isomer increased by up to 15%. This highlights the importance of the flow chemistry technique described herein, which allows for large-scale production of substantially pure α-isomer of 118, and thus large-scale production of substantially pure α-isomer form of M1P.

[0261] Example 4: Preparation and characterization of polymorph A of M1P (potassium salt hydrate)

[0262] This example shows the synthesis and characterization of polymorph A of M1P, which is the dipotassium salt dihydrate.

[0263] M1P Form A was prepared using the following procedure. About 500 mg of M1P was added to a vial. 3 mL of methanol was added to the vial under stirring at 5°C. After stirring at 5°C for about 5 days, the resulting suspension was filtered. The resulting solid was air-dried under ambient conditions for about 22 hours. About 420 mg of M1P Form A was obtained as a white solid with a yield of 84%. The characterization data of Form A are shown in Table 2.

[0264] Table 2: Characterization of polymorph A of M1P

[0265]

[0266]

[0267] Example 5: Preparation and characterization of M1P (potassium salt hydrate) polymorph B

[0268] This example illustrates the synthesis and characterization of polymorph B of M1P, which is the dipotassium salt trihydrate.

[0269] M1P Form B was prepared using the following procedure. About 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. 1.5 mL of the filtered solution was quickly added to 7.5 mL of methanol in a 20 mL glass vial. The clear solution immediately became a suspension, and the suspension was stirred at 25 ° C. After stirring at 25 ° C for about 6 days, the suspension was filtered through a 0.45 μm nylon membrane filter at 4,000 rpm. The obtained solid was air-dried for about 22 hours under ambient conditions. About 360 mg of M1P Form B was obtained as a white solid with a yield of 72%. The characterization data of Form B are shown in Table 3.

[0270] Table 3: Characterization of polymorph B of M1P

[0271]

[0272]

[0273] Example 6A: Preparation and characterization of M1P (potassium salt hydrate) polymorph C

[0274] This example illustrates the synthesis and characterization of polymorph C of M1P, which is the dipotassium salt trihydrate.

[0275] M1P Form C was prepared using the following procedure. About 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 to produce a suspension. About 5 mg of Form C seed crystals were added to the suspension. After stirring at 25 °C for about 6 days, the suspension was filtered through a 0.45 μm nylon membrane filter at 4,000 rpm. The resulting solid was air-dried for about 22 hours under ambient conditions. About 389 mg of M1P Form C was obtained as a white solid with a yield of 77%.

[0276] M1P Form C was also prepared using the following method. M1P potassium salt (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 sparger, and the temperature of the reactor was adjusted to between 20 and 30 °C. The solution was stirred for 1-2 hours until clear. The temperature was adjusted to 0 to 10 °C, and then MeOH (2.5-3.0 V) was slowly added. M1P Form C seeds (0.1%-2.0%X) were added, and the mixture was stirred at 0 to 10 °C for 14-20 h. Then, H2O:MeOH (volume ratio 1 / 3, 2.5-3.0X (target 2.7X)) was slowly added at 0 to 10 °C over 4 hours, and stirring was continued for 6 to 8 hours. Stirring and adding additional MeOH continued until the sample for XRPD analysis was confirmed to be consistent with the internal standard of Form C. Finally, the crystals were isolated by active filtration drying (AFD).

[0277] Table 4a: Characterization of polymorph C of M1P

[0278]

[0279]

[0280] Example 6B: Polymorph Screening

[0281] This example describes polymorph screens using several different solvent systems and various conditions. Overall, it was surprisingly observed that only certain solvent systems and certain conditions resulted in the production of M1P Form C (as described above in Example 6A).

[0282] Equilibration with solvent at 5°C for 2 weeks: About 50-150 mg of M1P (mixture of Form A and Form B) was stirred and equilibrated 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-methylTHF, 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). According to XRPD, Form C was only observed when water / methanol was used at 5:1, 3:2, 1:1, 2:3, 1:2, and 1:10, v:v. No XRPD was obtained for water and water / MeOH (10:1, v:v) samples. The XRPD of the remaining samples showed Form A, Form B or a mixture of Form A and Form B.

[0283] Equilibration with solvent at 25°C for 1 week: About 50-150 mg of M1P (mixture of Form A and Form B) was stirred and equilibrated 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-methylTHF, 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). According to XRPD, Form C was only observed when water / methanol was used at 5:1, 3:2, 1:1, 2:3, 1:2, and 1:10, v:v. No XRPD was obtained for water and water / MeOH (10:1, v:v) samples. The XRPD of the remaining samples showed Form B or a mixture of Form A and Form B.

[0284] Equilibration under temperature cycling: About 50-150 mg of M1P (mixture of Form A and Form B) was equilibrated in 0.04-0.40 mL of the following solvents for 10 cycles at a heating / cooling rate of 0.1°C / min under temperature cycling from 5°C to 50°C and 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 various ratios of water / methanol (10:1, 5:1, 3:2, 1:1, 2:3, 1:2, 1:5, and 1:10, v:v). According to XRPD, Form C was only observed when water / methanol was used at 3:2, 1:1, and 1:2, v:v. No XRPD was obtained for water and water / MeOH (10:1 and 5:1, v:v) samples because M1P was too soluble. XRPD for the remaining samples showed Form A or a mixture of Form A and Form B.

[0285] Crystallization from hot saturated solution by slow cooling: About 50 mg of M1P (mixture of Form A and Form B) was dissolved in a minimum amount of the following solvents at 50°C: 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 a rate of 0.1°C / min. The sample without precipitate was further cooled to -20°C at 5°C. XRPD was not performed on the above samples because M1P was too soluble.

[0286] Crystallization from hot saturated solution by rapid cooling: About 50 mg of M1P (mixture of Form A and Form B) was dissolved in a minimum amount of the following solvents at 50°C: 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. The sample without precipitate was further cooled to -20°C at 5°C. XRPD was performed only on water / MeOH (1:1, v:v), which showed Form C. XRPD was not performed on the remaining samples as M1P was too soluble.

[0287] Crystallization by adding antisolvent: About 1 g of M1P (a mixture of Form A and Form B) was dissolved in 2 mL of water at ambient temperature (about 20-22 ° C). The resulting solution was filtered. Then, 0.1 mL of the clear solution was transferred to a vial, and 3-5 times of the test antisolvent was slowly added to the clear solution. The sample without precipitate was further cooled to -20 ° C. The test antisolvents 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 studied by XRPD. XRPD was obtained only for the water / methanol system, which showed Form B. XRPD was not performed on the remaining samples mentioned above because M1P was too soluble.

[0288] Crystallization by back-addition of antisolvent: About 1 g of M1P (a mixture of Form A and Form B) was dissolved in 2 mL of water at ambient temperature (about 20-22°C). The resulting solution was filtered. Then, 0.1 mL of the clear solution was added to 5 times the test antisolvent in a vial. The sample without precipitate was further cooled to -20°C. The test antisolvents included: 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 studied by XRPD. Additional analyses were performed for samples with different XRPD patterns, including DSC, TGA, IC, KF, 1H-NMR and PLM. According to XRPD, it was observed that Form B was obtained in water / methanol, water / ethanol, water / isopropanol, water / acetone and water / 1,4-dioxane. Since M1P was too soluble, XRPD was not performed on the remaining samples mentioned above.

[0289] Crystallization by vapor diffusion: About 500 mg of M1P (a mixture of Form A and Form B) was dissolved in 0.5 mL of water at ambient temperature (about 20-22°C). The resulting solution was filtered. The clear solution was then transferred to a vial without a cap. The vial without a cap was placed in a larger vial. The anti-solvents tested were then added to the larger vial. The anti-solvents tested included: 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 capped and placed under ambient conditions. The precipitate was collected by centrifugal filtration. The solid portion (wet cake) was studied by XRPD. The XRPD of the water / methanol sample and the water / acetone sample showed a mixture of Form A and Form B. The XRPD of water / ethanol showed Form A. Since M1P was too soluble, XRPD was not performed on the remaining samples mentioned above.

[0290] Example 7: Water activity experiment

[0291] This example shows that M1P Form C is stable over a wide range of water activities. Generally, Form C is a more stable form compared to Form A and Form B. In addition, under certain specific conditions, Form A and Form B are converted to Form C. Water activity experiments were performed at 25°C in several different water / EtOH systems to determine the relative stability of M1P Forms A, B, and C under these conditions.

[0292] About 5 mg of each M1P Form A, Form B and Form C were combined and added to a saturated solution of 0.2 mL water / EtOH system. The resulting suspension was stirred at 5°C, 25°C or 50°C for 4 or 11 days. After 4 or 11 days, the solid (wet cake) was separated by centrifugal filtration and analyzed by XRPD to determine which form remained. The results are summarized in Table 5.

[0293] Table 5: Water activity experiments to determine the relative stability of Form A, Form B and Form C in solvent systems with different water activity (aw) values

[0294]

[0295]

[0296] Note: Water activity was calculated by UNIFAC method. “ / / ”: Not performed.

[0297] Example 8: Water adsorption and desorption experiments

[0298] The water sorption behavior of M1P Form C was studied at 25°C using dynamic vapor sorption (DVS). The relative humidity (RH) was increased from 0-80%, with a minimum equilibrium time of 60 min and a maximum equilibrium time of 360 min. As shown in Table 6, Form C adsorbed only 1.9% (w / w) HO at 80% RH. XRPD analysis was performed after the DVS experiment, and no crystalline form changes were observed, indicating the low hygroscopicity and favorable stability of Form C.

[0299] Table 6: DVS experiment using Form C at 25°C

[0300]

[0301]

[0302] Example 9: Stability of Form C

[0303] This example illustrates the stability of Form C in several stress tests. Form C was subjected to compression, dry grinding simulation, and wet granulation simulation. The results are summarized in Tables 7-9.

[0304] Compression study

[0305] About 10 mg of M1P Form C was compressed at 5 MPa or 10 MPa for 5 minutes using a hydraulic press. Potential polymorphic changes and crystallinity were evaluated by XRPD. According to the XRPD results of the compression study, no polymorphic changes were observed when compressed at 5 MPa and 10 MPa, and the crystallinity was reduced by showing broad peaks.

[0306] Dry Grinding Research

[0307] About 10 mg of M1P Form C was manually ground using a mortar and pestle for 3 min. Potential polymorphic changes and crystallinity were evaluated by XRPD. The XRPD results of the dry grinding study showed no polymorphic changes after a grinding time of 3 min, and the crystallinity decreased by showing broad peaks.

[0308] Wet granulation simulation

[0309] Ethanol was added dropwise to approximately 10 mg of M1P Form C until the sample was fully wetted. The wet sample was gently ground in a mortar and pestle. After granulation, the sample was dried at ambient conditions for 10 min. Potential polymorphic changes and crystallinity were evaluated by XRPD. The XRPD results of the wet granulation study showed no polymorphic changes in ethanol as the granulation solvent.

[0310] Example 10: Solubility of M1P Form C

[0311] This example illustrates 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 a soluble medium (pH 7.0 Tris buffer, 50 mM) was added. The obtained solution was stirred at 37 ° C and 400 rpm for 2 h and 24 h, and then centrifuged at 37 ° C and 14,000 rpm for 5 min. At each time point of 2 h and 24 h, the supernatant was analyzed by a pH meter to determine the solubility. M1P Form C exhibited a favorable solubility of more than 160 mg / mL.

[0312] Example 11: Polymorphic Characterization of M1P Form C by 12 Months Stability

[0313] M1P Form C was produced according to the above-mentioned example. XRPD was performed on samples collected at 1, 3, 6, 9 and 12 months and tested under the following four conditions:

[0314] -20℃±5℃,

[0315] 25℃±2℃, 60% relative humidity (RH)±5%RH,

[0316] 40℃±2℃, 75%RH±5%RH and

[0317] 5℃±3℃.

[0318] In each case, the XRPD spectra of the samples were consistent with the reference standard spectrum of M1P Form C, indicating that the samples remained in Form C at all time points tested. No changes in polymorphic form were observed at any of the temperature conditions studied.

[0319] Furthermore, no substantial changes were observed in the purity of Form C as determined by chromatography.

Claims

1. A continuous flow production method comprising: a) providing a compound of formula B-α-1 and a nucleophilic catalyst at a temperature of -30°C to -10°C to produce a pre-cooled solution, wherein the compound of formula B-α-1 is: b) continuously combining the pre-cooled solution and the phosphorylating agent in a first reactor at a temperature of -30°C to 5°C and a residence time of 30 seconds to 5 minutes to obtain an intermediate composition comprising a compound of formula (C-α-1): c) transferring the intermediate composition from the first reactor to a second reactor; as well as d) continuously combining the intermediate composition and an oxidant in the second reactor at a temperature of 0° C. to 30° C. and with a residence time of 10 minutes to 30 minutes to produce a reaction mixture comprising a compound of formula (D-α-1):

2. The method according to claim 1, wherein: The pre-cooled solution has less than 1% of the compound of formula (B-β-1): and / or The reaction mixture comprising the compound of formula (D-α-1) contains less than 2% of the compound of formula (D-β-1):

3. The method according to claim 1 or 2, wherein 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).

4. The method according to any one of claims 1 to 3, wherein the nucleophilic catalyst is 4,5-dicyanoimidazole.

5. The method according to any one of claims 1 to 4, wherein the phosphorylating agent is (BnO)2PN i Pr2.

6. The method according to any one of claims 1 to 5, wherein the oxidizing agent is H2O2.

7. The process according to claim 6, wherein 1 to 3 molar equivalents of H2O2 are used.

8. The method according to any one of claims 1 to 7, wherein an organic solvent is used in the pre-cooled solution.

9. The method according to any one of claims 1 to 8, wherein the phosphorylating agent is at least partially dissolved in an organic solvent.

10. The method of claim 8 or 9, wherein the organic solvent comprises a polar aprotic solvent.

11. The method according to any one of claims 8 to 10, wherein the organic solvent is acetonitrile, THF or 2-MeTHF.

12. The method of any one of claims 8-11, wherein the organic solvent comprises acetonitrile.

13. The method according to any one of claims 1 to 12, wherein the pre-cooled solution is provided at a flow rate of 0.5 mL / min to 1 L / min.

14. The method of any one of claims 1-13, wherein the phosphorylating agent is transferred at a flow rate of 0.5 mL / min to 1 L / min.

15. The method of any one of claims 1-14, wherein the oxidant is provided at a flow rate of 0.5 mL / min to 1 L / min.

16. The method of any one of claims 1-15, further comprising quenching the reaction mixture.

17. The method of claim 16, wherein the reaction mixture is quenched with an aqueous solution comprising a reducing agent.

18. The method of claim 17, wherein the reducing agent is Na2SO3.

19. The method of any one of claims 16-18, wherein the reaction mixture is quenched at a temperature of 0°C to 25°C.

20. The method according to any one of claims 16 to 19, further comprising extracting the compound of formula (D-α-1) into an organic solvent.

21. The method of claim 20, wherein the organic solvent comprises toluene.

22. The method according to claim 20 or 21, further comprising washing the organic solvent with water and then with a 10% Na2SO4 solution.

23. The method according to any one of claims 20 to 22, further comprising isolating the compound of formula (D-α-1) by evaporating at least a portion of the organic solvent.

24. The method of claim 23, wherein the compound of formula (D-α-1) is not further purified.

25. The method according to any one of claims 1-24, further comprising deprotecting the compound of formula (D-α-1) to produce a composition comprising the α 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 β isomer of mannose-1-phosphate or a salt thereof or a hydrate of any of the foregoing.

26. The method according to any one of claims 1 to 25, further comprising making a compound of formula (A-α-1): Reaction with DMAPA selectively removes the Bz group attached to the oxygen of the anomeric center to produce the compound of formula (B-α-1).

27. The method according to any one of claims 1 to 26, further comprising making a compound of formula (SM-α-1): Reaction with benzoyl chloride to produce the compound of formula (A-α-1).

28. The method according to any one of claims 1 to 27, further comprising selectively deprotecting the phosphate group in the compound of formula (D-α-1) to produce a compound of formula (E-α-1) having the following structure:

29. The method according to claim 28, further comprising deprotecting the compound of formula (E-α-1) to produce a compound of formula (G-α-1): or a salt thereof, or a hydrate of any of the foregoing.

30. A composition produced according to the method of any one of claims 1-29.

31. A composition comprising the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, wherein the composition has a purity of at least 96%.

32. The composition of claim 31, 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.

33. A composition according to any one of claims 30-32, wherein less than 1% of the composition is 34. A composition according to any one of claims 30-33, wherein less than 1% of the composition is 35. A composition according to any one of claims 30-34, wherein less than 1% of the composition is 36. The composition of any one of claims 30-35, comprising α-D(+)mannose-1-phosphate dipotassium salt; and Optionally further comprises at least one pharmaceutically acceptable carrier, excipient and / or stabilizer.

37. A method for preparing Form C of mannose-1-phosphate, comprising: a) combining a starting composition comprising (i) substantially pure alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (ii) substantially pure crystalline Form A of the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or (iii) substantially pure crystalline Form B of the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, or any combination of (i)-(iii); as well as b) stirring the suspension to produce a product composition comprising Form C of mannose-1-phosphate.

38. The method of claim 37, wherein the starting composition comprises the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, and has less than 2% of the beta isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing.

39. The method of claim 37 or 38, wherein the starting composition comprises a mixture of Form A and Form B of mannose-1-phosphate.

40. The method of any one of claims 37-39, wherein the starting composition comprises crystalline Form A of mannose-1-phosphate.

41. The method of any one of claims 37-39, wherein the starting composition comprises Form B of mannose-1-phosphate.

42. A crystalline form C of mannose-1-phosphate produced according to the method of any one of claims 37 to 41.

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

44. Form C according to claim 42 or 43, which exhibits an X-ray powder diffraction (XRPD) pattern comprising peaks at 11.6, 14.6, 17.8, 23.2, 27.7 and 30.7 ± 0.2° 2θ.

45. Form C according to claim 42 or 43, which exhibits an XRPD substantially similar to that of Figure 1.

46. ​​Form C according to any one of claims 42-45, which exhibits a TGA thermogram with a weight loss of about 5% to 8% in the temperature range of 101°C-150°C.

47. Form C according to any one of claims 42-45, which exhibits a TGA thermogram substantially similar to that of Figure 6.

48. Form C according to any one of claims 42 to 47, having an average particle size of 1 μm to 20 μm.

49. Form C according to any one of claims 42-48, wherein it is stable when compressed at 5 MPa and / or 10 MPa for 5 minutes.

50. Form C according to any one of claims 42-49, wherein it is stable when ground under dry conditions or wet conditions.

51. Form C according to any one of claims 42-50, wherein it is stable when stored in an open container at 40°C and 75% relative humidity for one week, or when stored in an open container at about 40°C and about 75% relative humidity for one week.

52. Form C according to any one of claims 42-50, wherein when stored under the following conditions: In a sealed container at about 60°C for one week; at about -20°C for up to about 12 months; up to about 12 months at about 25°C and about 60% relative humidity; at about 5°C for up to about 12 months; and / or At about 40°C and about 75% relative humidity for up to about 12 months, It is stable.

53. A composition comprising: at least 98% by weight of Form C according to any one of claims 42-52.

54. The composition of claim 53, wherein less than 1% of the composition is the beta isomer of mannose-1-phosphate potassium salt having the structure:

55. The composition of claim 53 or 54, wherein less than 0.5% of the composition is Form A and / or Form B of mannose-1-phosphate.

56. The composition of claim 55, wherein: Form A of mannose 1-phosphate exhibits an XRPD pattern comprising peaks at 20.3, 21.1, 23.5, 27.1, 28.1, and 29.0 ± 0.2 degrees 2θ; and Form B of mannose-1-phosphate exhibits an XRPD pattern comprising peaks at 8.3, 14.2, 19.8, 21.4, 23.9, and 27.5 ± 0.2° 2θ.

57. A composition comprising: (i) liposomes; and (ii) the α isomer of mannose-1-phosphate encapsulated in the liposome, or a salt thereof, or a hydrate of any of the foregoing.

58. The composition of claim 57, wherein the liposomes comprise one or more phospholipids conjugated to polyethylene glycol (PEG).

59. A composition comprising: A liposome having a lipid membrane encapsulating an intraliposomal compartment, wherein the liposome encapsulates the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing, in the intraliposomal compartment, and wherein the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, and an intraliposomal buffer comprising a buffer salt and optionally an acid, wherein the buffer salt has a pKa between 6 and 8.5; an extralipidic buffer comprising a buffer salt and a tonicity adjusting agent, wherein the buffer salt has a pKa between 6 and 8.5; and Optional free radical scavenging antioxidant.

60. The composition of claim 59, wherein the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG).

61. The composition of claim 59 or 60, wherein the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing is produced according to the method of any one of claims 1 to 25.

62. The composition according to any one of claims 57-61, wherein the alpha isomer of mannose-1-phosphate, or a salt thereof, or a hydrate of any of the foregoing is alpha-D(+) mannose-1-phosphate dipotassium salt.

63. The composition of any one of claims 57-62, wherein 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.

64. The composition of any one of claims 57-63, wherein the alpha isomer of mannose-1-phosphate potassium salt hydrate is Form C.

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

66. The composition of any one of claims 57-65, formulated for intravenous administration.

67. The composition of any one of claims 57-65, formulated for injection.

68. The composition of any one of claims 57-67, wherein the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 98% after storage of the composition at controlled room temperature.

69. The composition of any one of claims 57-68, wherein the purity of the alpha isomer of mannose-1-phosphate in the composition is at least about 95% after storage of the composition at ambient temperature.

70. The composition of any one of claims 53-69, further comprising: at least one pharmaceutically acceptable carrier, excipient and / or stabilizer.

71. A method of treating congenital disorders of glycosylation (CDG) in a subject in need thereof by administering the composition of any one of claims 30-36 and 53-70.

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