CRYSTALLINE CDM-NAG AND METHODS FOR PRODUCING SAME

FIELD OF THE INVENTION

The present invention provides a crystalline composition, which comprises CDM-

NAG. The present invention also provides a process of producing said crystalline composition, and an improved method of preparing NAG and ^" CDM-NAG.

BACKGROUND OF THE INVENTION

The ability of siRNA to silence specific genes has generated great interest in its use as a research tool and therapeutic agent for a wide spectrum of disorders that½clude cancer, infectious disease, and metablic conditions. Achieving efficient in vivo delivery of -siRNA to the appropriate target cell would be a major advance- in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. Rozema et aLhave developed a vehicle for the delivery of siRNA- to hepatocytes both in vitro and in vivo. Key features of this; vehicle include a membrane-active polymer conjugated to an endosomolytic agent (siRNA polyconjugate) reversibly masked until it reaches the acid environment of endosomes. Amine groups of the endosomolytic agent

(amphipathic poly( vinyl ether)) are masked with maleic anhydride, creating acid-labile maleamate bonds. These bonds are cleaved within the acidic environment of the endosome, unmasking the agent's amines and activating its endosomolytic capabilities. Rozema et al.

PNAS. (2007) 104:12982-12987

The siRNA-polymer conjugate is reversibly modified with maleic anhydride derivatives synthesized from carboxy dimethylmaleic anhydride (CDM)-containing N- acetylgalactosamine (NAG) groups. The NAG ligand is responsible forJhepatocyte targeting. Rozema et al. PNAS. (2007) 104:12982-12987.

An siRNA-polymer conjugate delivery vehicle can be easier to make with increased purity for therapeutic application. Crystallization and crystallization methods for siRNA-polymer conjugate delivery vehicle components, such as CDM-NAG, allow for easier CDM-NAG purification and a more pure CDM-NAG component product. SUMMARY OF THE INVENTION

It is an object of the present invention to provide a composition comprising crystalline CDM-NAG. The present invention also provides aiprocess of producing said crystalline composition, and an improved method of preparing NAG and CDM-NAG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Figure 1 shows the x-ray- diffractogram for CDM-NAG Form I. Y axis Illustrates the intensity and X axis illustrates- the 20 angle. DETAILED DESCRIPTION OF THE INVENTION

All references cited in the instant application are hereby incorporated by reference.

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of embodiments of the Invention.

For the purpose of this invention, the term "about" when used in the context of 2Θ peaks refers to a shift up to ±0.1 degrees (error). In one embodiment, all peaks in X-ray diffraction pattern shift up to +0.1 degrees, or up to -0.1 degrees. An X-ray diffraction pattern or peaks within that error is considered the same or substantially similar. The shift may vary depending on the calibration, sample or instrumentation.

Compositions

The present invention encompasses crystalline CDM-NAG. In one embodiment, the crystalline CDM-NAG is Form I, and characterized by an X-ray diffraction pattern substantially similar to that set forth in Figure 1 A as measured using CuKa radiation. In one embodiment, CDM-NAG Form I is characterized by an X-ray diffraction pattern comprising two or more characteristic peaks at about 4.0, 8.0, 11.2, 118, 16.4, 17.9, 18.6, 19.2, 20.4, 21.4, 22.7 and 24.2 degrees 2Θ as measured using CuKa radiation. In one embodiment, CDM-NAG Form I is characterized by an X-ray diffraction pattern comprising three or more characteristic peaks at about 4.0, 8.0, 1 1.2, 15.8, 16.4, 17.9, 18.6, 19.2, 20.4, 21.4, 22.7 and 24.2 degrees 2Θ as measured using CuKa radiation. In another embodiment, CDM-NAG Form I is characterized by an X-ray diffraction pattern comprising characteristic peaks at about 4.0, 8.0 and 20.4 degrees 2Θ as measured using CuKa radiation. In one embodiment, CDM-NAG Form I is characterized -by an-X-ray diffraction pattern comprising characteristic peak-s at about 15.8, 16.4,_17.9 and 18.6 degrees 2Θ as measured using Cu a radiation.

Crystallization with Organic Solvents

In one embodiment, the crystalline CDM-NAG is crystalli-zed from an organic solvent. The organic solvent may be one or more of isopropyl acetate (IP Ac), toluene, dichloromethane (DCM), 1 ,2-dicbJoroethane (DCE), cyelopentylmethyl ether, 2- methyltetrahydrofuran (2-MeTHF), trifluorotoluene, tetrahydrofuran, 1 ,2-dimethoxyethane (DME),- methylcyclohexane, 1,4-dioxane, chlorobenzene, methylcyclohexane, N,N- dimethylacetamide, dimethylformamide.and acetonitrile (MeCN). In one embodiment, the organic solvent is acetonitrile. In one embodiment, the organic solvent is 1 ,2-dimethoxyethane. In one embodiment, the organic solvent is 1 : 1 DCM/Tetrahydrofuran. In one embodiment, the organic solvent is 1 : 1 DCE/IPAc. In another embodiment, the organic solvent is 1 : 1 1_,2- dimethoxyethane/2-MeTHF. In another embodiment, the organic solvent is 1 :1

methylcyclohexane/ 1 ,4-dioxane. Jn another embodiment, the organic solvent is 1 : 1

chlorobenzene/DCM. In a further embodiment, the organic solvent is 1 : 1 1 ,4-dioxane/2- MeTHF. In a further embodiment, the organic solvent is 1 : 1 trifluorotoluene/chlorobenzene. r one embodiment, the organic solvent is 100% DCE. In one embodiment,, the organic solvent is- 100% DME. In one embodiment, the organic solvent is 1 :1 DCM/chlorobenzene. In another embodiment, Ihe organic solvent is N,N~dimethylacetamide. In another embodiment, the organic solvent is 3:1 1,2-dimethoxyethane/dimethylformamide.

In another embodiment, crystalline CDM-NAG Form I is crystallized from isopropylacetate. In another- embodiment, CDM-NAG Form I is crystallized from toluene. In another embodiment, CDM-NAG Form I is crystallized from dichloromethane. In another embodiment,. CDM-NAG Form I is crystallized from 1 ,2-dichloroethane. In another

embodiment, CDM-NAG Form I is crystallized from cyclopentyl metfiyl ether. In another embodiment, CDM-NAG Form I is crystallized from 2-methyltetrahydrofuran. In another embodiment, CDM-NAG Form I is crystallized from trifluorotoluene. In another embodiment, CDM-NAG Form I is crystallized from 50% dichloromethane and 50% tetrahydrafuran. In another embodiment, CDM-NAG Form I is crystallized from 50% dichloroethane and 50% isopropylacetate. In another embodiment, CDM-NAG Form I is crystallized from 50% dimethoxyethane and 50% 2-methyl tetrahydrafuran. In another embodiment, CDM-NAG Form I is crystallized from 50% Methyl-Cyclohexane and 50% 1, 4-dioxane. In another embodiment, CDM-N AG-Form I is crystallized from 50% chlorobenzene and 50% dichi romethane. In another embodiment, CDM-NAG Form I is crystallized from 50% 1, 4-dioxane and 50% ^~ 2- methyltetrahydrofuran. m another embodiment, CDM-NAG Form I is crystallized from 50% trifluorotoluene and 50% chlorobenzene. Crystallin CDM-NAG Form I can also be crystallized from one or more of isopropyi acetate (IPAG), toluene, dichloromethane (DCM), 1 ,2- dichloroethane (DCE), cyelopentylmethyl ether, 2-methyltetrahydrofuran (2-MeTHF), trifluorotoluene, tetrahydrofuran, 1,2-dimethoxyethane (DME), methylcyclohexane, 1, 4-dioxane, chlorobenzene, methylcyclohexane, and acetonitrile (MeCN). In one embodiment, the organic solvent is acetonitrile. In one embodiment, the organic solvent is 1,2-dimethoxyethane.

However, it should be apparent to a person skilled in the art that the crystallizations of the methods described herein can be carried out in any suitable solvents or solvent mixtures which may be readily selected by one of skill in the art of organic synthesis. Such suitable organic solvents, as used herein may include, by way of example and without limitation, chlorinated solvents, hydrocarbon solvents,, ether solvents, polar protic solvents and polar aprotic solvents. Suitable halogenated solvents include, but are not limited to carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane, tetrachloroethylene, tricliloroethylene, 1,1.1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane, 1,2- dichloroethane, 2-chloropropane, hexafluorobenzene, 1 ,2,4-trichlorobenzene, o-dichlorobenzene, chlorobenzene, fluorobenzene, fluorotrichloromethane, chlorotri-fluoromethane,

bromotrifluoromethane, carbon tetraftuoride, dichlorofluoromethane, chlorodifluoromethane, trifluoromethane, 1 ,2-dichlorotetrafluorethane and hexafiuoroethane. Suitable hydrocarbon solvents include, but are not limited to benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o-, or p-xylene, octane, indane, nonane. Suitable ether solvents include, but are not limited to dimethoxymethane,

tetrahydrofuran, 1,3-dioxane, 1, 4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol diisopropyl ether, anisole, or t-butyl methyl ether.

Suitable polar protic solvents include, but are not limited to methanol, ethanol, 2- itroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanoI, ethylene glycol, 1-propanol, 2-propanol, 2- methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanoi, diethylene glycoL 1-, 2-, or 3- pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenal, and glycerol. Suitable polar aprotic solvents include, but are not limited to dimethylformamide (DMF), dimetfeylacetamide (DMAC), l,3-dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidinone (D PU), l,3-dimethyl-2-imidazolidmene (DMI), N- methylpyrrolidinone (NMP), forma ide, N-methylacetamide, N-methylformamide, acetonitnle (ACN), dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone-, ethyl acetate, isopropyl acetate, t-butyl acetate, sulfolane, Ν,Ν-dimethylpropionamide, nitromethane, nitrobenzene, he-xamethylphosphoramide .

Synthesis

The present invention also provides an improved synthesis method for preparing NAG and CDM-NAG, -which results in a higher purity NAG and CDM-NAG end product.

In one embodiment, the invention provides a process for producing NAG

represented by the structure , comprising the steps of:

I) Glycosylating 2) Deacetylating to form

In one embodiment, step l) is performed in the presence of an organic solvent selected from dichlorometharie _f dichloroethane and 2-inethyltetrahydrofuran and a lewis acid selected from trifluoromethanesulfonic acid, trifluoromethanesulfonate salt, borontrifluoride diethyl ethercomplex and tin (IV) chloride. In one embodiment, the trifluoromethanesulfonate salt is Copper(II) trifluoromethanesulfonate or trimethylsilyl trifluoromethanesulfonate. The combination of a select organic solvent and a lewis acid during this reaction provides the advantage of producing predominantly the desired beta isomer.

In another embodiment, step 3) is performed in the presence of

trifluroethanol and palladium on carbon under ¾. The selection of trifluroethanol as the reaction solvent provides the advantage of minimizing the reaction of carbon dioxide with the amino group of NAG, thereby improving the purity of the intermediate product NAG.

In a further embodiment, the invention provides a process of preparing CDM-

NAG of the following stracturer

comprising the steps of :

reacting NAG with to form CDM-NAG.

In one embodiment, the above reaction is performed in the presence of

dimethylformamide and isopropylacetate. In another embodiment

reacting CDM of the structure with pivaloyl chloride. In another embodiment, the reaction is performed in the presence of N-methylmorpholine and

isopropylacetate. The improved synthesis method for CDM-NAG provides higher yields and purity of the end product.

UTILITY

The CDM-NAG component is usefuLas a reversible linker (CDM) and a hepatocyte targeting agent (NAG) for a nucleic. acid-polymer conjugate delivery vehicle. Most specifically, the CDM-NAG component is useful as a reversible linker (CDM) and a hepatocyte targeting agent (NAG) for an siRNA-polymer conjugate delivery vehicle. To the applicant's knowledge, the first disclosure of a polymer-based nucleic acid delivery vehicle that contains a reversible linker component (derivatives of maleic anhydride and malic acid) and a hepatocyte targeting component (galactose) is found in PCT Publication Number WO 00/34343 to Mosaic Technologies, Inc. A detailed synopsis of general polymer synthesis is found in US

2004/0156909. Further detailed synopsis of CDM-NAG incorporated into a nucleic acid- polymer and siRNA-polymer conjugate delivery vehicle is found in WO 2008/022309. The synthesis of nucleic acids and chemically modified nucleic acids is described in WO

2007/022369.

embodiment, the instant invention comprises a nucleic acid-pelymer conjugate delivery vehicle that is obtained from crystalline CDM-NAG.

In another embodiment, the instant invention comprises a siRNA-polymer conjugate delivery vehicle that is obtained from crystalline CDM-NAG.

In another embodiment, the instant invention comprises a siRNA-polymer conjugate delivery vehicle that is obtained from crystalline CDM-NAG Form I. EXPERIMENTAL DETAILS SECTION-

EXAMPLE 1

Synthesis of CDM-tsfAG

-NAG can be synthesized according tojihe method outlined below.

CDM-NAG

NAG

Acetylation of galactosamine hydrochloride

To a- 3 -necked, 12 L flask fitted with N ₂ inlet, temperature probe, and addition funnel was added 1 (452 g, 2.10 mol) and pyridine (2035 ml 25.2 mol), and the ^~ mixture was cooled witlran ice bath. Acetic anhydride- (1781 ml, 18.9 mol) was added s-iowly over 30 min maintaining the temperature below 5°C. After stirred overnight (17 hr) at-RT, the mixture was cooled with an ice bath and slowly added water (5.0 L) maintaining- the temperature below 2°C. After aging for Thr in the ice bath, the mixture was filtered to collect solid which was washed with water (10 L). The white solid thus obtained was dried in a -vacuum oven with N ₂ .sweep until the water level was below 500 ppm. (674 g, 1730 mmol) Cbz Protection

CbzCI

.0,

HO' NH ₅ ^' NHCbz

To a 3 -necked, 3 L flask equipped with an overhead stirrer, addition funnel, N ₂ inlet, and temperature probe was added 3 (250 ml, 2340 mmol) and 2-Methyl-THF (1010 ml), and the mixture was cooled within ice- bath. A solution of benzyl chloroformate (167 ml, 1125 mmol) ½~2-Methyl-THF (323 ml) was added slowly over 40 min, maintaining the temperature below t0°C. After aging 1 hr at RT, the resulting milky mixture was added hydrochloric acid (1 M in EtOAc, 90 ml, 90 mmol) and aged for 30 min. White solid was removed by filtration through a pad of Solka Floc/Celite, and the filtrate was concentrated under vacuum to afford 4 as a light yellow oil (228 g, quantitative).

Giycosylation

In a 12 L flask was placed 2 (440 g, 1130 mmol), 4 (297 g, 1243 mmol, LI eq.), and anhydrous DCM (4.4 L, 10 vols) under nitrogen atmosphere. The mixture was added trifluoromethanesulfonic acid (TfOH, 17.3 g, 1 .2 mL, 113 mmol, 0.1 eq.) via a syringe at RT, and then the resulting mixture was warmed to a gentle reflux. After 18 hr, LC showed the reaction was not complete. Additional TfOH (5.1 mL, 0.05 eq.) was introduced via a syringe, and the mixture was refluxed further. After 9 hr, the reflux condenser was replaced with a distillation head, and the mixture was concentrated to about half of the original volume under vacuum-. The resulting DCM- solution was washed successively with aqueous solution of 2CO3 (10 wt%, 780 mL), water (1.0 L), andNaCl solution (20 wt% ₅ 0.75-L), and then dried over anhydrous MgS0 ₄ . After filtration, the filtrate -was placed ^" in a 12 L flask, and the solvent was switched to 2-Me-THF by distillation of DCM with addition of 2-Me-THF until the final volume was ca. 2.64 L, and the residual DCM was below 5% judged ?y 1H-NMR. White solid ^" precipitated slowly during the solvent switch. The resulting slurry was added heptane (Γ320 mL, 3 vols) slowly over 1 hr maintaining the internal temperature at 35°C. After the addition was completed, the -slurry was stirred at the same temperature for 1 hr then allowed to cool to RT slowly. The solid was collected by filtration, washed with 2-Me-THE/heptane (5/3, v/v, 2.0 L, 4.5 vols), and -dried under vacuum with N ₂ sweep affording 5 as a white microcrystalline solid (514 g, 1130 mmol). Deacetylation

In aJ2 L flask was placed 5 (510 g, 879 mmol), ₂ CG ₃ (3 g, 22 mmol, 0.025 eq.), and anhydrous MeOH ^" (5.10 L), and the resulting, mixture was stirred at RT under N ₂ for 7 hr, by which time the reaction mixture became homogeneous. The mixture was added Amberlyst 15 acidic resin (17.6 g, 88 mmol equivalent), and the mixture was stirred at RT for 30 min. The resin was removed by filtration, and the filtrate was concentrated under vacuum until the final volume was 1.40 L. Close to the end of the distillation, the contents in the reaction vessel slowly solidified forming a thick slurry. Maintaining the internal temperature at 35°C, the slurry was added MTBE (4.0 L, 8 vols) slowly over 2 hr. During the addition, the internal temperature increased up to 48°C due to the exotherm. After the addition was completed, the reaction mixture was stirred at 35°C for 1 hr and allowed to cool to RT overnight. The white solid was collected by filtration, rinsed with MeOH/MTBE (1/6, v/v, 2.1 L, 4.1 vols), and dried under vacuum with N ₂ sweep affording 6 as a white powder (364 g, 806 mmol). Hydrogenolysis

A solution of 6 (10.0 g, 21.7 mmol) in anhydrous TFE (30 mL) was stirred in the presence of dry 10 wt% palladium on carbon (0.5 mol%, 115 mg) under ¾ (50 psig) at RT for 5 hr, after which the catalyst was removed by filtration through a pad of Celite (10 g) washing with TFE (30 ml, 3 vols). 1H-NMR analysis showed that the crude contained ca. 16 mol%-of the ca bamic acid impurity. The filtrate was placed in a flask equipped with an overhead stirrer, -a. thermocouple, and a heating mantle, and concentrated under -150 Torr for 1 hr then at 50 Torr until the final volume reached 27 ml maintaining the internal temperature not to exceed 25°C. At the end of the distillation the impurity level was reduced- < 1 mol% judged by 1H-NMR analysis. (If there is still significant impurity left, the distillation should be continued with the addition of fresh TFE).

In a 500ml 3-necked flask equipped with an overhead stirrer and-a thermocouple was placed TFE (6.7 ml = 1 vol), MTBE (26.8 ml - 4 vols), and crystalline NAG (74 mg, 1 % seed load), and the mixture was stirred for 15 min at RT (21°C). The mixture was then added a solution of 7 in TFE (27 ml = 6.7 g NAG + 3 vol TFE) and MTBE (80 ml = 12 vol)

simultaneously using two syringe pumps over 3 hrs maintaining the solvent ratio. The resulting slurry was aged at RT for 1 hr and filtered with the aid of MTBE (27,ml - 4 vols). The solid was dried in a vacuum oven with N ₂ sweep at35°C for overnight.

C DM -NAG

NAG Preparation of CDM-Piv

To a slurry of CDM (20g, 0.108 mol, 1.0 equiv) (in dry iPAc ( F= 50-70 ppm, 5 vol) was added neat Pi Cr(13.36g, 0.11 mol, 1.02 equiv) at -20 °C. While stirring rigorously (at least 700-800 rpm), neat NMM (11 ,21g, 0.1-1 mol, 1.02 equiv) was added over 30-45 min. The temperature was maintained between -20°C and -5°C during the addition.

The resulting slurry was then aged for another 30 min at -5°C, at whic - a 92: 6.1 : 1.85 LCAP ratio (205 nra) of DP: CDM-anhydr-ide dimer: SM was obtained.

The reaction was further allowed to age for 2 more hours at 5°C to give a mixture containing 92.6 LCAP DP, 7 LCAP CDM-dimer and 0.39 SM. The reaction was judged complete -and the slurry was quickly filtered through a fritted funnel and the solid was washed with dry, cold MTBE (KF <50 ppm, 2 vol wi ^' SM).

Preparation of CPM-NAG

To a solution of CDM-Piv in iPAc/MTBE obtained above (21.23 mL, 0.77M, 1,05 equiv) at -5°C was added solid NAG (5g, 15.5 mmol, 1 equiv), followed by dr DMF (KF <50 ppm, 17.5 mL, 3.5 vol). The resulting slurry was then aged at O^C for at least 5 hours, until -a clear light yellow solution was obtained. At this point, the-reaction was typically complete, as analyzed by RPLC (Atlantis HILIC silica column, MeCN:0.1 % H3PCVH2O, 205 nm).

Once the reaction was judged complete, the mixture was concentrated at T<20°C to remove..most volatiles (iPAc, MTBE). The crude mixture was then loaded onto the C-18 reverse phase column liquid chromatography (RPLC on Kromasil or Sunfire columns) for purification (methods will be sent separately) and the collected fractions were lyophilized to give- the product as white amorphous solid.

Alternatively, once the reaction was judged complete, the mixture was cooled to - 10°C - -5°C and 2 vol of EtOAc (wrt SM-NAG) was added, followed by 3 vol of ¾0 (wrt NAG) slowly. At the end of water addition, the resulting biphasic layers were agitated for 5 min at 0°C and allowed to settle. The aqueous layer was separated, washed 3x with 2.5 vol of DCM (wrt NAG), and then purified by C-l 8 reverse phase column chromatography (RPLC on

Kromasil or Sunfire columns) and the collected fractions were lyophilized to afford the product as white amorphous solid. Crystallization

88-90 LCAP, CAD 96.5-97 LCAP, CAD

To dry DME or MeCN (20 vol) at 25°C under N ₂ was added amorphous solid CDM-NAG (22,.6g) all at once. The resulting slurry was seeded with authentic crystalline materials (1%) and then aged at 25°C for 15 hours. At- the end of age, the water content in the solution was brought down from 500 ppm to 200 ppm by azeotropic drying with the same solvent at the same concentration, while maintaining the temperature below 25°C. After confirming a full turnover from amorphous to the crystalline phase, the white slurry was then filtered and the wetcake was washed with 3 vol of the same (cold) solvent (0°C). The filtered solid was then dried under N ₂ atmosphere at RT to give anhydrous crystalline CDM-NAG (Form I) as analyzed by XRPD experiments.

Form I was also crystallized in IP Ac, Toluene, DCM, DCE, Cyclopentyl methyl ether, 2-MeTHF, Trifluorotoluene, 50% DCM and 50% THF, 50% DCE and 50%IPAc, 50% DME and 50% 2-MeTHF, 50% Methyl-Cyclohexane and 50% 1, 4-dioxane, 50% chlorobenzene and 50% DCM, 50% 1, 4-dioxane and 50% 2-MeTHF, 50% FT and 50% chlorobenzene.

Other crystalline forms were identified from the polymorph screen. Form II was crystallized under 100% DME, and Form II-I was crystallized under 100% N,N- dimethylacetamide, or 3:1 DME/DMF.

Characterization of Crystalline CDM-NAG

An XRPD pattern of the crystalline CDM-NAG was generated on a Philips Analytical X'Pert PRO X-ray Diffraction System with PW3050/60 console using a continuous scan from 4 to 40 degrees 2Θ. Copper K l and Ka2 radiation was used as the source-. The experiment was run under ambient conditions. The diffraction peak positions were referenced by silicon which has a 2Θ value of 28.443 degree.

The diffraction peaks of Form I are listed in Table 1 below: Table 1

Stability of Crystalline CDM-NAG Form I

The stability of crystalline CDM-NAG Form I was monitored using the following analytical procedures: impurities by HPLC and water by F. Form I CDM-NAG crystalline material was stored at 40°C/75%RH condition. CDM-NAG amorphous material was stored at 25°C/60%RH condition. The available stability data indicate that the crystalline CDM-NAG Form I is stable when stored at 40°C/75%RH for at least nine weeks, and has a better stability profile compared to -CDM-NAG amorphous material.

The purity of CDM-NAG was analyzed by a reversed phase HPLC method using an Atlantis Hilic Silica column (150 X 4.6mm, 3 μη particle size) or equivalent. The mobile phase components are 0.1% (v/v) trifluoroacetic acid in water (A) and 0.05% (v/v) trifluoroacetic acid in acetonitrile (B). The mobile phase composition is to hold at 95% B initially for 3 min, then a gradient from 95% B to 60% B in 17 minutes, then 60% B to 20% B in 5 minutes, then 20% B to 5% B in 5 minutes. The system was then rerequilibrated at 95% B for 10 minutes. The flow rate was 1.0 mL/min, the injection volume was 10 L and the sample tray temperature was maintained at 5°C. The column temperature was maintained at 10°C. Detection was by UV at 205 nm or by CAD detection. The retention time for CDM-NAG was approximately 10-12 minutes. The sample solution was prepared in acetonitrile to a final concentration of 0.3 mg/mL. Table 2: Summary of Stability studies for Crystalline CDM-NAG Form I

Table 3: Summary of Stability studies for Amorphous CDM-NAG