Stabilized 1alpha-hydroxy vitamin d

The invention provides a stabilized Ια-hydroxy vitamin D (SHVD) which is particularly well suited for pharmaceutical formulations.

2001278956 15 Jun 2006

STABILIZED la-HYDROXY VITAMIN D

This invention relates a stabilized la-hydroxy vitamin D (SHVD) which is particularly well suited for pharmaceutical formulations.

In this specification where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date publicly available, known to the public, part of the common general knowledge or known to be relevant to an attempt to solve any problem with which this specification is concerned.

Vitamin D compounds having a hydroxy group at the la-position have had considerable attention in recent years because of their strong vitamin D activity. These activated vitamin D compounds are, however, also known as being chemically unstable, particularly under exposure of light and in the presence of oxygen, and as having poor storage stability at higher temperatures. The compounding of activated vitamin D into a pharmaceutical formulation exacerbates these stability problems.

Despite recognition and study of various aspects of the problem as well as prior art attempts to stabilize pharmaceutical compositions of activated vitamin D, the prior art has produced very little in the way of a la-hydroxy vitamin D form that has a specific chemical and physical profile which provides for a stabilized compound useful for pharmaceutical formulations. It has now been found that a stabilized la-hydroxy vitamin D with heretofore unknown technical properties is surprisingly stable compared to known forms of activated vitamin D.

FIG. 1 is an exemplary reaction scheme for the synthesis of la-OH-D2.

FIGS 2A and 2B are HPLC chromatograms showing the preparation of SHVD in accordance with the present invention.

The present invention relates to a stabilized la-hydroxy vitamin D form with superior technical properties and superior stability (SHVD). SHVD is particularly well adapted for use in pharmaceutical compositions or formulations. Accordingly, the present invention will now be described in detail with respect to such endeavors; however, those skilled in the art will appreciate that such a description of the invention is meant to be exemplary only and should not be viewed as limitative on the full scope thereof.

2001278956 15 Jun 2006 la

In the following description of the method of the invention, process steps are carried out at room temperature and atmospheric pressure unless otherwise specified.

As used herein, the termvitamin Dis intended to include vitamins D3 and D4 and related analogs. The temi'la-hydroxy vitamin Drefers to a vitamin D compound or analog that has a substituted hydroxy group in at least the carbon-la position of the A ring

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-2of the compound. Those compounds that are substituted only with a hydroxy in the laposition, e.g., la-hydroxyvitamin D₂ and Ια-hydroxyvitamin D3, are pro-drugs as they undergo further hydroxylation to form metabolically active vitamin D compounds. Those compounds that are hydroxylated in the 24 or 25 position as well as the la position are typically metabolically active vitamin D compounds, i.e., la, 24-dihydroxyvitamin D₂ and la, 25-dihydroxyvitamin D3. The term “active vitamin D” or “activated vitamin D” referees to a vitamin D compound that is hydroxylated in at least the la, 24 or 25 position. As used herein, the term “stabilized Ια-hydroxy vitamin D” or “SHVD” is not meant to include certain commercially available vitamin D compounds, i.e., excludes lahydroxyvitamin D3, la,25-dihydroxyvitamin D₃, 1,24-dihydroxyvitamin D₃, 22-oxa-l,25dihydroxyvitamin D3 (OCT), paricalcitol (a 19-nor-vitamin D₂) and calcipotriol. The term “non-SHVD” refers to Ια-hydroxyvitamin D compounds that do not meet the purity profile and other characteristics described herein for SHVD.

SHVD of the present invention is crystalline, substantially solvent free, storage stable and well-suited for modem therapy formulations. Studies have demonstrated that SHVD with the technical properties found in the present invention is surprisingly stable, particularly its reduced rate of conversion to the corresponding previtamin form on heat challenge or elevated temperature. SHVD in accordance with the present invention shows virtually no degradation after 6 years storage at -70°C. This corresponds to excellent storage stability when formulated in a pharmaceutical dosage form, e.g., a soft gelatin capsule. The SHVD of the present invention does not require low oxygen or inert atmosphere for storage as has been advanced by some prior art stabilized formulations.

SHVD of the present invention is also characterized in that it has an ultraviolet spectrum peak maximum (k_max) at about 265 nm with a molar extinction coefficient, Σ₂65ηπι of 17490 and a minimum (λπϋη) at about 227 nm, and has a specific purity profile. SHVD has a purity equal to or greater than 98% weight-base (i.e., at least 98%) by high performance liquid chromatography (HPLC), has residual solvents of 0.5% or less, has total impurities of 1.5% or less, and has no single impurity greater than 0.5% by HPLC. The impurities include czs-la-OHTL and ύώ-1β-0ΗΟ₂. While not wanting to be bound by any particular theory, it is believed that the remarkable stability of SHVD is related to its being substantially solvent free and substantially free of impurities.

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-3The SHVD in accordance with the present invention is further characterized by a substantially reduced rate of conversion to the corresponding previtamin upon heat challenge when compared to the same non-SHVD. This stability of SHVD with respect to conversion to the corresponding previtamin D is especially surprising. A la-hydroxy vitamin D compound is in thermal equilibrium with its previtamin form, i.e., they are thermal isomers of each other.

Byway of example, the equilibrium for la-OH-D₂ is illustrated below.

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The la-hydroxyprevitamin D₂ has a characteristic ultraviolet spectrum peak maximum (Z_max) at 259 nm. Storage of the Ια-hydroxy vitamin D form in solution or at elevated temperatures produces the Ια-hydroxy previtamin D form. The isomers are in equilibrium with each other even at room temperature. Increased temperature shifts the equilibrium to the formation of the previtamin.

The SHVD of the present invention shows reduced rate of conversion to lahydroxy previtamin D compared to corresponding non-SHVD compounds under the same conditions, e.g., a heat challenge. The heat challenge is suitably an elevated temperature held in air for a sustained period, which period may be up to 10 days. The elevated temperature is suitably about 60° C. Upon such heat challenge the SHVD of the present

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-4invention has a lower rate of conversion compared to that for a non-SHVD compound under the same conditions.

Ια-hydroxy vitamin D compounds may be prepared by any of the known methods of synthesis. It has been found that certain synthetic pathways provide a compound that, upon purification, may have the superior technical properties of the SHVD of the present invention. An exemplary synthetic scheme is shown in Fig. 1 illustrating the synthesis of Ια-hydroxyvitamin D2 (also known as doxercalciferol). Generally, the synthesis includes conversion of a starting material vitamin D (i.e., the compound that is to be hydroxylated in the la-position) to the cyclovitamin form, hydroxylation of the cyclovitamin in the laposition, reconversion of the hydroxylated cyclovitamin to the cis and trans forms of the vitamin, and conversion of the trans form to the cis form. The hydroxy group in the 3position is protected, suitably by conversion to a tosylate as a first step in the synthesis.

By way of example, as shown is Fig. 1, vitamin D2 (or ergocalciferol) is the starting material. Vitamin D₂ is first tosylated in the 3-position to form vitamin D₂ 3-tosylate. The tosylate is then converted to the cyclovitamin that is then Ια-hydroxylated. The hydroxylated cyclovitamin is then converted to cis and trans Ια-hydroxyvitamin D2. The trans isomer is irradiated with ultraviolet light to convert to the cis isomer, i.e., lahydroxyvitamin D2.

The resulting product is purified by column chromatography. The column-purified Ια-hydroxyvitamin D2 is then recrystallized from an organic solvent, e.g., methyl formate. The recrystallized crystals are then vacuum oven dried to a residual solvent content of &lt;0.5% to provide SHVD. The SHVD in accordance with the present invention is particularly satisfactory when successive (generally at least two) recrystallizations are performed. Suitable solvents, in addition to methyl formate, include ethyl formate, ethyl acetate, acetone, methylethylketone, hexane, 2-propanol-hexane, pentane, heptane, diethyl ether, diisopropyl ether, methanol, ethanol acetonitrile, and combinations thereof.

The SHVD of the present invention is suitably used in pharmaceutical formulations, such as for oral use, e.g., soft gelatin capsules, solutions, tablets. The concentration of active ingredient in convenient unit dosage form is 0.5 pg to 25 pg, with a weekly dose of between 1 pg and 100 pg/week. In a soft gelatin formulation, the capsule fill suitably contains SHVD of the present invention which is dissolved in a

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-5pharmaceutically acceptable oil, e.g., fractionated coconut oil, and includes an antioxidant which may be, for example, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or vitamin E. The capsule shell suitably contains gelatin, glycerin, titanium dioxide and coloring agent. The fill is typically about 58-59% by weight of the whole capsule.

The present invention is further explained by the following examples which should not he construed by way of limiting the scope of the present invention.

In the examples, UV spectra were taken on a Shimadzu UV 160V, IR spectra on a Analect DS-20, and NMR on a SEOL NMR spectrometer operating at 400 MH3 or like instruments known in the art.

Preparation of stabilized Ια-hydroxyvitamin D2 (SHVD2)

Example 1: Preparation of ία-hydroxy vitamin Tb crude drug substance fl) Preparation of ergocalciferol tosylate(2)

To 100 g of ergocalciferol (vitamin D2) was added 3.1 g of 4dimethylaminopyridine, 100 mL anhydrous pyridine, and 340 mL anhydrous dichloromethane. The flask contents were stirred until the internal reaction temperature was &lt; 5°C. Then, 122.6 g of p-toluenesulfonyl chloride was added, and the mixture stirred until all the solids were dissolved. The reaction was allowed to proceed with slow stirring under argon. After 32 hours, the completion of the reaction was monitored by TLC (silica gel plates; ethyl acetate in hexanes; 20% phosphomolybdic acid in ethanol).

Over a period of approximately 20 minutes, the reaction mixture was dripped into a chilled beaker containing saturated sodium bicarbonate. The reaction mixture was rinsed into a separatory funnel with 900 mL isopropyl ether. The mixture was extracted with isopropyl ether by shaking, and the organic and aqueous layers separated. The isopropyl ether extracts were washed with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride. The combined isopropyl ether extracts were dried over 250 g of anhydrous magnesium sulfate and filtered. The isopropyl ether extracts were combined and concentrated in vacuo. The crude tosylate was suitable for use in the solvolysis below.

(2) Preparation of 6(R)-methoxy-3,5-cyclovitamin D?(3)

To a stirred mixture of 1950 mL of dry methyl alcohol and 500 g of sodium bicarbonate was added the ergocalciferol tosylate from Step 1 dissolved and rinsed in 250 mL dichloromethane. The stirred mixture was refluxed under an argon atmosphere for 18WO 02/06218

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-622 hours with an internal temperature of about 56 °C. After about 20 hours, the extent of reaction was monitored by TLC (silica gel; solvents 20% phosphomolybdic acid in ethanol).

The reaction mixture was cooled to 25-30°C, and the sodium bicarbonate was removed by filtration. The reaction flask, filtered sodium bicarbonate and filtration flask were rinsed with 1000 mL of isopropyl ether. The organic filtrate was concentrated in vacuo, maintaining the temperature of the bath at 50 °C. The semi-solid residue was diluted with isopropyl ether. This isopropyl ether solution was washed three times with water and once with saturated sodium chloride, and then dried over magnesium sulfate. The magnesium sulfate was removed by filtration and rinsed with isopropyl ether. The combined organic filtrate and the isopropyl ether rinse of the filtration flask were concentrated in vacuo for 4-18 hours to yield the 6(R)-methoxycyclovitamin.

(3) Preparation of la-tivdroxv-6fR)-methoxv-3.5-cvclovitaniin Dg(4)

To a stirred suspension of 12.3 g of freshly ground selenium dioxide in 1500 mL of 1,2-dichioroethane was added 81 mL of anhydrous /-butyl hydroperoxide (5-6 M in decane). After stirring under positive argon atmosphere for 3-4 hours at ambient temperature, 18 mL of pyridine was added. The 6(R)-methoxy-3,5-cyclovitamin D2 from Step 2, dissolved in 1,2-dichloroethane, was added dropwise into the chilled reaction flask followed by a rinse of 1,2-dichloroethane. The reaction continued with stirring at an internal temperature of &lt;5°C, and the reaction progress was monitored at 15-minute intervals by TLC. The reaction was considered complete when the starting 6(R)-methoxy3,5-cyclovitamin D₂ was either no longer visible or did not appear to change between two consecutive monitoring thin layer chromatograms.

The reaction was quenched with 10% sodium hydroxide. The aqueous and organic layers were separated, and the aqueous layer was extracted with isopropyl ether. This isopropyl ether extract was added to the organic layer from the reaction mixture. The combined extracts were dried with anhydrous magnesium sulfate and filtered. The flask and residual magnesium sulfate were rinsed with isopropyl ether, and the combined isopropyl ether extracts and isopropyl ether rinse of the filtration flask were concentrated in vacuo.

The product was dissolved in 500 mL of 5% ethyl acetate in hexanes, purified on a silica gel (60/230-400 Mesh) in a column (75 mm I.D.), and eluted with approximately

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-74000 mL 5% ethyl acetate in hexanes, followed by 8000 mL 20% ethyl acetate in hexanes under positive argon pressure. Fractions of approximately 200 mL each were collected during the elution. The fractions were analyzed by silica gel TLC. The fractions containing the la-hydroxy-6(R)-methoxy-3,5-cyclovitamin D2 were identified, combined, and concentrated in vacuo.

(4) Preparation of cis- and trans- Ια-hydroxwitamin D₂(5 and 6)

A stirred solution of the la-hydroxy-6(R)-methoxy-3,5-cyclovitamin D2 from Step 3 in 120 mL of dimethyl sulfoxide and 130 mL of glacial acetic acid was heated to about 60° C and stirred at this temperature under an argon atmosphere for 60 minutes. The reaction was monitored periodically by silica gel TLC for the consumption of the reactant la-hydroxy-6(R)-methoxy-3,5-cyclovitamin D2. Upon completion, the reaction was cooled to 20 - 25°C. The reaction was quenched by pouring the mixture and an isopropyl ether rinse into saturated sodium bicarbonate and water held at a temperature &lt;10°C. Isopropyl ether was added and the mixture was stirred. This mixture was then rinsed into a separatory funnel with 950 mL isopropyl ether and extracted by shaking. The aqueous and organic layers were separated. The aqueous layer was extracted with isopropyl ether. The organic layer from the reaction mixture was combined with this isopropyl ether extract and washed with saturated sodium bicarbonate three times, water three times, and saturated sodium chloride once. The organic extracts were dried over magnesium sulfate, filtered, and the cake magnesium sulfate and filtration flask were rinsed with isopropyl ether. The combined isopropyl ether filtrates were concentrated in vacuo, maintaining the temperature of the bath at about 35 °C. Residual solvent was removed from the remaining residue by vacuum.

The product, dissolved in 350 mL dichloromethane, was purified on silica gel (60/230-400 mesh) in a column (75 mm I.D.) eluted with 40% ethyl acetate in hexanes under argon. Fractions of approximately 125 mL were collected. The fractions were analyzed by silica gel TLC eluted with 40% ethyl acetate in hexanes. The fractions containing cis- and trans-la-OH-D) were identified, combined, and concentrated in vacuo.

(5) Preparation of cfe-la-hvdroxyvitamin D?(5)

The cis- and Zrans-la-OH-D2 mixture from Step 4 and 1.1 g of 9-acetylanthracene were dissolved in 2000 mL methyl alcohol and transferred with methyl alcohol rinses totaling 2000 mL to a photolysis chamber and diluted with additional methyl alcohol to a

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-8total of 13.7 L. The photolysis was run to completion, approximately 2-6 hours. The product solution and 400 mL methyl alcohol rinse were concentrated in vacuo, maintaining the temperature of the bath around 35 °C.

The residual solvent was removed under high vacuum. The product, dissolved in dichloromethane, was purified on silica gel (60/230-400 mesh) in a column (75 mm I.D.) eluted with 40% ethyl acetate in hexanes and ethyl acetate under argon pressure. Fractions (125 mL) were collected with 40% ethyl acetate in hexanes elution, then fractions (200 mL) were collected with 100% ethyl acetate elution. The fractions were analyzed by silica gel TLC eluted with 40% ethyl acetate in hexanes. The fractions containing the czs-laOH-Di were identified, combined, and concentrated in vacuo, The residual solvent was removed under high vacuum. A typical yield for this step was 15-30 g (15-30% overall yield from the starling material, ergocalciferol).

Example 2: Purification of crude drug substance to yield SHVD₂

The column-purified ezs-la-hydroxyvitamin D₂ (crude drug substance) was successively recrystallized from methyl formate.

For each recrystallization, the column-purified crude drug substance (usually 1530 g) was resuspended in 3500 mL methyl formate in a round bottom flask. This flask was attached to a rotary evaporator with a chilled condenser and slowly rotated in a water bath at 40°C until the solids dissolve. The solution was concentrated in the rotary evaporator, maintained at 40°C until crystals began to form. When crystals were observed, the flask was placed in a -20°C freezer to cool for 48-72 hours.

After the crystals obtained from the recrystallization had air dried for 5 - 10 minutes, the crystals were milled to a powder, and then, transferred to a crystallization dish. The dish with crystals was placed in a vacuum oven set at about 55 °C for 72 -120 hours. The amount of drug substance obtained (SHVD₂) was usually 5 - 10 g, which represents a yield of 5 -10 % of the starting material, ergocalciferol.

Evidence that the purification procedure improved the purity of the crude drug substance was demonstrated by the HPLC chromatogram in FIG. 2. The first chromatogram (A) was obtained with column-purified crude drug substance. Chromatogram (B) shows the final product. As shown in FIG. 2, the levels of the

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-9impurities with retention times of approximately 37.7 and 41 minutes were reduced by recrystallization.

Example 3: Determination of residual solvents of SHVD2

A method for quantitating residual solvents with toluene was used. The target analytes included methanol, methyl formate, methylene chloride, hexane, diisopropyl either, 1,1-dichloroethane and ethyl acetate.

A gas chromatographic method was used with helium as the carrier gas, split injections/run, and run in single ion mode (SIM mode). It required a 60 m x 0.25 mm ED, DB-624 capillary column with a film thickness of 1.4 pm, a split/splitless injector, a gas chromatograph capable of ramping from 50 °C to 250 °C, and a mass selective detector. The chromatograph was set to detect ions 31, 32, 43, 45, 49, 60, 61, 62, 84, 86 and 87 at a dwell time of 25 ms per ion.

An external calibration was used, consisting of a blank, matrix blank and a 4-point curve. A curve was prepared at concentration of 1 pg/g, 10 pg/g and 50 pg/g. A 10 mg sample of ezs-la-OH-D₂ was prepared in toluene. Results from the samples were calculated using the linear curve generated above. The lower limit for all analytes was 100 pg/g. The results indicated that the amount of residual solvents in the cz's-I0C-OH-D2 (SHVD₂) was &lt;0.5%

Example 4: Storage stability testing of STIVD₂

The stability of samples from four lots of SHVD2 was examined at 6-month intervals during long-term storage at -70°C. All lots were stored in the container-closure system selected as packaging for the drug substance. Stability was assessed by examining samples for changes in (1) weight percent I01-OH-D2 content, (2) percent related substances content, and (3) impurity profile, using HPLC. In addition, all lots were examined for changes in UV spectrum by UV spectrophotometry.

Results of this study showed no significant differences in mean weight % la-OHD₂ content between baseline and 6 or 12 months. The weight % la-OH-D2 content for all lots examined was >98.0% at all time points. Impurity profile chromatography

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-10demonstrated no new peaks at 6 or 12 months relative to baseline. The percent related substances remained &lt;2.0%.

Example 5: Preparation of SHVD2 soft gelatin capsule fill

BHA was added to fractionated coconut oil (FCO), heated and stirred until the BHA was completely dissolved. This solution was then cooled. SHVD₂ was accurately weighed into a suitable container, dehydrated alcohol was added, and the solution was stirred until the SHVD2 was dissolved. FCO was weighed into a stainless steel mixing tank. The BHA/FCO solution was added to the FCO while stirring. Then SHVD2/dehydrated alcohol solution was added and the solution was stirred until uniform. The vessel containing the SHVD₂/dehydrated alcohol solution was rinsed with dehydrated alcohol and the rinses were added to the FCO/SHVD₂/dehydrated alcohol mix. The mixing tank containing this mix was sealed, a vacuum was applied, and the contents were mixed (deaerated). The resulting solution was transferred through filters to receivers and blanketed with nitrogen.

Example 6: Stability testing: SHVD₂ v. non-SHVD₂

Approximately 1 mg of SHVD₂ or non-stabilized Ια-hydroxyvitamin D₂ (nonSHVD₂) is placed in each of 9 (12 X 75mm) test tubes. The test tubes are placed in an oven at about 60°C. One test tube of each of the two vitamin D₂ preparations is removed at 0, 3, 6, 9, 12, 18, 24, 36 and 48 hours of incubation. The contents in each test tube are dissolved in ethanol to produce a 1 mg/mL solution. Each solution (10pL) is analyzed by the following HPCL conditions: a YMC Pack C8 column (4.6 x 250 mm, 5pm) with a mobile phase of acetonitrile: methanol: water (60:20:20) at a flow rate of 1.0 mL/min. The rate of appearance of the peak migrating with a relative retention time of approximately 0.9 compared with the Ια-hydroxyvitamin D₂ peak is greater with the non-SHVD₂ than with SHVD₂.

Example 7: Stability testing: SHVD₂ v. commercially available la-OH-D₂

Approximately 1 mg of SHVD₂ or commercial Ια-hydroxyvitamin D₂ is placed in each of 2 (12 x 75 mm) test tubes. One test tube from each of the two preparations is

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-11placed in an oven at about 60°C. For analysis, the contents in each test tube are dissolved in ethanol to produce a 1 mg/mL solution. Each solution (10pL) is analyzed by the following HPLC conditions: a YMC Pack C8 column (4.6 x 250 mm, 5 am) with a mobile phase of acetonitrile: methanol: water (60:20:20) at a flow rate of 1.0 mL/min. The rate of appearance of the peak migrating with a relative retention time of approximately 0.9 compared with the Ια-hydroxyvitamin D₂ peak is greater with the commercial lahydroxyvitamin D₂ than with SHVD₂.

Example 8: Stability testing of SHVD₂ v. non-SHVD₂

Approximately 1 mg of stabilized Ια-hydroxyvitamin D₂ (doxercalciferol) (SHVD₂) was placed in each of two test tubes (12 x 75 mm). One sample was placed in a dessicator containing an atmosphere of ethanol vapors to increase the residual solvent content of the material to form non-stahilized doxercalciferol (non-SHVD₂). The material in the dessicator was heated at 60°C for 48 hours. The other sample was not subjected to the ethanol atmosphere, thereby retaining its stabilized form, but was heated at 60° C for 48 hours. The contents in each test tube were dissolved in ethanol to produce a 1 mg/mL solution. Each solution (10 pL) was analyzed by the following HPLC conditions: a YMC Pack C8 column (4.6 x 250 mm, 5 pm) with a mobile phase of acetonitrile: methanol: water (60:20:20) at a flow rate of 1.0 mL/min. The rate of appearance of the peak migrating with a relative retention time of approximately 0.9 compared with the doxercalciferol peak is greater with the non-SHVD₂ material than with SHVD₂.

Example 9: Stability testing: la,24(S)-dihydroxyvitamin D₂ containing > 0.5% residual solvents (a non-SHVD₂)

Approximately 1 mg of la,24(S)-dihydroxyvitamin D₂ containing >0.5% residual solvents (non-SHVD) was placed in a capped vial and heated at 60°C for 48 hours. The contents of the vial were dissolved in ethanol to produce a 1 mg/mL solution. The solution (10pL) was analyzed by the following HPLC conditions: a YMC Pack Pro C18 column (4.6 x 150 mm, 5 pm) with a mobile phase of acetonitrile: water (50:50) at a flow rate of 1.0 mL/min. After heating the non-SHVD, a compound with a relative retention time of approximately 0.85 compared with standard la,24(S)-dihydroxyvitamin D₂ was observed.

- 12 In summary, the present invention provides SHVD, a stabilized Ια-hydroxyvitamin D form with superior technical properties ande superior stability. The novel form of the present invention is crystalline, substantially solvent free, stable and well suited for modern therapy formulations.

While the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate the various modifications, including varioations, additions and omissions, which may be made in what has been described. Accordingly, it is intended that these modifications also be encompassed by the present invention and that the scope of the present inventio be limited solely by the broadest interpretation that lawfully can be accorded the appended claims.

The word ‘comprising’ and forms of the word ‘comprising’ as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions.

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