Technical Field

The present invention relates to the field of 7-dehydrocholesterol.

Background of the invention

7-Dehydrocholesterol is a key intermediate used in the synthesis for cholecalciferol (=vitamin D3).

7-Dehydrocholesterol, however, has limited storage stability and degrades during extended storage.

Summary of the invention

Therefore, the problem to be solved by the present invention is to increase the storage stability of 7-dehydrocholesterol, respectively to decrease the degra dation of 7-dehydrocholesterol upon storage. It has been, surprisingly, found that 7-dehydrocholesterol-hemihydrate shows polymorphism. 7-Dehydrocholesterol- hemihydrate offers a solution to this problem. 7-Dehydrocholesterol-hemihydrate can be easily formed from 7-dehydrocholesterol. Furthermore, 7-dehydrocholes terol can easily be regenerated in high yields from 7-dehydrocholesterol-hemi- hydrate. It has been found that 7-dehydrocholesterol-hemihydrate shows significantly increased storage stability, respectively shows a significant decrease in the degradation.

Further aspects of the invention are subject of further independent claims. Particularly preferred embodiments are subject of dependent claims.

Detailed description of the invention

In a first aspect, the present invention relates to 7-dehydrocholesterol- hemihydrate.

The ability of a substance to exist in more than one crystalline form is generally referred to as polymorphism and these different crystalline forms are usually named "polymorphs" and may be referred to by certain analytical proper ties such their X-ray powder diffraction (XRD) patterns. In general, polymorphism reflects the ability of a molecule to change its conformation or to form different intermolecular and intramolecular interactions. This can result in different atom arrangements that are reflected in the crystal lattices of different polymorphs. However, polymorphism is not a universal feature of solids, since some molecules can exist in one or more crystal forms while other molecules cannot. Therefore, the existence or extent of polymorphism for a given compound is unpredictable.

The different polymorphs of a substance show different crystal lattice energies and thus each polymorph typically shows one or more different physical properties in the solid state, such as density, melting point, colour, stability, dissolution rate, flowability, compatibility with milling, granulation and compacting and/or uniformity of distribution [See, e.g., P. DiMartino, et al., J. Thermal Anal. 48:447-458 (1997)]. The capacity of any given compound to occur in one or more crystalline forms (i.e. polymorphs) is unpredictable as are the physical properties of any single crystalline form. The physical properties of a polymorphic form may affect its suitability in pharmaceutical formulations. For example, those properties can affect positively or negatively the stability, dissolution and bioavailability of a solid-state formulation, which subsequently affects suitability or efficacy of such formulations in treating disease. Likewise, the physical properties of a polymorphic form may also affect the further processability of a compound

An individual polymorph having one or more desirable properties can be suitable for the development of a pharmaceutical formulation having desired property(properties). Existence of a compound with a polymorphic form(s) having undesirable properties can impede or prevent development of the polymorphic form as a pharmaceutical agent.

It has been first found that 7-dehydrocholesterol (=DHC) shows polymor phism. Particularly, it has been found that 7-dehydrocholesterol-hemihydrate is a specific polymorph of 7-dehydrocholesterol. 7-Dehydrocholesterol-hemihydrate is a crystalline compound in which water is stoichiometrically bound to 7-dehydro cholesterol. In fact, 7-dehydrocholesterol-hemihydrate comprises one molecule of water (- 'bound water") per two molecules of 7-dehydrocholesterol in its crystalline structure. The bound water is also called "water of crystallization" or "water of hydration" by the person skilled in the art.

It has been found that 7-dehydrocholesterol-hemihydrate exhibits maxima of the intensities (in counts per second) measured using X-ray powder diffraction (XRD) in the 2 theta (2Q) range of

3.07 - 3.15 °,

6.26 - 6.34°,

6.52 - 6.60°,

13.10 - 13.18°,

16.23 - 16.31 °,

18.95 - 19.03°, and

19.40 - 19.48°.

The X-ray powder diffraction (XRD) has been measured in the reflection mode at 295 K using CuKal as radiation source. The measurement has been carried out in the range of 2 - 50° 2Q.

The most important feature of the maxima is their position, and not the value of the intensity. Therefore, the maxima are referred to as 2 theta maxima in the following. The exact intensity (in counts per second) of the maxima mentioned above may vary between the individual XRD measurements. However, the intensity (in counts per second) of the 2 theta maxima can be used for further characterization of 7-dehydrocholesterol-hemihydrate. The 2 theta maximum in the range of 16.23 - 16.31 ° has particularly the highest intensity (in counts per second) in the whole measured powder X-ray diffractogram (XRD) of the composition.

Furthermore, the intensity (in counts per second) of the 2 theta maximum in the range 3.07 - 3.15° is typically at least 10 %, particularly at least 20 %, of the intensity (in counts per second) of the 2 theta maximum in the range of 16.23 - 16.31 °.

Furthermore, the intensity (in counts per second) of the 2 theta maximum in the range of 19.40 - 19.48° at least 10 %, preferably at least 20 %, more preferably 55 - 75 %, of the intensity (in counts per second) of the 2 theta maximum in the range of 3.07 - 3.15 °.

The powder X-ray diffractogram (XRD) of 7-dehydrocholesterol- hemihydrate as measured is shown in figure 1. See the experimental part for further details.

7-Dehydrocholesterol-hemihydrate can be particularly formed from 7- dehydrocholesterol. Particularly, it can be formed by crystallization or precipita tions from solutions of 7-dehydrocholesterol containing water. Particularly 7- dehydrocholesterol-hemihydrate is obtained by crystallization from a solution 7- dehydrocholesterol in a suitable hydrocarbon, particularly an alkane, preferably hexane or heptane, and water. The crystallization is particularly realized by cooling a hot (i.e. in the range of between 35°C to 60°C) solution of 7- dehydrocholesterol in heptane or hexane and water.

More particularly 7-dehydrocholesterol-hemihydrate is obtained by crystallization from a solution 7-dehydrocholesterol in a suitable polar solvent with a boiling point which is significantly lower than 80°C, particularly acetone or methyl ethyl ketone, and water. The crystallization is particularly realized by cooling a hot (i.e. in the range of between 35°C to 55°C) solution of 7-dehydrocholesterol in acetone and water.

It has been found that 7-dehydrocholesterol-hemihydrate is formed even in an excess of water. Particularly, it has been observed that under all variations of conditions of the experiments undertaken, no formation of 7-dehydrocholesterol- hydrate (DHC · hteO) (corresponding to a molecular ratio of DHC/water = 1 :1 ) has been ever observed.

Despite the fact that 7-dehydrocholesterol-hemihydrate is formed even in an excess of water, it is very advantageous to remove the excess of water to obtain 7-dehydrocholesterol-hemihydrate which is void of excess (i.e. unbound) water.

Particularly, it has been observed that the 7-dehydrocholesterol-hemi- hydrate can be prepared by a process comprising the following steps:

a) providing an initial composition consisting essentially of a mixture of 7- dehydrocholesterol and water wherein the molar ratio of 7-dehydro- cholesterol to water is between 1.8:1 and 0.1 :1

b) removing water from the mixture of step a) to an extend that a

composition is formed in which the molar ratio of 7-dehydrocholesterol and water is strictly between 2.1 :1 and 1.9:1 , preferably 2:1.

Any amounts of 7-dehydrocholesterol mentioned in this document are determined by High-Performance Liquid Chromatography (HPLC).

Specifically, the amounts of DHC are determined by High-Performance Liquid Chromatography (HPLC) using a HPLC Agilent 1200 with a HPLC column Supelcosil ABZ+/Sigma of 250 mm length, 4.6 mm internal diameter, 5 micrometre particle size, measured at 30°C, using a detector DAD at wavelength of 212 nm, 270 nm and 300nm. The eluent was acetonitrile (A) / methyl tert. -butyl ether (B) in a gradient program:

0 min A/B= 70/30 I ml/min

25 min A/B= 50/50 I ml/min

28 min A/B= 90/10 1 ml/min

30 min A/B= 90/10 1 ml/min

The calibration has been performed by dissolving 5 exactly weighed samples of the crystal in the range of 1 to 20 mg in a solvent consisting of acetonitrile / methyl tert. -butyl ether 60/40.

Under the above measuring conditions, the 7-dehydrocholesterol-hemi- hydrate dissociates into the 7-dehydrocholesterol and water. Any amounts of water mentioned in this document are determined by Karl Fischer Titration. Particularly, Metrohm 874 / Metrohm 851 using a Metrohm Pt double electrode and Hydranal Medium K titer solution were used.

Said initial composition is composed of crystals wetted by water. "Wetted" in this context means that at the surface of the crystal some free, liquid water (- 'unbound water") is observable.

Therefore, in step a) the molar ratio of 7-dehydrocholesterol to water as determined by HPLC resp. Karl Fischer titration, as mentioned before is between 1.8:1 and 0.1 :1. This corresponds to a weight fraction of water of between 2.5 and 32 % by weight.

The unbound water is then removed in step b).

In step b) the unbound water is removed from 7-dehydrocholesterol-hemi- hydrate. The water in the crystal structure of 7-dehydrocholesterol-hemihydrate is bound (- 'bound water" or "water of crystallization")) by hydrogen bonds within the crystal lattice. Flowever, the level of binding force of water to the 7-dehydrocholes- terol in the 7-dehydrocholesterol-hemihydrate is limited. It is important to note that, for the purpose of this invention, it is exactly this reversibility of binding of water to DFIC which is important. Flence, for obtaining optimal results the removal of excess (i.e. unbound) water is preferably removed under very mild removal conditions to avoid removal of bound water.

For example, the removal of water can be by simple drying, i.e. exposure to air at ambient pressure. This is typically performed by blowing a gas mixture, particularly air, over the crystals. The removal can be accelerated by using warm gas respectively air. Particularly, the step b) of water removal can be performed by drying the crystals after filtration of the crystals on the filter paper, or on a Nutsche filter (Biichner funnel) or on a glass frit (sintered glass) filter.

In another embodiment, the water is removed by heating under reduced pressure. Typically, the water is removed by heating to a temperature of between 50 and 80°C, particularly of between 60 and 70°C, and a pressure of between 0.1 and 15 mbara, particularly of between 1 and 10 mbara.

It is important to stress, that, particularly when performed under elevated temperature and/or reduced pressure, care needs to be taken to assure that the removal is not performed so that too much water (i.e. also part of the bound water = water of crystallization) is removed. Therefore, it is essential that the removal of water in step b) is done so that the excess of water (i.e. the unbound water, i.e. water wetting the crystals) is removed but not more, so that the amount of the molar ratio of 7-dehydrocholesterol and water, determined as indicated above by HPLC and Karl Fischer titration, is between 2.1 :1 and 1 .9:1 , particularly between 2.05 : 1 and 1 .95 : 1 , preferably 2.0:1 .0. This corresponds to a weight fraction of water of between 0.022 and 0.024, particularly between 0.0223 and 0.0234, preferably of 0.0228.

In an even further embodiment, the amount of the molar ratio of 7- dehydrocholesterol and water, determined as indicated above by HPLC and Karl Fischer titration, is between 2.0:1 and 1 .9:1 , particularly between 2.0 : 1 and 1 .95 : 1 , preferable 2.0:1 .0. This corresponds to a weight fraction of water of between 0.0228 and 0.024, particularly between 0.0028 and 0.023, preferably of 0.0228.

It has been observed, that when the ratio of 7-dehydrocholesterol to water is higher than 2.1 the storage stability strongly decreases. In other words, when the ratio of 7-dehydrocholesterol to water is higher than 2.1 , the degradation during storage strongly increases, i.e. 7-dehydrocholesterol is degraded

significantly into degradation products. The decomposition of 7-dehydrocholes terol -hem i hydrate can be evidenced by changes in the powder X-ray diffractogram (XRD). It has been observed in the present invention that the formation of 7-de- hydrocholesterol-hemihydrate is a very efficient method of decreasing the degradation of 7-dehydrocholesterol upon storage, respectively of increasing the storage stability of 7-dehydrocholesterol. The formation of the of 7-dehydrocholesterol-hemihydrate has been described above in great detail.

The term "stabilization" in this application is understood in this document, 7-dehydrocholesterol is stabilized towards degradation during storage. In other words, a low degradation leads to high stabilization and vice versa. This is reflected in that the increase of the storage stability, respectively the decrease of degradation upon storage, is characterized in that the weight ratio DHC20w/DHCo is more than 0.80, particularly more than 0.90. The term DHC20 _W is the amount of 7-dehydrocholesterol after storage of 7-dehydrocholesterol in contact with air and the term DHCo is the amount of 7-dehydrocholesterol in the same sample before storage.

The difference between DHC20 _W and DHCo is due to a degradation of DHC during the storage. Hence, the higher the weight ratio DHC20wl DHCo is, the lower is the amount of any degradation products being formed during the time of storage and, hence, the higher the storage stability is.

It has been particularly useful that the degradation upon storage, respectively the storage stability, is assessed by determining said weight ratio DHC20W / DHCo for a sample being stored at 4°C in contact with air for 20 weeks.

The amount of 7-dehydrocholesterol is determined by HPLC as mentioned before.

Hence, in a further aspect, the present invention related to a method of decreasing the degradation of 7-dehydrocholesterol upon storage of at least 1 week, said method comprises the steps

a) forming 7-dehydrocholesterol-hemihydrate;

b) storing 7-dehydrocholesterol-hemihydrate for an extended time period which is at least 1 week, preferably at least 4 weeks, more preferably at least 20 weeks, before releasing 7-dehydro ^_i cholesterol from 7- dehydrocholesterol-hemihydrate.

Particularly, it has been observed that when more than the amount of unbound water is removed the stability significantly drops. When the removal of any unbound and bound water is essentially complete, 7-dehydrocholesterol anhydrate (DHC anhydrate) is obtained which has been shown to be very unstable and prone to chemical degradation. Said 7-dehydrocholesterol anhydrate has a powder X-ray diffractogram (XRD) which has a significantly different crystal structure as evidenced by significantly different powder X-ray diffractogram (XRD).

A powder X-ray diffractogram (XRD) of the DHC anhydrate is shown in figure 2. See the experimental part for more details.

Said 7-dehydrocholesterol anhydrate shows characteristic 2 theta maxima in the range of

2.63 - 2.93°

5.45 - 5.75°, and

16.11 - 16.41 °.

The 2 theta maximum in the range of 2.63 - 2.93°has the highest intensity (in counts per second) in the whole powder X-ray diffractogram (XRD) of the 7- dehydrocholesterol anhydrate. The X-ray powder diffraction (XRD) has been measured in the reflection mode at 295 K using CuKcd as radiation source in the range 2-50 ° 2Q.

Therefore, in a further aspect, the invention relates to a composition which is obtained from the removal of water from an initial composition consisting essentially of a mixture of 7-dehydrocholesterol and water,

wherein the mixture of 7-dehydrocholesterol and water with an initial molar ratio of 7-dehydrocholesterol to water is 1.8:1 and 0.1 :1 ,

to a final molar ratio of 7-dehydrocholesterol to water of between 2.1 :1 and 1.9:1 , preferable 2:1

wherein the amounts of 7-dehydrocholesterol are determined by High- Performance Liquid Chromatography (HPLC) and the amounts of water are determined by Karl Fischer Titration characterized in that a powder X-ray diffractogram (XRD) of said composition shows 2 theta maxima in the range of 3.07 - 3.15 °,

6.26 - 6.34°,

6.52 - 6.60°,

13.10 - 13.18°,

16.23 - 16.31 °, 18.95 - 19.03°, and

19.40 - 19.48°.

The X-ray powder diffraction (XRD) has been measured in the reflection mode at 295 K using CuKal as radiation source in the range 2-50 ° 2 theta.

All the details are already discussed above in great details for the formation of the 7-dehydrocholesterol-hemihydrate.

Said composition is particularly free of 7-dehydrocholesterol anhydrate. Hence, in the powder X-ray diffractogram (XRD) of said composition particularly no significant, preferably no, 2 theta maxima in the range of

2.63 - 2.93° and

5.45 - 5.75°

are detected.

As mentioned above, 7-dehydrocholesterol-hemihydrate shows an increased storage stability, respectively a decrease degradation, as compared to 7-dehydrocholesterol.

Therefore, it is very advantageous to store and to transport 7-dehydrocho lesterol -hem i hydrate rather than 7-dehydrocholesterol.

A "package" (in German " Packung ") as defined in this document is the combined physical object consisting of a packaging and the packaged good.

The "packaging" (in German "Verpackung"), as defined in this document, also referred as "containment", is the physical object which has an inner hollow space which serves to take up a packaged good and is a physical barrier towards the outer space of the packaging and the environment around the packaging respectively the package. A "transport packaging" is any packaging which is suitable for transport purposes.

The "packaged good" (in German " Packgut ") as defined in this document is the material which is stored in the hollow space of the packaging.

Hence, in a further aspect, the invention relates to a package (1 ) consisting of a transport packaging (2) and 7-dehydrocholesterol-hemihydrate, as already described above in great detail, as a packaged good (3) or part of the packaged good (3), which is localized in the inner space of the transport packaging (2).

The transport packaging is any packaging used for transport. Examples for such packaging are bags, big bags, barrels, canister, drums, cans, bottles, containers, tanks. The transport packaging is preferably sealed and more preferably made out of plastic or metal or of a composite material, particularly a metalized polymer material such as a foil or a paper or cardboard coated by metal or plastic film.

In one of the embodiments, the transport packaging consists of a sealable packaging, i.e. it has at least one opening, and a seal. The seal can be part of the sealable packaging, e.g. linked by a flexible part, or be a separate part. The sealable packaging is filled by the packaging good and then the seal is sealing the opening in the sealable packaging. After the transport of the package to a different location, the seal is removed and the packaging good can be partially or completely removed from the packaging. Preferably the seal can be re-used for sealing the packaging again. Non-limiting examples for seals are caps, lids, valves, cover plates or adhesive foils.

A specific example for such a preferred embodiment is a bottle ("sealable packaging") which a cap ("seal"). 7-dehydrocholesterol-hemihydrate can be filled into the opening of the bottle into the inside of the bottle. A cap is put, preferable screwed, onto the bottle so that the bottle is completely sealed. After the transport, e.g. shipping, of the sealed bottle filled with 7-dehydrocholesterol-hemihydrate ("package") to a different location, the cap can be removed, the 7-dehydrocholes- terol-hemihydrate can be taken out as packaged goof from the bottle. The open bottle can be closed again by putting the cap again onto the open bottle.

In another embodiment, 7-dehydrocholesterol-hemihydrate is put as packaged good inside the packaging through a temporary opening and then sealed for transport, for example by welding or gluing. After the transport of the package to a different location, the package is broken and the packaging good can be partially or completely removed from the packaging.

A specific example for such a preferred embodiment is a bag which has an opening. 7-dehydrocholesterol-hemihydrate can be filled into the opening of the bag which can be closed, e.g. by thermoplastic welding, to seal the packing. After transport, e.g. shipping, of the sealed bag filled with 7-dehydrocholesterol- hemihydrate ("package") to a different location, bag can be opened, for example by a knife, so that 7-dehydrocholesterol-hemihydrate can be taken out as packaged good from the bag.

The 7-dehydrocholesterol-hemihydrate is placed inside the packaging prior to the transport. Typically, the cavity, i.e. the inner space of the transport packaging, is not completely filled with 7-dehydrocholesterol-hemihydrate.

Therefore, typically a part (e.g. up to 10 %, sometimes up to 20 % by volume) of the volume of the inner space of the transport packaging is filled up with air.

The packaging is suitable for transport.

The transport is typically a transport by car, truck, ship or plane. The invention is very advantageous for long distance transports such as

intercontinental transports. It is particularly also very advantageous for transports of several days to months, particularly in or through tropic climate zones.

The increased storage stability, respectively decreased degradation, can be particularly advantageous in that the needs of special conditions for transport such as cooling may be strongly reduced or even removed when the method of the present invention is used.

It is important to realize that when 7-dehydrocholesterol-hemihydrate is dissolved in a respective solvent, the crystal dissolves and dissociates into 7- dehydrocholesterol and water, and, hence, 7-dehydrocholesterol is released. Any solvent in which 7-dehydrocholesterol, respectively 7-dehydrocholesterol-hemi- hydrate, can be dissolved or solubilized at ambient or elevated temperatures, typically up to 100°C, can be used as respective solvent.

Preferably, the respective solvent is selected from the group consisting of linear, branched or cyclic alcohols, preferably with less than 10 carbon atoms, acetone, methyl ethyl ketone, tetrahydrofuran (THF), methyl tetrahydrofuran (2- MTHF), cyclopentyl methyl ether (CPME), methyl tert.- butyl ether (MTBE), C1-4 alkyl esters of acetic acid, chlorobenzene, alkanes and mixtures thereof.

Particularly preferred is the solvent selected from the group consisting of isopro panol, butanol, chlorobenzene and the mixtures thereof.

Therefore, 7-dehydrocholesterol is again available for further reaction. Flence, the method of stabilization, respectively of decreasing the degradation, of 7-dehydrocholesterol by formation of 7-dehydrocholesterol-hemihydrate is a very efficient manner and is a way of regenerating the starting material, i.e. 7-dehydro- cholesterol-hemihydrate, again on demand very easily. Therefore, 7-dehydro cholesterol can be easily stored without significant degradation also over an extended period of time and only later can be transformed if desired further to other compounds such as vitamin D3 upon irradiation of 7-dehydrocholesterol.

Hence the method as described above comprises preferably a step g) which is performed after step b)

g) releasing 7-dehydrcncholesterol from 7-dehydrocholesterol-hemihydrate.

The preferred method of releasing 7-dehydrcncholesterol from 7-dehydro- cholesterol-hemihydrate is adding a solvent which is suitable for dissolving or solubilizing 7-dehydrocholesterol-hemihydrate in order to dissociate 7-dehydro- cholesterol-hemihydrate into 7-dehydrocholesterol and water, and, hence, to release 7-dehydrocholesterol. Examples for such suitable solvents for the release are given above in this document.

This invention offers also a very efficient and economical way for the synthesis of vitamin D3 where the key intermediate 7-dehydrocholesterol is produced in a different place to where the 7-dehydrocholesterol is used for further chemical reactions. The invention allows particularly a transport between

production sites without significant degradation during transport or storage.

In a preferred embodiment, an additional step a') is performed between step a) and b) and an additional step b' is performed in step b) after storage and before the release of 7-dehydrcncholesterol from 7-dehydrocholesterol-hemihydrate a') preparing a package (1 ) consisting of a transport packaging (2) and 7-dehydro- cholesterol-hemihydrate a as a packaged good (3) or part of the packaged good (3), which is localized in the inner space of the transport packaging (2) b'ίϋ') opening the package (1 ) and removing 7-dehydrocholesterol-hemihydrate from the inner space of the transport packaging (2).

The details and preferred embodiments of the features of these steps have been discussed above in great detail. Figures

Figure 1 shows a XRD of 7-dehydrocholesterol-hemihydrate.

Figure 2 shows a XRD of of 7-dehydrocholesterol anhydrate.

Figure 3 shows a XRD of 7-dehydrocholesterol-hemihydrate. Figure 3 is the same as figure 1 zoomed in the 2 theta range of 2-20°.

Figure 4 shows a XRD of of 7-dehydrocholesterol anhydrate. Figure 4 is the same as figure 2 zoomed in the 2 theta range of 2-20°.

Figure 5 shows a representation of the ratio DHCi2w/DHCo versus FhO/DFIC before storage (see examples).

Figure 6 shows a schematic representation of a package (1 ) which consists of a transport packaging (2) and a packaged good (3).

The packaged good (3), i.e. 7-dehydrocholesterol-hemihydrate, is localized in the inner space of the transport packaging (2). The inner space is not completely filled with 7-dehydrocholesterol-hemihydrate. The remaining volume is filled with air. Figure 6a shows a schematic representation of a transport packaging (2) of figure 6, which has an inner space (4). The inner space (4) can be filled at least partially with the packaging good, i.e. 7-dehydrocholesterol-hemihydrate, to form a package (1 ) as shown in figure 6.

Figure 7 shows a schematic representation of one embodiments of a package (1 ) which consists of a transport packaging (2) and a packaged good (3). The transport packaging consists of a sealable container (6) and a seal (5).

The packaged good (3), i.e. 7-dehydrocholesterol-hemihydrate, is localized in the inner space of the transport packaging (2). The inner space is not completely filled with 7-dehydrocholesterol-hemihydrate. The remaining volume is filled with air. Figure 7a shows a schematic representation of such a transport packaging (2) of figure 7. The sealable container (6) has an opening through which the packaged good (3), i.e. 7-dehydrocholesterol-hemihydrate, can be put into the inner space (4) of the sealable container (6). After a partially filling of the cavity, i.e. the inner space (4), the seal (5) can be put onto the opening of the sealable container (6) to seal the packing and to form a package (1 ) as shown in figure 7.

List of reference signs

1 Package (German: Packung)

2 Transport packaging (German: Verpackung)

3 Packaged good (German: Packgut )

4 Inner space of the transport packing (2)

5 Seal (German: Verschluss)

6 Sealable container (German: verschliessbarer Behalter)

Examples

The present invention is further illustrated by the following experiments.

Determination of 7-dehvdrocholesterol-hemihvdrate

The amount of 7-dehydrocholesterol in a sample has been determined by High-Performance Liquid Chromatography using a HPLC Agilent 1200 with a HPLC column Supelcosil ABZ+/Sigma of 250 mm length, 4.6 mm internal diameter, 5 micrometre particle size, measured at 30°C, using a detector DAD at wavelength of 212 nm, 270 nm and 300nm. The eluent was acetonitrile (A) / methyl tert. -butyl ether (B) in a gradient program:

0 min A/B= 70/30 1 ml/m in

25 min A/B= 50/50 1 ml/min

28 min A/B= 90/10 1 ml/min

30 min A/B= 90/10 1 ml/min

The calibration has been performed by dissolving 5 exactly weighed samples of the crystal in the range of 1 to 20 mg in a solvent consisting of acetonitrile / methyl tert. -butyl ether 60/40. Deternnination of water

Water in a sample has been determined by Karl Fisher Titration by means of Metrohm 874 / Metrohm 851 using a Metrohm Pt double electrode and

Hydranal Medium K titer solution.

Formation of 7-dehvdrocholesterol-hemihvdrate

8.8 g 7-dehydrocholesterol has been dissolved in a mixture of 221 g acetone and 11 g water under stirring and heating to 50°C. Then the solution was cooled to 0°C over several hours under stirring so that white crystals have been precipitated. These crystals have been filtered over a Nutsche filter.

The filter cake had a wet appearance due to excess water. Flence, some air has been flowed through the filter cake to remove the majority of excess of water to yield visually dry 7-dehydrocholesterol-hemihydrate crystals.

The filter cake had a wet appearance due to excess water. Flence, some air has been flowed through the filter cake to remove the majority of excess of water to yield visually dry 7-dehydrocholesterol-hemihydrate crystals.

Drying

A sample 1 of the crystals has been isolated and its ratio of 7-dehydrocho- lesterol to water has been determined using the method shown above.

Then the crystals from the filter cake have been put in a round bottom flask and have been exposed to a vacuum of 5 mbara at a heating temperature of 60°C. After different specific times of drying, individual samples (2, Ref.1-2) have been taken and the amounts of 7-dehydrocholesterol ( DHCo ) and water have been determined according the method shown above.

Storage

For the storage test, all samples (1 ml) have then been filled into 10 ml brown glass vials which then have been closed by a plastic cap. The samples then have been stored during 12 weeks at a temperature of 4°C in contact with air in the vial. Then the amount of 7-dehydrocholesterol ( DHCi2w ) has been determined using the method shown above.

The results are compiled in table 1.

Table 1 :Storage stability, resp. degradation of 7-dehydrocho- lesterol-hemihydrate.

It is important to stress that it was observed that the sample 1 and 2 showed a molar ratio of DHC/H2O of 2.0 after storage. This corresponds exactly to the molar ration of DHC/ H2O of 7-dehydrocholesterol-hemihydrate. Hence, it can be concluded that in sample 1 and 2 the excess of hydrate has been completely removed during storage to form the storage stable of 7-dehydrocholesterol-hemi- hydrate.

Figure 5 shows a graphic representation of the molar ratio of H2O to DHC versus the ratio of DHCi2w to DHCo. In this representation the stability effect, respectively the impact on degradation, can be visualized very clearly.

It shows clearly that 7-dehydrocholesterol-hemihydrate is very storage stable, whereas when a ratio of H2O/DHC being significantly lower than 0.5 is obtained, the stability decreases very strongly.

This means that 7-dehydrocholesterol-hemihydrate has a very low degradation, whereas when a ratio of MeOH/DHC being significantly lower than 0.5 is obtained, the degradation increases very strongly.

The 7-dehydrocholesterol anhydrate having a H ₂ 0/DHC-ratio of 0 is very unstable, i.e. after 12 weeks storage at 4°C in contact with air only about 16 % of the amount of DHC present before storage remains, i.e. about 84 % of the DHC has been degraded. DHC, i.e. in the form of the 7-dehydrocholesterol ansolvate, exhibits an extremely high degradation.

Crystal structures

The 7-dehydrocholesterol-hemihydrate and the 7-dehydrocholesterol anhydrate have been investigated by XRD before storage. The X-ray powder diffractogram (XRD) has been measured in the reflection mode at 295 K using CuKal as radiation source. The measurement has been carried out in the range of 2 - 50° 2Q.

The diffractograms are shown in figure 1 and figure 3 for 7- dehydrocholesterol-hemihydrate and in figure 2 and figure 4 for 7- dehydrocholesterol anhydrate.

Table 2. Characteristic maxima of the XRD of 7-dehydrocholesterol- hemihydrate.

Table 3. Characteristic maxima of the XRD of 7-dehydrocholesterol- anhydrate.

The XRD of 7-dehydrocholesterol-hemihydrate remains unchanged after storage.