Method of purifying macrolides

ES 2 297 481 T3

DESCRIPTION

Method to purify macrolides.

The present invention relates to a method for purifying macrolides, especially tacrolimus, ascomycin, sirolimus, everolimus, or pimecrolimus, by a separation method using sorption resins.

Related requests

This application claims the benefits of US Provisional Application Serial No. 60 / 490,070, filed July 24, 2003, and US Provisional Application Serial No. 60 / 539,363, filed January 26, 2004, the contents of which they are incorporated herein by reference.

Background of the invention

Macrolides are multi-membered lactone rings that have one or more deoxysugars as substituents. Erythromycin, azithromycin, and clarithromycin are macrolides that have bacteriostatic and / or bactericidal activity.

Tacrolimus (FK 506) is also a macrolide antibiotic that is also an immunosuppressive agent. More potent than cyclosporine, tacrolimus is known to have a selective inhibitory effect on T lymphocytes.

Pimecrolimus is a macrolactam and ascomycin derivative known to inhibit the production of pro-inflammatory cytokines by T cells and mast cells. The Merck Index 1331 (Maryadele J. O'Neil et al. Eds., 13^to ed. 2001). Pimecrolimus is used in a way known as an immunosuppressant. Id.

Sirolimus, another macrolide, has been reported to be an immunosuppressant. Sirolimus has been administered with cyclosporine and corticosteroids after transplantation to prevent graft rejection. Martindale: The Complete Drug Reference 568 (Sean C. Sweetman ed., Pharmaceutical Press 33rd ed. 2002).

Everolimus, a derivative of sirolimus, has been reported to be an immunosuppressant used in organ transplantation. Martindale at 539.

Macrolides are typically obtained by fermentation, although synthetic routes are known for some. Macrolides, as obtained, typically contain various impurities that can be detected by various means, for example high pressure liquid chromatography (HPLC). The presence of impurities in a pharmaceutical compound is undesirable, and health authorities in many jurisdictions (for example, the Food and Drug Administration of the United States of America) have established guidelines that refer to acceptable levels of impurities in drugs. The need and commercial utility of methods to reduce the level of impurities in any drug are self-evident.

Song, Z. et al., (J. Org. Chem., 1999, 64, 1859-1867) describes a procedure using HP-20S resin and acetonitrile-water eluent for the separation of synthetic macrolide L733.735 from ascomycin, the latter being a starting material in the synthesis of L733,735.

Summary of the invention

In one aspect, the present invention relates to a method of removing impurities from (ie, reducing the level of impurities in) a macrolide selected from tacrolimus, ascomycin, sirolimus (rapamycin), everolimus, and pimecrolimus. The method comprises the following steps:

a) provide a loading of the macrolide having an initial level of impurities,

b) loading the charge onto a bed of sorption resin,

c) eluting the loaded sorption resin bed with an eluent, eluent which comprises water and tetrahydrofuran, and additionally, when the macrolide is tacrolimus, the eluent may comprise water, acetonitrile and phosphoric acid, to obtain an effluent, and

d) collecting at least a fraction of the effluent comprising the macrolide.

Detailed description of the invention

As used herein, the term "room temperature" refers to a temperature of 0 ° to 40 ° C, preferably 10 ° to 35 ° C.

As used herein, the term "reduced pressure" refers to a pressure less than 760 mm Hg (101.325 kPa).

ES 2 297 481 T3

As used herein, the term "antisolvent" refers to a substance, normally liquid at room temperature, in which the macrolide is, at best, barely soluble.

As used herein, the term "impurity" refers to any compound that has a retention time different from the desired macrolide. The different retention time can be measured, for example, by the HPLC method described here below.

As used herein, the terms RRT0.95 and RRT1.25 refer to ascomycin and dihydrotacrolimus, respectively, which are impurities of tacrolimus, having relative retention times (relative to tacrolimus) of around 0.95 and 1, 25 in HPLC analysis, such as described below.

As used herein in connection with mixtures or combinations of liquids, the term volume percent or volume percent (vol-%) refers to the volume fraction calculated as follows (illustrated for species A):

vol -% A = WtA x Pa / (WtA x Pa + Wtn x Pb) where:

Wt_TO and Wt_B are the weights in grams of species A and B, respectively, and

Pa and p_B are the densities, in g / ml, of species A and B, respectively.

In one embodiment, the present invention provides a chromatographic method for separating macrolides, selected from tacrolimus, ascomycin, sirolimus, everolimus, and pimecrolimus, from the impurities contained therein (ie, to reduce the level of impurities in the macrolide). Separation (reduction) is accomplished by loading the macrolide onto a sorption resin bed, and eluting with an eluent containing THF and water. When the macrolide is tacrolimus, the eluent can comprise water, acetonitrile, and phosphoric acid. When the macrolide is tacrolimus, the reduced impurities include at least ascomycin and dihydrotacrolimus, the quantification of which by HPLC is described below. When the macrolide is ascomycin, the reduced impurities include at least tacrolimus. The macrolide used can come from any source.

In the practice of the present invention, the reduction (separation) is effected by eluting with an eluent a bed of sorption resin, loaded with a macrolide charge, to obtain an effluent. Sorption resins useful in the practice of the present invention are well known in the art, and are preferably nonionic, cross-linked styrene-divinylbenzene materials, but may be chemically modified. Acrylic-type sorption resins are also known. Sorption resins have very porous structures, the surfaces of which can absorb - and then desorb - various chemical species. The absorption and desorption are influenced by the environment, for example by the solvent used. In the presence of polar solvents (for example water), sorption resins show hydrophobic behavior. When using non-polar solvents (eg hydrocarbons), sorption resins can show some polar behavior. Sorption resins typically have a macroreticular structure, and have specific surfaces of at least 300 μm.²/ g.

Sorption resins useful in the practice of the present invention include Amberlite® XAD resins, available from Rohm and Haas; XAD 4, XAD 7 HP, XAD 16 HP, XAD 761, and XAD 1180, to name just a few. Also useful are Diaion sorption resins, available from Mitsubishi: HP10, HP 20, HP 21, HP 30, HP40, HP 50, SP 800, SP 825, SP 850, SP 875, SP 205, SP 206, SP 207 , HP1MG and HP2MG, to name just a few. Amberlite® XAD 1180 is an example of a preferred sorption resin for use in the practice of the present invention. Amberlite XAD 1180 is a macroreticular cross-linked aromatic polymer. It is a cross-linked, hydrophobic, non-ionic polymer, whose adsorbent properties derive from its patented macroreticular structure (which contains both a continuous polymeric phase and a continuous porous phase), its high specific surface area, and the aromatic nature of its surface. The specific surface is 500 m2 / g or higher. Porosity is 0.60 ml / ml or higher. The PDS 0205 A Product Data Sheet - Jan.98-1 / 2 gives additional information on this resin.

In a first stage of the method of the present invention, the macrolide charge is loaded onto a bed of sorption resin. The filler can be provided as a solution of the macrolide in an organic solvent, combined with an antisolvent.

Alternatively, the macrolide filler is adsorbed onto (deposited on) a filler portion of the sorption resin, prior to loading onto the sorption resin bed. A solution of the macrolide in an organic solvent, optionally containing water, is combined with a portion of the sorption resin and with an antisolvent. The sorption resin can be the same as that used to prepare the bed, or it can be a different sorption resin. The loading portion of the sorption resin can be 33% to 50% of the bed volume. The filler portion is then juxtaposed to a wet sorption bed, to provide a bed loaded with the filler.

The organic solvent used to prepare the solution from which the charge is loaded or deposited is preferably selected from the group consisting of tetrahydrofuran (THF), acetone, acetonitrile (ACN), methanol,

ES 2 297 481 T3 ethanol, n-butanol, n-propanol, iso-propanol, esters (for example ethyl acetate), and dipolar aprotic solvents such as dimethylformamide (DMF). Most preferably, the organic solvent is THF, acetone, or ACN. When the macrolide is tacrolimus, the preferred solvents are THF and ACN. Preferably, the antisolvent is water or a straight or branched chain alkane, or a cycloalkane, such as hexane, heptane or cyclohexane. The addition of an antisolvent reduces the solubility of the macrolide in the solution, and is believed to facilitate adsorption of the sample onto the loading portion of the sorption resin. The antisolvent is added slowly to avoid large concentration gradients that can result in partial mass precipitation of the macrolide, which can lead to fouling and plugging. Preferably, the solvent: antisolvent ratio is 40% or less.

The combination of macrolide solution, sorption resin loading portion, and antisolvent can be done in any convenient vessel equipped with a stirrer (eg, stirred tank reactor).

In a particular embodiment, the sorption resin loading portion is contained in a column and is contacted with a flow of macrolide solution that passes through the column in a recirculating system. An antisolvent is gradually introduced into the solution stream flowing through and around the sorption resin loading portion, whereby the macrolide sample is gradually adsorbed onto the sorption resin loading portion.

By way of example, when the macrolide is tacrolimus, the solution can be around 100 g / l, and the volume of antisolvent can be at least five times the volume of the solution. The apparent volume of the loading portion of the sorption resin can be approximately equal to the volume of the solution. The skilled person will know how to optimize the ratios, by normal experimentation, to obtain adsorption of the macrolide on the loading portion of the sorption resin.

After the adsorption is substantially complete, which can be monitored by tracking the concentration of macrolide remaining in the solution, the feedstock is separated from the remaining solution. The separation can be by filtration. When using the recirculating column method to obtain the feed load, the column is simply decoupled from the recirculating system.

In a subsequent step of this embodiment, the filler portion now loaded with macrolide is juxtaposed with a prepared bed of wet sorbing resin. The bed is confined in a suitable vessel. Preferably, the bed is confined in a column, preferably of circular cross section. To prepare the bed, the desired amount of sorption resin is suspended with water or with a mixture of water and a solvent (eg THF or ACN). A combination of water with solvent is advantageous when the bed has a large diameter. The suspension is then transferred to the desired vessel, preferably a cylindrical column such as used for column chromatography. The water (or the combination of water and solvent) is removed to leave a bed of wet sorption resin. The practice for preparing and packaging chromatographic columns is well known to the skilled person and related personnel, and known practices are readily adapted to the practice of the present invention.

The filler portion can be juxtaposed to the wet sorption resin bed simply as a layer thereon. When the fed load is prepared in a recirculating system, the vessel containing the fed load can be coupled to the container containing the wet sorption resin bed by any means that establishes fluid communication between them.

The separation of the macrolide (i.e., tacrolimus, ascomycin, sirolimus, everolimus, or pimecrolimus) and impurities, thereby reducing the level of impurities in the macrolide, is accomplished by passing an eluent through the feed, and subsequently through the sorption resin bed juxtaposed to it and in fluid communication with it.

The eluent includes water and THF. Additionally, when the macrolide is tacrolimus, the eluent can comprise water, acetonitrile, and phosphoric acid. A preferred eluent, especially when the macrolide is tacrolimus, is essentially a mixture of THF and water having 20% ​​by volume to 50% by volume, 31% by volume to 40% by volume, THF. When an organic solvent such as methanol, acetonitrile, acetone or n-butanol is used with the THF-water eluent, the THF content is less than 38% by volume, preferably between 4 and 38% by volume. Another preferred eluent for tacrolimus is a mixture of acetonitrile and water having 30% by volume to 70% by volume, most preferably 40% by volume to 65% by volume, of acetonitrile and phosphoric acid, preferably 0.0005 to 0.003 parts. of phosphoric per 1 part of eluent.

The eluent is eluted through the loading portion and the sorption resin bed juxtaposed thereto, at a rate that depends on the total cross-sectional area of ​​the bed (measured perpendicular to the flow of the eluent). Preferably, the flow rate (relative to the cross-sectional area) is less than 25 cm / h, preferably less than 15 cm / h. Lower elution rates increase time, but improve separation efficiency. A preferred elution rate for increased separation efficiency is about 90 ml / hour.

The eluent exiting the sorption resin bed (i.e., the effluent) is collected in one or more fractions, as is normal for the skilled person using separation methods, such as chromatography, the former depending on the preferred retention of the chemical species in a stationary phase (for example, a static bed). An inorganic acid, such as phosphoric acid, can be added to the effluent.

ES 2 297 481 T3

Preferably, after eluting the bed with an amount of eluent, the bed is brought into fluid communication with a second bed, so that the effluent from the first bed elutes through the second bed. After elution of the first and second beds, the second bed can and is preferably decoupled from the first bed (ie, fluid communication is broken), and the elution is continued through the second bed alone. The eluent is a mixture of TFA and water having 33% by volume to 35% by volume THF, and is the preferred eluent.

Optionally, additional columns can be connected to the system.

The concentration and composition of the fractions can be monitored by any convenient means. The detection and quantification of impurities in a macrolide, in particular ascomycin and dihydrotacrolimus in tacrolimus, can be carried out by the HPLC method described here below.

Depending, among others, on the column load and on the composition and flow rate of the eluent, a main fraction (the fraction of interest) of effluent is collected that includes more than 60%, preferably between 60% by weight and 90% in weight of the macrolide originally present in the solution. When the macrolide is tacrolimus, and the eluent is THF-water (31 to 40% by volume THF), the main fraction is collected so that the final isolated product has 0.1 area% or less (by HPLC described more below) of impurity RRT0.95.

If desired, the macrolide separated from impurities, and therefore having a reduced level of impurities, can be isolated from the effluent by any conventional means (eg extraction, lyophilization, evaporation, addition of antisolvent). As useful antisolvents, mention may be made of water, alkanes and cycloalkanes. Isolation methods can be combined. For example, the antisolvent can be combined with a concentrated eluent.

A preferred method of isolation includes the concentration of the main fraction at 70 ° C or less, preferably 60 ° C or less, preferably at the pressure of 760 mm Hg, up to about 50% of its initial volume, whereby product crystals. Preferably, before concentration, an acid, 1 to 10 ml per liter of eluent, is added to stabilize the macrolide.

Optionally, the concentrated main fraction is kept at room temperature for a standing time. When using a standing time, the preferred standing time is 1-4 days. Macrolide crystals having a reduced amount of impurities are recovered by any conventional means, for example nitration (gravity or vacuum).

Further reduction of impurities can be achieved by subjecting the recovered product to various additional treatments according to the method of the present invention.

The reduction of impurities in a macrolide, achieved by the method of the present invention, can be monitored by the HPLC method described hereinafter.

In another embodiment, the macrolide is tacrolimus, and at least ascomycin and dihydrotacrolimus impurity levels are reduced. The levels of other impurities are also reduced. The method includes the following steps: preparing a tacrolimus fed filler comprising a tacrolimus solution with or without a filler portion of a sorption resin, especially a macroreticular resin such as Amberlite® XAD 1180 and Diaion HP 20; loading the feed into a wet sorption resin, especially Amberlite® XAD 1180 and Diaion HP 20, which can be contained in a vessel, especially a column; eluting the loading portion and the sorption resin with an eluent which is a mixture of tetrahydrofuran (THF) and water, 20% by volume to 50% by volume, especially 31% by volume to 40% by volume THF, or a mixture of acetonitrile (ACN), water and phosphoric acid, in which acetonitrile is present in an amount from 30% by volume to 70% by volume, and most especially 40% by volume to 65% by volume; collect at least a main fraction (fraction of interest) of eluent, which contains more than 60%, preferably between 60% and 90% of the initial tacrolimus (depending on the initial purity), and, optionally, isolate from the main fraction the tacrolimus having a reduced amount of impurities, for example by concentrating the main fraction (s), for example under reduced pressure, in the presence of an acid, and optionally recovering the product thus obtained.

In yet another embodiment, the macrolide is ascomycin, and tacrolimus impurity levels are at least reduced. The levels of other impurities are also reduced. The method includes the following steps: preparing an ascomycin feedstock comprising an ascomycin solution with or without a filler portion of a sorption resin, especially a macroreticular resin such as Amberlite® XAD 1180 and Diaion HP 20; loading the feedstock to the wet sorption resin, especially Amberlite® XAD 1180 and Diaion HP 20, which can be contained in a vessel, especially a column; eluting the loading portion and the sorption resin with an eluent which is a mixture of tetrahydrofuran (THF) and water, 20% by volume to 50% by volume, especially 31% by volume to 40% by volume THF; collect at least a main fraction (fraction of interest) of the eluent, which contains more than 60%, preferably between 60% and 90% of the initial ascomycin (depending on the initial purity), and, optionally, isolate the ascomycin that has a reduced amount of impurities from the main fraction, for example by concentrating the main fraction or fractions, for example under reduced pressure in the presence of an acid, and optionally recovering the product thus obtained.

ES 2 297 481 T3

In yet another embodiment, the macrolide is sirolimus. The method for removing impurities from sirolimus includes the following steps: preparing a sirolimus feedstock comprising a sirolimus solution with or without a filler portion of a sorption resin, especially a macroreticular resin such as Amberlite^® XAD 1180 and Diaion HP 20; charge the feed charge to a wet sorption resin, especially Amberlite^® XAD 1180 and Diaion HP 20, which can be contained in a vessel, especially a column; eluting the loading portion and the sorption resin with an eluent which is a mixture of tetrahydrofuran (THF) and water, 20% by volume to 50% by volume, especially 31% by volume to 40% by volume THF; collect at least a major fraction (fraction of interest) of eluent, which contains more than 60%, preferably between 60% and 90% of the initial sirolimus (depending on the initial purity), and optionally isolate sirolimus which has an amount reduction of impurities from the main fraction, for example by concentrating the main fraction or fractions, for example under reduced pressure in the presence of an acid, and optionally recovering the product thus obtained.

In yet another embodiment, the macrolide is everolimus. The method for removing impurities from everolimus includes the following steps: preparing an everolimus feedstock comprising a solution of everolimus with or without a filler portion of a sorption resin, especially a macroreticular resin such as Amberlite^® XAD 1180 and Diaion HP 20; charge the feed charge to the wet sorption resin, especially Amberlite^® XAD 1180 and Diaion HP 20, which can be contained in a vessel, especially a column; eluting the loading portion and the sorption resin with an eluent which is a mixture of tetrahydrofuran (THF) and water, 20% by volume to 50% by volume, especially 31% by volume to 40% by volume THF; collect at least a major fraction (fraction of interest) of eluent, which contains more than 60%, preferably between 60% and 90% of the initial everolimus (depending on the initial purity), and optionally isolate everolimus which has an amount reduction of impurities from the main fraction, for example by concentrating the main fraction or fractions, for example under reduced pressure in the presence of an acid, and optionally recovering the product thus obtained.

In yet another embodiment, the macrolide is pimecrolimus. The method for removing impurities from pimecrolimus includes the following steps: preparing a pimecrolimus feedstock comprising a solution of pimecrolimus with or without a filler portion of a sorption resin, especially a macroreticular resin such as Amberlite® XAD 1180 and Diaion HP 20; charge the feed charge to a wet sorption resin, especially Amberlite^® XAD 1180 and Diaion HP 20, which can be contained in a vessel, especially a column; eluting the loading portion and the sorption resin with an eluent which is a mixture of tetrahydrofuran (THF) and water, 20% by volume to 50% by volume, especially 31% by volume to 40% by volume THF; collect at least a main fraction (fraction of interest) of eluent, which contains more than 60%, preferably between 60% and 90% of the initial pimecrolimus (depending on the initial purity), and optionally isolate pimecrolimus that has an amount reduction of impurities from the main fraction, for example by concentrating the main fraction or fractions, for example under reduced pressure in the presence of an acid, and optionally recovering the product thus obtained.

Chromatographic conditions

<td>Column:</td><td>ZORBAX SB-C18 75 x 4.6 mm; 3.5 pm</td>

<td>Pre-column:</td><td>SymmetryShield PP18 3.9 x 20 mm; 5 pm</td>

<td>Eluent:</td><td>A: Measure 200 ml of acetonitrile into a 2000 ml volumetric flask, then dilute to volume with distilled water, to a total volume of 2000 ml. Then add 100 µl of 50% acetic acid. B: Add 100 µl of 50% acetic acid to 2000 ml of acetonitrile.</td>

GRADIENT TABLE

<td>Time (min.)</td><td>Eluent "A" (% w / w)</td><td>Eluent "B" (% w / w)</td><td>Flow rate (ml / min.)</td>

<td> 0</td><td> 60</td><td> 40</td><td> 2,3</td>

<td> 15</td><td> 55</td><td> 45</td><td> 2,3</td>

<td> 25</td><td> 30</td><td> 70</td><td> 1,8</td>

<td> 25,1</td><td> 60</td><td> 40</td><td> 1,8</td>

<td> 27</td><td> 60</td><td> 40</td><td> 1.8</td>

ES 2 297 481 T3

<td>Flow:</td><td>2.3 ml / min.</td>

<td>Detection wavelength:</td><td>210 nm</td>

<td>Injected volume:</td><td>20 pl</td>

<td>Sample solvent:</td><td>acetonitrile</td>

<td>Temp. column unit:</td><td>60 ° C</td>

<td>Analysis time:</td><td>27 min.</td>

<td>Tacrolimus retention time:</td><td>approx. 14 min.</td>

The retention times of the impurities ascomycin (RRT0.95) and dihydrotacrolimus (RRT1.25) are given relative to tacrolimus, and are expressed as a percentage of area relative to the area of ​​all peaks in the chromatogram.

The retention times of the tacrolimus impurity (RRT1.00) are given relative to ascomycin, and are expressed as a percentage of area relative to the area of ​​all peaks in the chromatogram.

The method of the present invention can be exemplified by the following non-limited examples.

Example 1

The area percentages refer to the area percentage of HPLC chromatograms obtained by the method described hereinabove.

The procedure below was carried out at a temperature of 28 ° C to 32 ° C.

A bed of sorption resin (Amberlite® XAD 1180) was prepared in a column (45 cm diameter) using water: THF to load the column (approx. 1001 wet sorption resin).

Water (86 L) was slowly added, with stirring, to a solution of tacrolimus (1227 g) in acetonitrile (10 L), in which the sorption resin (Amberlite® XAD 1180; 9 L) was suspended with stirring. The tacrolimus used contained about 2.6 area% RRT0.95, and about 2.9 area% RRT1.25. When the water addition was complete, the sorption resin feed was collected by filtration.

The collected feedstock was loaded (juxtaposed) as a layer on top of the wet sorbing resin bed.

The column was eluted first with approx. 1800 µl of a first eluent consisting of THF / water (33% by volume of THF). The column was then eluted with a second eluent consisting of THF / water (40% by volume THF). The elution rate was about 11 to 13 l / hr (6.9 to 8.2 cm / hr). A main fraction was collected, approx. 460 μl, containing around 820 g of tacrolimus (yield 67%). A previous fraction, approx. 801, containing approx. 190 g of tacrolimus.

The main fraction (460 L) was combined with phosphoric acid, 85% (460 ml), and concentrated under reduced pressure to a volume of about 2301. The concentrate was kept at room temperature for one day. (Note: Longer standing times were attempted in subsequent experiments. The crystals obtained were more easily filtered than those obtained here). The crystals were washed with hexane, and dried at 40 ° C.

The product isolated from the main fraction had about 0.1 area% RRT0.95, and about 1.7 area% RRT1.25.

The product isolated from the previous fraction had about 3 area% RRT0.95, and about 0.3 area% RRT1.25.

Example 2

The general procedure of Example 1 was repeated to investigate the effect of eluent composition and flow rate.

Through these experiments, it could be established that reducing the elution flow rate increases the separation efficiency of the chromatography. Increasing the elution flow rate reduces the efficiency of the chromatography. A flow rate of 25 cm / cm².hour (instead of 6.9-8.2 cm / cm².hour) resulted in a significant reduction in efficiency, but a major fraction having the quality of that described in Example 1 could be collected.

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Furthermore, it was established that the first eluent of 34% by volume of THF (instead of 33% by volume) increased the chromatography performance. The yield was 69%. The RRT0.95 impurity level of the main fraction was 0.10 area%.

When an eluent having 31% by volume THF was used, the elution time of tacrolimus was increased. The mentioned eluent concentrations (31%, 33%, 34%, 40% by volume of tetrahydrofuran) were found to be usable for the elution of tacrolimus, without increasing the solvent concentration.

These additional experiments also established that water: tetrahydrofuran: solvent mixtures were equally effective. The tested solvents used for the water: tetrahydrofuran: solvent eluents were methanol, acetonitrile, acetone, n-propanol, and n-butanol. In all cases, an adequate quality was obtained.

Example 3

The area percentages refer to the area percentage of the HPLC chromatograms obtained by the method described herein above.

The procedure below was carried out at 20 ° C to 25 ° C.

A bed of sorption resin (Diaion SP 207) was prepared in a column (3.2 cm diameter) using water to charge the column (approx. 550 ml of wet sorption resin).

Tacrolimus (7.2 g) was dissolved in a mixture of acetonitrile (30 ml) and water (20 ml). Tacrolimus contained about 2.6 area% RRT0.95 (ascomycin), and about 2.9 area% RRT1.25 (dihydrotacrolimus).

The tacrolimus solution was loaded as a layer on top of the wet sorption resin bed.

The column was eluted with approx. 8 l of eluent consisting of acetonitrile / water / phosphoric acid (600: 400: 1). The elution flow rate was 90 ml / hour.

Fractions 32-45 were combined. The pooled fractions contained 1.9 g of tacrolimus. The impurity content of the pooled fractions was about 2.9 area% RRT0.95 (ascomycin), and about 1.2 area% RRT1.25 (dihydrotacrolimus).

The described purification process is suitable for the reduction of dihydrotacrolimus. Preferably, the eluent has an acetonitrile content of about 30% to 70%, preferably about 40% to 65%.

The inorganic acid content is used to prevent decomposition of tacrolimus during chromatography. Preferably the inorganic acid is phosphoric acid. Preferably, the content of phosphoric acid is between about 0.0005 to 0.003 parts of acid per 1 part of eluent.

The purification process described increases the efficiency of the processes described by Examples 1 and 2.

Example 4

Two columns for chromatography were prepared according to Example 1. Before chromatography, 3000 g of active substance containing tacrolimus were adsorbed onto a XAD 1180 sorption resin according to the following procedure. The tacrolimus was dissolved in 15 L of acetone. Sorption resin (33 L) was added to the solution, and 90 L of water was added slowly to the solution / resin mixture, with continuous stirring. The sorption resin feed charge was placed on top (juxtaposed) as a layer on top of the sorption resin contained in the first column.

The first column was eluted with a mixture of tetrahydrofuran: water (34% by volume THF). The elution flow rate was 15 l / hour. Fractions of 20 L each were collected. The volume of each fraction was 20 l. After elution of 35^to fraction, the second column was connected (fluidly coupled) in series with the first column, and elution was continued on the columns in series.

After the 95th fraction eluted, the first column was disconnected, and elution was continued in the second column only. The appropriate purified fractions were combined.

The major part of THF was removed from the combined fractions by evaporation under reduced pressure. The concentrate was extracted with ethyl acetate, and the phases were separated. The separated ethyl acetate phase was concentrated under reduced pressure (approx. 1 part tacrolimus and 1 part ethyl acetate). Cyclohexane and water were slowly added to the concentrated ethyl acetate extract. The precipitated tacrolimus was recovered from the mixture at 0-30 ° C. The crystals were filtered and dried.

ES 2 297 481 T3

The starting material contained approx. 0.5 area% ascomycin (RRT0.95) and approx. 1.3% dihydrotacrolimus (RRT1.25). The crystals produced contained less than 0.1 area% ascomycin, and approx. 0.4 area% dihydrotacrolimus.

Example 5

Tacrolimus was dissolved in a mixture of water: tetrahydrofuran (67 volumes: 33 volumes). The solvent concentration achieved was approx. 30 g / liter. The solution was passed over XAD 1180 sorption resin. The sorption resin adsorbed the tacrolimus.

After adsorption, the elution of tacrolimus continued as in Example 1.

Example 6

Tacrolimus was dissolved in a mixture of water: tetrahydrofuran (67 volumes: 33 volumes). The solvent concentration achieved was approx. 30 g / liter. The solution was passed over HP20 sorption resin. Sorption resin adsorbs tacrolimus.

After adsorption, the elution of tacrolimus continues as in Example 1.

Example 7

The procedure below was carried out at a temperature of 28 ° C to 32 ° C.

A bed of sorption resin (Amberlite® XAD 1180) was prepared in a column (45 cm diameter) using water: THF to charge the column (approx. 1001 wet sorption resin).

Water (86 L) was added slowly, with stirring, to a solution of ascomycin (1227 g) in acetonitrile (10 L), in which the sorption resin (Amberlite® XAD 1180; 9 L) was suspended with stirring. The ascomycin used contained RRT1.00 (tacrolimus). When the water addition was complete, the sorption resin feed was collected by filtration.

The collected feedstock was loaded (juxtaposed) as a layer on top of the wet sorption resin bed.

The column is first eluted with approximately 18001 of a first eluent consisting of THF / water (33% by volume THF). The column was then eluted with a second eluent consisting of THF / water (40% by volume THF). The elution flow rate is around 11 to 13 l / hr (6.9 to 8.2 cm / hr). A main fraction was collected, approx. 460 μl, containing ascomycin. A previous fraction, approx. 80 μl, containing ascomycin.

The main fraction (4601) was combined with phosphoric acid, 85% (460 ml), and concentrated under reduced pressure to a volume of about 230 L. The concentrate was kept at room temperature for one day. The crystals were washed with hexane and dried at 40 ° C.

Example 8

Two columns were prepared for chromatography according to example 1.

Before chromatography, 3000 g of active substance, containing ascomycin, was adsorbed on the XAD 1180 sorption resin according to the following procedure. Ascomycin was dissolved in 15 L of acetone. Sorption resin (33 L) was added to the solution, and 90 L of water was slowly added to the solution / resin mixture, with continuous stirring. The sorption resin feed charge was topped (juxtaposed) as a layer on top of the sorption resin contained in the first column.

The first column was eluted with a mixture of tetrahydrofuran: water (34% by volume THF). The elution flow rate is 15 l / hour. Fractions of 20 L each were collected. The volume of each fraction was 20 l. After elution of the 35th fraction, the second column was connected (fluidly coupled) in series to the first column, and elution was continued in the series columns.

After the 95th fraction eluted, the first column was disconnected, and elution was continued in the second column only. The appropriate purified fractions were combined.

Most of the THF was removed from the combined fractions by evaporation under reduced pressure. The concentrate was extracted with ethyl acetate, and the phases were separated. The separated ethyl acetate phase was concentrated under reduced pressure (approx. 1 part ascomycin and 1 part ethyl acetate). Cyclohexane and water were slowly added to the concentrated ethyl acetate extract. Precipitated ascomycin was recovered from the mixture at 0-30 ° C. The crystals were filtered and dried.

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Example 9

The procedure below was carried out at a temperature of 28 ° C to 32 ° C.

A bed of sorption resin (Amberlite® XAD 1180) was prepared in a column (45 cm diameter) using water: THF to load the column (approx. 1001 wet sorption resin).

Water (86 L) was added slowly, with stirring, to a solution of sirolimus (1227 g) in acetonitrile (10 L), in which the sorption resin (Amberlite® XAD 1180; 9 L) was suspended with stirring. Used sirolimus contains impurities. When the water addition was complete, the sorption resin feed was collected by filtration.

The collected feedstock was loaded (juxtaposed) as a layer on top of the wet sorption resin bed.

The column is eluted first with approx. 1800 µl of a first eluent consisting of THF / water (33% by volume of THF). The column was then eluted with a second eluent consisting of THF / water (40% by volume THF). The elution flow rate is around 11 to 13 l / hr (6.9 to 8.2 cm / hr). A main fraction was collected, approx. 460 l, containing sirolimus. A previous fraction, approx. 80 l, containing sirolimus.

The main fraction (4601) was combined with phosphoric acid, 85% (460 ml), and concentrated under reduced pressure to a volume of about 2301. The concentrate was kept at room temperature for one day. The crystals were washed with hexane and dried at 40 ° C.

Example 10

Two columns were prepared for chromatography according to example 1.

Before chromatography, 3000 g of active substance, containing sirolimus, was adsorbed on the XAD 1180 sorption resin according to the following procedure. Sirolimus was dissolved in 15 L of acetone. Sorption resin (33 L) was added to the solution, and 90 L of water was slowly added to the solution / resin mixture, with continuous stirring. The sorption resin feed charge was topped (juxtaposed) as a layer on top of the sorption resin contained in the first column.

The first column was eluted with a mixture of tetrahydrofuran: water (34% by volume THF). The elution flow rate is 15 l / hour. Fractions of 20 L each were collected. The volume of each fraction was 20 l. After elution of 35^to fraction, the second column was connected (fluidly coupled) in series to the first column, and elution was continued on the columns in series.

After the 95th fraction eluted, the first column was disconnected, and elution was continued in the second column only. The appropriate purified fractions were combined.

Most of the THF was removed from the combined fractions by evaporation under reduced pressure. The concentrate was extracted with ethyl acetate, and the phases were separated. The separated ethyl acetate phase was concentrated under reduced pressure (approx. 1 part sirolimus and 1 part ethyl acetate). Cyclohexane and water were slowly added to the concentrated ethyl acetate extract. The precipitated sirolimus was recovered from the mixture at 0-30 ° C. The crystals were filtered and dried.

Example 11

The procedure below was carried out at a temperature of 28 ° C to 32 ° C.

A bed of sorption resin (Amberlite® XAD 1180) was prepared in a column (45 cm diameter) using water: THF to load the column (approx. 1001 wet sorption resin).

Water (86 L) was added slowly, with stirring, to a solution of everolimus (1227 g) in acetonitrile (10 L), in which the sorption resin (Amberlite^® XAD 1180; 9 l) was suspended with stirring. The everolimus used contains impurities. When the water addition was complete, the sorption resin feed was collected by filtration.

The collected feedstock was loaded (juxtaposed) as a layer on top of the wet sorption resin bed.

The column is eluted first with approx. 1800 µl of a first eluent consisting of THF / water (33% by volume of THF). The column was then eluted with a second eluent consisting of THF / water (40% by volume THF). The elution flow rate is around 11 to 13 l / hr (6.9 to 8.2 cm / hr). A main fraction was collected, approx. 460 l, containing everolimus. A previous fraction, approx. 80 l, containing everolimus.

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The main fraction (4601) was combined with phosphoric acid, 85% (460 ml), and concentrated under reduced pressure to a volume of about 230 L. The concentrate was kept at room temperature for one day. The crystals were washed with hexane and dried at 40 ° C.

Example 12

Two columns were prepared for chromatography according to example 1.

Before chromatography, 3000 g of active substance, containing everolimus, was adsorbed on the XAD 1180 sorption resin according to the following procedure. The everolimus was dissolved in 151 acetone. Sorption resin (33 L) was added to the solution, and 90 L of water was added slowly to the solution / resin mixture, with continuous stirring. The sorption resin feed charge was topped (juxtaposed) as a layer on top of the sorption resin contained in the first column.

The first column was eluted with a mixture of tetrahydrofuran: water (34% by volume THF). The elution flow rate is 15 l / hour. Fractions of 20 L each were collected. The volume of each fraction was 20 l. After elution of 35^to fraction, the second column was connected (fluidly coupled) in series to the first column, and elution was continued on the columns in series.

After the 95th fraction eluted, the first column was disconnected, and elution was continued in the second column only. The appropriate purified fractions were combined.

Most of the THF was removed from the combined fractions by evaporation under reduced pressure. The concentrate was extracted with ethyl acetate, and the phases were separated. The separated ethyl acetate phase was concentrated under reduced pressure (approx. 1 part everolimus and 1 part ethyl acetate). Cyclohexane and water were slowly added to the concentrated ethyl acetate extract. The precipitated everolimus was recovered from the mixture at 0-30 ° C. The crystals were filtered and dried.

Example 13

The procedure below was carried out at a temperature of 28 ° C to 32 ° C.

A bed of sorption resin (Amberlite® XAD 1180) was prepared in a column (45 cm diameter) using water: THF to load the column (approx. 1001 wet sorption resin).

Water (861) was added slowly, with stirring, to a solution of pimecrolimus (1227 g) in acetonitrile (101), in which the sorption resin (Amberlite® XAD 1180; 91) was suspended with stirring. The pimecrolimus used contains impurities. When the water addition was complete, the sorption resin feed was collected by filtration.

The collected feedstock was loaded (juxtaposed) as a layer on top of the wet sorption resin bed.

The column is eluted first with approx. 1800 1 of a first eluent consisting of THF / water (33% by volume of THF). The column was then eluted with a second eluent consisting of THF / water (40% by volume THF). The elution flow rate is about 11 to 13 1 / hr (6.9 to 8.2 cm / hr). A main fraction was collected, approx. 460 μl, containing pimecrolimus. A previous fraction, approx. 80 μl, containing pimecrolimus.

The main fraction (4601) was combined with phosphoric acid, 85% (460 ml), and concentrated under reduced pressure to a volume of about 230 L. The concentrate was kept at room temperature for one day. The crystals were washed with hexane and dried at 40 ° C.

Example 14

Two columns were prepared for chromatography according to example 1.

Before chromatography, 3000 g of active substance, containing pimecrolimus, was adsorbed onto the XAD 1180 sorption resin according to the following procedure. The pimecrolimus was dissolved in 15 1 acetone. Sorption resin (33 L) was added to the solution, and 90 L of water was added slowly to the solution / resin mixture, with continuous stirring. The sorption resin feed charge was topped (juxtaposed) as a layer on top of the sorption resin contained in the first column.

The first column was eluted with a mixture of tetrahydrofuran: water (34% by volume THF). The elution flow rate is 15 l / hour. Fractions of 20 1 each were collected. The volume of each fraction was 20 1. After elution of the 35th fraction, the second column was connected (fluidly coupled) in series to the first column, and elution was continued in the series columns.

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After the 95th fraction eluted, the first column was disconnected, and elution was continued in the second column only. The appropriate purified fractions were combined.

Most of the THF was removed from the combined fractions by evaporation under reduced pressure. The concentrate was extracted with ethyl acetate, and the phases were separated. The separated ethyl acetate phase was concentrated under reduced pressure (approx. 1 part pimecrolimus and 1 part ethyl acetate). Cyclohexane and water were slowly added to the concentrated ethyl acetate extract. The precipitated pimecrolimus was recovered from the mixture at 0-30 ° C. The crystals were filtered and dried.