FIELD OF THE INVENTION

The present invention relates to new crystalline forms of tulathromycin, to new tulathromycin salts and processes for their preparation.

BACKGROUND OF THE INVENTION

Tulathromycin is a macrolide antibiotic used to treat bovine respiratory disease (BRD) in cattle and swine respiratory disease in pigs. It is marketed by Pfizer Inc. under the tradename Draxxin™. According to EPAR-Scientific discussion document for Draxxin™ tulathromycin consists of two isomers, tulathromycin A (CP-472,295) and tulathromycin B (CP-547 , 272) . Tulathromycin drug substance typically contains less than 1% tulathromycin B, whereas tulathromycin drug product contains 8 to 13% tulathromycin B. In solution, the two isomers form a stable equilibrated mixture which is considered as the active substance .

Tulathromycin B

Tulathromycin A Tulathromycin A is described in EP988310, whereas tulathromycin B is described in EP1131331. Crystalline anhydrous, monohydrate and sesquihydrate form of tulathromycin are described in EP1189912. Polymorphs of tulathromycin diphosphate salt are described in EP1189913.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an X-ray powder diffraction pattern of crystalline form I of tulathromycin prepared according to Example 4.

Figure 2 is an X-ray powder diffraction pattern of crystalline form II of tulathromycin prepared according to Example 5.

Figure 3 is an X-ray powder diffraction pattern of crystalline form III of tulathromycin prepared according to Example 1.

Figure 4 is a FT-IR spectrum of amorphous tulathromycin besylate prepared according to Example 6.

Figure 5 is a FT-IR spectrum of amorphous tulathromycin mesylate prepared according to Example 7.

Figure 6 is a FT-IR spectrum of amorphous tulathromycin esylate prepared according to Example 8.

Figure 7 is a FT-IR spectrum of amorphous tulathromycin (-)-

10-camphosulfonate prepared according to Example 9.

Figure 8 is a FT-IR spectrum of tulathromycin bis (phosphate ) prepared according to Example 10.

Figure 9 is a FT-IR spectrum of

(2R, 3S, R, 5R, 8R, 10R, 11R, 12S, 13S, 14R) -11- ( ( (2S, 3R, 4S, 6R) -4-

(dimethylamino) -3-hydroxy-6-methyItetrahydro-2H-pyran-2- yl) oxy) -2-ethyl-3, 4, 10-trihydroxy-13- ( ( ( 3S, 4S, 6R, 8R) -8- methoxy-4, 8-dimethyl-l , 5-dioxaspiro [2.5] octan-6-yl) oxy) -

3, 5, 8, 10, 12, 14-hexamethyl-l-oxa-6-azacyclopentadecan-15-one as used in Example 10.

Figure 10 is a FT-IR spectrum of tulathromycin bis (phosphate ) prepared according to Example 13. The X-ray powder diffraction patterns were obtained by Philips P 3040/60 X' Pert powder diffTactometer, X'celerator detector at CuKa radiation, 1.54178 A, 3°<2Θ<33°.

FT-IR spectra of KBr discs were recorded over the wave number range of 4000-400 cm ^"1 on Perkin Elmer FT-IR spectrometer Spectrum GX at a resolution of 4 cm ^"1 .

DESCRIPTION OF THE INVENTION

The present invention relates to new crystalline forms of tulathromycin designated herein as form I, II and III. All three forms are anhydrous and non-hygroscopic and are suitable for the preparation of pharmaceutical compositions comprising solid tulathromycin.

Tulathromycin crystalline form I is characterized by an X-ray powder diffraction pattern having peaks at about 5.9, 6.7, 8.2, 13.4 and 16.4 ± 0.2 degrees two-theta. Tulathromycin crystalline form I can be further characterized by X-ray powder diffraction peaks at about 14.5, 14.7 and 19.3 ± 0.2 degrees two-theta. The X-ray powder diffraction pattern of tulathromycin crystalline form I is shown in Figure 1.

Tulathromycin crystalline form I can be prepared by a process comprising the steps of:

a) exposing tulathromycin crystalline form III to 35-45 °C, preferably about 40°C and 65-85% RH (relative humidity), preferably about 75% RH for at least 10 days;

b) heating tulathromycin to 70-90°C, preferably about 80°C; and

c) cooling tulathromycin to room temperature.

Tulathromycin crystalline form II is characterized by an X-ray powder diffraction pattern having peaks at about 6.4, 8.0, 13.0, 16.0 and 18.9 ± 0.2 degrees two-theta. Tulathromycin crystalline form I can be further characterized by X-ray powder diffraction peaks at about 9.2 and 14.9 ± 0.2 degrees two-theta. The X-ray powder diffraction pattern of tulathromycin crystalline form II is shown in Figure 2.

Tulathromycin crystalline form II can be prepared by a process which comprises heating tulathromycin crystalline form III to at least 60°C (and preferably less than 150°C) for at least 50 minutes .

Tulathromycin crystalline form III is characterized by an X- ray powder diffraction pattern having peaks at about 5.9, 6.4, 6.7, 8.0, 8.2 and 13.0 ± 0.2 degrees two-theta. Tulathromycin crystalline form III can be further characterized by X-ray powder diffraction peaks at about 13.4, 16.0, 16.4 and 18.9 ± 0.2 degrees two-theta. The X-ray powder diffraction pattern of tulathromycin crystalline form III is shown in Figure 3.

Tulathromycin crystalline form III can be prepared by a process comprising the steps of:

a) preparing a solution of tulathromycin in a solvent;

b) adding an antisolvent to the solution;

c) cooling the mixture of step b) to form a precipitate; and d) recovering the obtained precipitate.

A solvent can be selected from the group consisting of ketones, halogenated hydrocarbons, alcohols, esters and/or mixtures thereof. Ketones can be selected from C1-C4 ketones such as methyl ethyl ketone (MEK) , methyl isobutyl ketone ( IBK) , acetone and/or mixtures thereof. Halogenated hydrocarbons can be selected from C1-C5 chlorinated hydrocarbons such as dichloromethane , chloroform, dichloroethane and/or mixtures thereof. Alcohols can be selected from C1-C6 alcohols such as methanol, ethanol, isopropanol, n-butanol, 1-pentanol, 2-pentanol, 1-hexanol and/or mixtures thereof. Esters can be selected from ethyl acetate, isopropyl acetate and/or mixtures thereof. Preferably, the solvent is selected from the group consisting of methyl ethyl ketone, methyl isobutyl ketone, acetone, ethyl acetate and/or mixtures thereof. Most preferably, the solvent is methyl ethyl ketone.

An antisolvent can be selected from the group consisting of hydrocarbons, ethers and/or mixtures thereof. Hydrocarbons can be selected from C5-C10 alkanes or cycloalkanes such as hexane, heptane, octane, cyclohexane, cycloheptane and/or mixtures thereof. Ethers can be selected from dimethyl ether, diethyl ether, methyl tert-butyl ether, diisopropyl ether and/or mixtures thereof. Preferably, the antisolvent is selected from the group consisting of n-hexane, n-heptane, cyclohexane, cycloheptane and/or mixtures thereof. Most preferably, the antisolvent is n-heptane.

A further aspect of the present invention concerns tulathromycin crystalline forms I, II and III obtained by the processes of the invention disclosed herein.

A further aspect of the present invention concerns tulathromycin crystalline forms I, II and III for use in a medicament .

Further aspects are directed to tulathromycin crystalline form I characterized by the X-ray powder diffraction pattern as shown in Figure 1, tulathromycin crystalline form II characterized by the X-ray powder diffraction pattern as shown in Figure 2, and tulathromycin crystalline form III characterized by the X-ray powder diffraction pattern as shown in Figure 3. Tulathromycin crystalline forms I, II and III can be used for the preparation of pharmaceutical compositions such as e.g. tablets, capsules and injectable solutions. Injectable solutions can be prepared, for example, according to methods described in EP1250343.

The average particle size of tulathromycin crystalline forms I, II and III can range from about 0.1 to 600 μιη, more preferably 1 to 250 μτα and most preferably 10 to 50 μπι. If smaller particles are needed, the obtained crystalline form of tulathromycin can be sieved, milled or micronized optionally together with other excipients. In the case of agglomerates ultrasonication can be used.

Tulathromycin crystalline forms I, II and III can be used for the preparation of other crystalline or amorphous forms of tulathromycin or its salts, such as crystalline forms disclosed in EP1189912 and EP1189913, which can be further used for the preparation of pharmaceutical compositions.

The present invention also relates to new tulathromycin salts, namely tulathromycin besylate, tulathromycin mesylate, tulathromycin esylate and tulathromycin (-)-lO- camphorsulfonate . These salts can be prepared from tulathromycin and corresponding acid. Tulathromycin used for preparation of these salts can be in an amorphous or crystalline form such as crystalline form I, II and III according to present invention.

It was found that the products and processes of the invention as disclosed herein have advantageous properties, including their preparation and use. The invention is illustrated by reference to the following examples. However, the examples are not intended to limit the scope of the claims in any way. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the purpose and interest of this invention. The temperatures mentioned in the Examples 1-3 refer to reactor content temperature (Tr) , the same goes for cooling rates.

EXAMPLES

Example 1: Preparation of tulathromycin crystalline form III

Tulathromycin base (12.0 g) and methyl ethyl ketone (18 ml) are charged into a glass reactor, equipped with condenser and mechanical stirrer. The mixture is heated to 80 °C and maintained until a clear solution is formed. Then 30 ml of n- heptane with temperature 35 °C is added to the solution. The solution is then thermostated at 50 °C. After this temperature is reached, the crystallization mixture is further cooled with average cooling ramp 1 °C/min to final crystallization temperature, 20 °C. After the crystallization mixture is cooled to 20 °C, it is further maintained at this temperature for the next 15 hours. Obtained product is isolated with suction filtration using Btichner funnel and nitrogen atmosphere. The isolated product is dried in a vacuum dryer under reduced pressure (<100 mbar) and temperature 40 °C for 12 hours.

Yield: 3.75 g

Example 2: Preparation of tulathromycin crystalline form III

Tulathromycin base (12.0 g) and methyl ethyl ketone (18 ml) are charged into a glass reactor, equipped with condenser and mechanical stirrer. The mixture is heated to 80 °C and maintained until a clear solution is formed. Then 30 ml of n- heptane with temperature 35 °C is added to the solution. The solution is then thermostated at 50 °C. After this temperature is reached, the crystallization mixture is further cooled with average cooling ramp 1 °C/min to final crystallization temperature, -10 °C. After the crystallization mixture is cooled to -10 °C, it is further maintained at this temperature for the next 15 hours. Obtained product is isolated with suction filtration using Buchner funnel and nitrogen atmosphere. The isolated product is dried in a vacuum dryer under reduced pressure (<100 mbar) and temperature 40 °C for 12 hours.

Yield: 5.34 g

Chromatographic purity:

Tulathromycin A = 98.0 area %

Tulathromycin B < 0.9 area %

The product purity is assessed by high pressure liquid chromatography (HPLC) with a column of the type X-Bridge Shield RP-18, (75 x 4.6) mm, 2.5 m particles; detector: UV 205 ran; flow rate: 1.0 ml/min; injection volume: 5 μΐ; mobile phase: A: 0.01M phosphate buffer pH=10; B: acetonitrile ; Gradient: 0'=20%B, 15'=75%B, 20'-25'= 80%B, 27 ' -30' =20%B .

Example 3: Preparation of tulathromycin crystalline form III

Tulathromycin base (16.0 g) and methyl ethyl ketone (20 ml) are charged into a glass reactor, equipped with condenser and mechanical stirrer. The mixture is heated to 80 °C and maintained until a clear solution is formed. Then 60 ml of n- heptane with temperature 35 °C is added to the solution. The solution is then thermostated at 50 °C. At this temperature of solution the reactor jacket temperature is set to 50 °C and vacuum is applied to distill approx. 50 vol. % of solvents. During the vacuum distillation the temperature of the reactor content is not lower than 30 °C. The suspension is formed during distillation phase. Furthermore after distillation phase is completed, 80 ml of n-heptane with temperature 35 °C is added and further stirred for the next 30 minutes (reactor jacket temperature is set to 40 °C) . The obtained suspension is cooled with average cooling rate of 0.10 °C/min to final crystallization temperature, 0 °C. After the crystallization mixture is cooled to 0 °C, it is further maintained at this temperature for the next 10 hours. Obtained product is isolated with suction filtration using Buchner funnel and nitrogen atmosphere. The isolated product is dried in a vacuum dryer under reduced pressure (<100 mbar) and temperature 40 °C for 12 hours .

Yield: 13.2 g

Average particle size: 70-80 μπ\

Chromatographic purity:

Tulathromycin A = 97.3 area %

Tulathromycin B < 0.6 area %

Particle size of tulathromycin was determined by SEM Zeiss Ultra Plus microscope at 500x magnification and with Smart SEM software .

Example 4: Preparation of tulathromycin crystalline form I

The sample of tulathromycin crystalline form III is exposed to 40 °C and 75 % RH for 14 days. After the stress stability this sample is placed into sample pan for DSC analysis (cca 3 mg) and heated up to 80 °C. After the sample is cooled to room temperature, X-ray powder diffraction pattern is recorded and the result is tulathromycin crystalline form I.

Average particle size: 20-30 μπι Example 5: Preparation of tulathromycin crystalline form II

The sample of tulathromycin crystalline form III is placed into sample holder for X-Ray analysis (cca 200 mg) and heated up to 70°C for 1 hour. After 1 hour X-ray powder diffraction pattern is recorded at 70°C and the result is tulathromycin crystalline form II.

Examples 6-9: Preparation of tulathromycin salts

0.5 g of tulathromycin is dissolved in 2 ml of ethanol at 50°C, then corresponding acid (see Table 1 below) is added and ethanol is evaporated at 50 °C to obtain tulathromycin salt in an amorphous form. Examples are summarized in Table 1.

Table 1:

Example 10: Preparation of tulathromycin bis (phosphate )

A 250 mL round bottom flask was charged with (2R, 3S, 4R, 5R, 8R, 10R, 11R, 12S, 13S, 1 R) -11- ( ( (2S, 3R, 4S, 6R) -4- (dimethylamino) -3-hydroxy-6-methyltetrahydro-2H-pyran-2- yl) oxy) -2-ethyl-3, 4, 10-trihydroxy-13- ( ( ( 3S, 4S, 6R, 8R) -8- methoxy-4 , 8-dimethyl-1 , 5-dioxaspiro [2.5] octan-6-yl ) oxy) - 3,5,8, 10, 12, 14-hexamethyl-l-oxa-6-azacyclopentadecan-15-one (11.5 g; chromatographic purity: 94.1 area%; melting point: 124.7-127.2 °C; Karl-Fischer (Ph. Eur. 2.5.12): 8.80%), propan-2-ol (60 mL) and n-propylamine (31 mL) . The reaction mixture was stirred at 55 °C for approximately 31 h. The solvents were removed in vacuum. The crude product was dissolved in a mixture consisting of EtOH (abs.; 115 mL) and H20 (9 mL) . While stirring this solution, a solution of orthophosphoric acid (3.53 g) in EtOH (abs.; 52 mL) was added, dropwise. The resulting suspension was left stirring overnight. The next day the suspension was filtered and the collected solid washed with 75 mL of fresh EtOH (abs.). The resulting material was dried under reduced pressure at 35°C for 6,5 h. After this time the material was left open in air to equilibrate with air moisture after its mass stabilizes it weighed 14,372 g tulathromycin bis (phosphate ) .

Chromatographic purity: 96.6 area % (Tulathromycin A)

Melting point: 177.4-184.9 °C

Karl-Fischer: 11.63%

Example 11: Preparation of tulathromycin solution in dichloromethane

A 100 mL round bottom flask was charged with 13,5 g of the product prepared in the Example 10 and dichloromethane (52 mL) . While stirring a solution of potassium carbonate (5,83 g) in water (19 mL) was poured in the flask containing the dichloromethane suspension. After stirring for 10 minutes (evolution of carbon dioxide, two clear layers form) . The layers were separated and the aqueous layer was washed twice with dichloromethane. From the combined organic layer (82 mL) approximately 5 % (4.1 mL) were taken and the solvents removed under vacuum (the resulting solid was used for analysis purposes). The remaining 77.9 mL were used in the next step. Chromatographic purity: 96.2 area % (Tulathromycin A) Example 12: Preparation of tulathromycin crystalline form III

The above prepared solution of tulathromycin base in dichloromethane was concentrated in a glass reactor using vacuum distillation until oily residue was obtained (9.6 g) . Further, methyl ethyl ketone (12.5 ml) is charged into a glass reactor, equipped with condenser and mechanical stirrer. The mixture is heated to 80 °C and maintained until a clear solution is formed. Then 37 ml of n-heptane with temperature 35 °C is added to the solution. The solution is then thermo stated at 50 °C. At this temperature of solution the reactor jacket temperature is set to 50 °C and vacuum is applied to distill approx. 60 vol. % of solvents. During the vacuum distillation the temperature of the reactor content is not lower than 30 °C, mainly between 35 - 40 °C. The suspension is formed during distillation phase. Furthermore after distillation phase is completed, 50 ml of n-heptane with temperature 35 °C is added and further stirred for the next 30 minutes (reactor jacket temperature is set to 40 °C) . The obtained suspension is cooled with average cooling rate of 0.10 °C/min to final crystallization temperature, 0 °C. After the crystallization mixture is cooled to 0 °C, it is further maintained at this temperature for the next 12 hours. Obtained product is isolated with suction filtration using Buchner funnel and nitrogen atmosphere. The isolated product is dried in a vacuum dryer under reduced pressure (<100 mbar) and temperature 40 °C for 24 hours,

(yield: 5.2 g)

Chromatographic purity 99.1 area % (Tulathromycin A)

Tulathromycin B < 0.2 area % Example 13: Preparation of tulathromycin bis (phosphate)

A 100 mL round bottom flask was charged with (2R, 3S,4R, 5R, 8R, 10R, 11R, 12 S, 13S, 1 R) -11- ( ( (2S, 3R, 4S, 6R) -4- (dimethylamino) -3-hydroxy-6-methyltetrahydro-2ii-pyran-2- yl) oxy) -2-ethyl-3, 4, 10-trihydroxy-13- ( ( (3S, S, 6R, 8R) -8- methoxy-4, 8-dimethyl-l, 5-dioxaspiro [2.5] octan-6-yl) oxy) - 3,5,8, 10, 12, 14-hexamethyl-l-oxa-6-azacyclopentadecan-15-one (5,746 g; chromatographic purity: 93.1 area %; melting point: 116.6-122.2 °C; Karl-Fischer: 8.34%), propan-l-ol (30 mL) and n-propylamine (15,5 mL) . The reaction mixture was stirred at 56 °C for approximately 31 h. The solvents were removed in vacuum. The crude product was dissolved in a mixture consisting of EtOH (abs.; 57 mL) and H20 (4,5 mL) . While stirring this solution, a solution of orthophosphoric acid (1.339 g) in EtOH (abs.; 26 mL) was added, dropwise. The resulting suspension was left stirring overnight. The next day the suspension was filtered, washed with 35 mL of fresh EtOH (abs.). This material was dried at 35°C for 7 h. After which time it is left open in air to equilibrate with air moisture after its mass stabilizes it weighed 5.906 g tulathromycin A bis (phosphate) .

Chromatographic purity: 97.0 area % (Tulathromycin A)

Melting point: 169.8-175.1 °C

Karl-Fischer: 13.49%