PREPARATION THEREOF

FIELD OF THE INVENTIONS

The present invention relates to a solid state form of Tiacumicin B and to a process for preparing said solid state form. The process according to this invention is more efficient than methods known in the art and is easily scalable for commercial production.

BACKGROUND

Tiacumicin B belongs to the family of Tiacumicins; Tiacumicins, produced by fermentation of Dactylosporangium aurantiacum, are a group of unsaturated 18-membered macrocyclic of the macrolide class that differ in the type of substituents of the unsaturated ring. Tiacumicin B has the structure shown below in the following Formula (I):

Tiacumicins and in particular Tiacumicin B show activity against a variety of bacterial pathogens including Clostridium difficile.

The Lipiarmycins are another class of product closely related to the Tiacumicins. In particular Tiacumicin B is identical to Lipiarmycin A3.

Lipiarmycin was first isolated from Actinoplanes deccanensis in the 1970s. The earliest patent by Lepetit U.S. Pat. No. 3,978,21 1 already described an anhydrous crystalline form of Lipiarmycin. Even if the PX D are not reported in said patent, however the melting point and infrared spectra are reported. This is sufficient to identify univocally the crystalline form (herein defined as Form I). In the above mentioned patent example of crystallization a procedure is described with anhydrous organic solvents leading anhydrous crystalline form of Tiacumicin B, as demonstrated by DSC data related to a product obtained with the same procedure and included in the present invention (Figure 1).

US 7,378,508 and its European counterpart EP 2 125 850 Bl disclose different crystalline forms of Tiacumicin B, namely Form A, Form B and amorphous. US 2013/0303472 claims three additional crystalline forms of which two are solvated (forms Z and Zl) and one anhydrous non solvated -form (form C).

The processes described in the prior art suffer for some drawbacks: in US 7,378,508 the procedure requires about 3 to about 14 days, therefore it is not suitable for industrial application. In patent application US 2013/0303472, more suitable processes are described, but on the other hand they require high stressed drying conditions. It is known, as described in US 2008/0176927, that Tiacumicins have stability issues and therefore high temperature drying (> of 40°C) lead to degradation of the molecule. Therefore neither the procedure described in the US 7,378,508, nor the procedure described in the application US 2013/0303472 are suitable for commercial production.

It is known that the occurrence of active ingredients in different crystal polymorphs (polymoφhism) is of great significance both for development of preparation processes and for development of formulations. Different crystal polymorphs of a chemical compound, in addition to appearance (crystal habit) and hardness, also differ in numerous further physicochemical properties, like for example stability, filterability, solubility and hygroscopicity. Thus a given crystalline form of a particular API often constitutes an important determinant of the API's ease of preparation, stability, solubility, shelf-life, ease of formulation, rate of the dissolution in the gastrointestinal tract and other fluids, and in vivo bioavailability. Choice of a crystalline form will depend on a comparison of physical properties variables of the different forms.

The present inventions provides an improved crystallization method for Tiacumicin B that surprisingly results in a new polymorphic form namely Form II. The method according to the inventions is simpler than those described in the prior art and preserves the quality of the product. This provides a new opportunity to improve and facilitate the handling and storage of Tiacumicin B as active ingredients.

SUMMARY

The object of this invention is a new form of Tiacumicin B called Form II.

Crystalline Form II of Tiacumicin B has the DSC diagram shown in Figure 4, characterized by a endothermic peak at 144.67°C in the DSC diagram.

Form II has the X ray powder diffractogram (X PD) shown in Figure 5, characterized by a system of main diffraction peaks expressed in 2-theta degree [°] at 3.31± 0.2 - 6.65 ± 0.2 -7.38 ± 0.2 - 8.3 ± 0.2 - 9.92 ± 0.2 - 10.42 ± 0.2 - 12.07 ± 0.2 - 12.8 ± 0.2 - 13.22 ± 0.2-13.95 ± 0.2 -14.22 - 14.26 ± 0.2 - 15.62 ± 0.2 - 18.6 ± 0.2 - 19.77 ± 0.2 - 20.92 ± 0.2.

Another object of the present invention is a method for obtaining Form II of Tiacumicin B by means of crystallization of crude Tiacumicin B and heating at 60°C-80°C in a suitable organic solvent.

BRIEF DESCRIPTION OF FIGURES

Fig. 1 : DSC diagram of Tiacumicin B Form I

Fig. 2: XRPD diffractogram of Tiacumicin B Form I

Fig. 3 : IR spectrum of Tiacumicin B Form I

Fig. 4: DSC diagram of Tiacumicin B Form II

Fig. 5: XRPD diffractogram of Tiacumicin B Form II DETAILED DESCRIPTION OF THE INVENTIONS

Crude Tiacumicin B can be obtained according to known methods described in the art (U.S. Pat. No. 3,978,21 1 ; U.S. Pat. No. 7,507,564; MI2012A001406; MI2012A000560).

Crude Tiacumicin B can be obtained for example trough a process comprising the steps of:

a) Filtration of the fermentation broth

b) Absorption to a HIC resin followed by elution

c) Insolubilization to afford crude Tiacumicin B

It has now been found that, depending on the crystallization conditions of the crude product, Tiacumicin B can be obtained in two distinct polymorphic forms, herein called Form I and Form II.

Polymorphic form I of Tiacumicin B is the form known to the skilled person which is cited in the prior art (U.S. Pat. No. 3,978,21 1); Polymorphic form II of Tiacumicin B is the object of this invention.

The two different polymorphic modifications are selectively obtainable by suitably adjusting the conditions applied in the crystallisation process.

It has also found that the two different polymorphs can be inter-converted from Form I to Form II and vice versa by applying suitable re-crystallisation conditions.

In particular, by operating as described in the U.S. Pat. No. 3,978,21 1 an anhydrous crystalline Tiacumicin B with the following characteristics is obtained:

1) a DSC diagram as shown in Figure 1, characterised by a endothermic peak at 179,81°C an XRPD diffractograms shown in Figure 2, characterised by a system of main peaks expressed in 2-theta degrees [°] at 3,3± 0,2 - 6,74 ± 0,2 - 7,83 ± 0,2 -9,94 ± 0,2 - 10,14 ± 0,2 - 12,1 ± 0,2 - 13,22 ± 0,2 - 13,76 ± 0,2 - 14,5 ± 0,2 - 14,97 ± 0,2 - 18,78 ± 0,2 - 19,9± 0,2 - 20,22 ± 0,2 - 21 ± 0,2.

2) the IR spectrum shown in Figure 3 3) a melting point of 173°C.

The data clearly demonstrate that the product obtained following the process reported in literature corresponds to Form I of anhydrous crystalline Tiacumicin B, known to the prior art.

It is also evident that the obtained Form I of Tiacumicin B differs considerably from the form we obtained and called Form II, in terms of the chemical-physical properties analysed (DSC, X PD diffractograms).

Object of the present invention is the polymorphic Form II of Tiacumicin B which in pure anhydrous crystalline form has the following characteristics:

1) a DSC diagram as shown in Figure 4, characterised by a endothermic peak at 144,67°C

2) an XRPD diffractograms shown in Figure 5, characterised by a system of main peaks expressed in 2-theta degrees [°] 3,31± 0,2 - 6,65 ± 0,2 -7,38 ± 0,2 - 8,3 ± 0,2 - 9,92 ± 0,2 - 10,42 ± 0,2 - 12,07 ± 0,2 - 12,8 ± 0,2 - 13,22 ± 0,2 - 13,95 ± 0,2 - 14,22-14,26 ± 0,2 - 15,62 ± 0,2 -18,6 ± 0,2 - 19,77 ± 0,2 - 20,92 ± 0,2.

Another object of this invention is a process for preparing the crystalline Form II of Tiacumicin B as defined above, comprising the steps of:

a) dissolving Tiacumicin B in an organic solvent under stirring at the reflux temperature, until the product has completely dissolved;

b) cooling the solution first at room temperature and then at 4°C to complete crystallisation.

Preferably the solvent is a polar organic solvent, such as propanol, acetone, acetonitrile and isopropanol, preferably the solvent is isopropanol.

Crude Tiacumicin B is obtained by methods known in the art (U.S. Pat. No. 3,978,21 1 ; U.S. Pat. No. 7,507,564; MI2012A001406; MI2012A000560).

A further object of the present invention is a pharmaceutical composition comprising the crystalline Form II of Tiacumicin B as above defined and a pharmaceutically acceptable excipient. Said pharmaceutical composition can be used in treating or preventing bacterial infections in a mammal.

Example 1: Preparation of Tiacumicin B Form I (comparative example)

Crude Tiacumicin B was dissolved in methanol at a concentration of 200 g/1. The resulting solution was loaded onto a column packed with HP20SS resin (1 1-50x5 cm) that had previously been equilibrated with 5 Bed volumes (BV) of acetate buffer at pH 5,0. The column was washed with 10 BV of acetonitrile in acetate buffer pH 6,0 (the % of acetonitrile ranges from 10% to 30%). Tiacumicin B was eluted with 5 BV acetonitrile in acetate buffer pH 6,0 (the % of acetonitrile ranges from 30% to 45%). The column eluate was divided into fractions and each fraction was analyzed by HPLC to assess purity. Fractions with a purity greater than 95% were pooled and concentrated; the concentrate was extracted with ethyl acetate. The ethyl acetate layer was washed with 2 volumes of water, concentrated to yield a oil. The resulting oil from the previous step was dissolved in methanol; the solution is warmed and added of diethyl ether and n-pentane. The solution was cooled and filtered. The solid was dried under vacuum over night.

XRPD diffractograms system of main peaks expressed in 2-theta degrees [°] at 3,3 ± 0,2 - 6,74 ± 0,2 - 7,83 ± 0,2 - 9,94 ± 0,2 - 10,14 ± 0,2 - 12,1 ± 0,2 - 13,22 ± 0,2 - 13,76 ± 0,2 - 14,5 ± 0,2 - 14,97 ± 0,2 - 18,78 ± 0,2 - 19,9 ± 0,2 - 20,22 ± 0,2 - 21 ± 0,2. Melting point of 173°C.

Example 2: Preparation of Tiacumicin B Form II

Crude Tiacumicin B was added of isopropanol at a concentration of about 300 g/1. The resulting suspension, placed under stirring, was heated at 65°C; The solid product completely dissolved. The solution was cooled at room temperature and then at 4°C. The solid obtained was dried under vacuum over night.

XRPD diffractograms system of main peaks expressed in 2-theta degrees [°]

3,31 ± 0,2 - 6,65 ± 0,2 - 7,38 ± 0,2 - 8,3 ± 0,2 - 9,92 ± 0,2 - 10,42 ± 0,2 - 12,07 ± 0,2 - 12,8 ± 0,2 -1 3,22 ± 0,2 - 13,95 ± 0,2 - 14,22-14,26 ± 0,2 - 15,62 ± 0,2 - 18,6 ± 0,2 - 19,77 ± 0,2 - 20,92 ± 0,2. Endothermic peak at 144,67°C.