A Quick and Easy Evaporative Crystallisation Screen for Drug Candidate Polymorphism

During drug development initial identification of a new Active Pharmaceutical Ingredient (API) usually yields an amorphous form of the compound. However, compounds which are crystalline in nature often adopt a number of crystalline forms or polymorphs. The different physical characteristics of these polymorphs can impact on the manufacturing process as well as the efficacy of the drug and initial screening undertaken on an amorphous form may be misleading, as crystallisation may change properties such as dissolution rate and biological activity. Polymorph screening is therefore an important stage in the drug development process, the aim being to identify the different crystalline structures that a drug may adopt. Information gained is used to optimise the physical properties of the drug compound, to ensure efficacy, and provide formulation and manufacturing consistency.

In this study Medicinal Chemists from one of our pharmaceutical customers in Japan evaluated the Exalt Controlled Crystallisation system from Genevac as a method of polymorph screening in their drug discovery programme. To evaluate the process a widely available compound, Piroxicam, which is known to form three different polymorphs [1] was chosen.

Method

Exalt is a method for evaporative crystallisation developed by Genevac which enables solutions of API in a wide range of solvents to be evaporated at the same time, and all at the same slow rate, producing crystals. Exalt uses a special holder for vials which allows a selection of baffles to be placed on top of each vial to slow the evaporation rate of volatile solvents (Figure 1). The size and number of baffles are selected to be most restrictive for the most volatile solvents, and least restrictive to the less volatile solvents. The holder is then placed in a Genevac HT series evaporator which cycles at atmosphere and at a slightly reduced pressure for the duration of the evaporation process.

Solutions of Piroxicam were prepared in a range of solvents (see Table 1) to yield a solution of 2mg/ml. 3ml of each solution was placed into a vial and capped with a tower, containing baffles, as recommended by Genevac [2]. The lower volatility of six solvents meant that no tower was required. In addition, to ensure complete evaporation by the end of the run, three of these solvents also required a reduction in initial volume (concentration of these solutions was corrected to yield 6mg per vial). The complete holders were then placed in to a Genevac HT-4X evaporator, running the Exalt programme, for 72 hours.

Results

Table 1 lists the 20 solvents screened, the tower configuration, and physical appearance after 72hr evaporation. Seven vials had not fully evaporated and required further evaporation in the HT4X. Subsequently 19 out of the 20 solutions yielded a crystalline solid suitable for X-Ray Diffraction (XRD) analysis. Analysis of XRD results (Figure 2) indicates that the Exalt screening method was able to identify polymorphs of types I, II and III (Table 2).  For three solvents results were inconclusive and whilst these were thought to be solvates further investigation would be required to confirm this.

Table 1. Solvents screened, Exalt configuration, and physical appearance of product after 72 hours evaporation.

No.    Solvent    Volume    Tower    Physical Appearance

after 72hr

1    Dichloromethane    3ml    #21    Powder

2    Tertiary Butyl Methyl Ether    3ml    #17    Solid

3    Acetone    3ml    #16    Crystal

4    Methyl Acetate    3ml    #16    Solid

5    Chloroform    3ml    #14    Solid

6    Tetrahydrofuran    3ml    #12    Solution (1ml)

7    Hexane    3ml    #15    Solution (0.5ml)

8    Methanol    3ml    #10    Needle crystal

9    Cyclo Hexane    3ml    #9    Powder

10    Ethyl Acetate    3ml    #10    Solution (0.5ml) and needle crystal

11    Methyl Ethyl Ketone    3ml    #10    Solution (0.5ml)

12    Acetonitrile    3ml    #4    Solution (0.5ml)

13    1,2-Dimethoxy Ethane    3ml    #1    Solution (0.5ml)

14    Ethanol    3ml    None    Solid

15    Isopropyl Acetate    3ml    None    Candy

16    Heptane    3ml    None    Powder

17    Isopropyl Alcohol    1ml    None    Powder

18    Toluene    2ml    None    Solution (0.5ml)

20    1,4-Dioxane    1ml    None    Solid

21    Benzene    1ml    None    Cubic crystal

Table 2. Crystalline Polymorphisms as identified by XRD analysis.

No.    Solvent    Crystal Polymorph Form

1    Dichloromethane    I & II

2    Tertiary Butyl Methyl Ether    I

3    Acetone    I

4    Methyl Acetate    I

5    Chloroform    III

6    Tetrahydrofuran    I

7    Hexane    I

8    Methanol    I

9    Cyclo Hexane    I

10    Ethyl Acetate    Not identified

11    Methyl Ethyl Ketone    Not identified

12    Acetonitrile    I

13    1,2-Dimethoxy Ethane    Not identified

14    Ethanol    II

16    Heptane    I

17    Isopropyl Alcohol    I

18    Toluene    I

20    1,4-Dioxane    I

21    Benzene    I

Conclusion

Exalt Controlled Crystallisation allows the quick and easy screening of an API with minimal compound (6mg per vial). Piroxicam was screened using 20 solvents, identifying three polymorphic forms, utilising just 150mg of compound. In addition the method is non-destructive meaning, where crystals are not formed, the compound may be re-dissolved for further use.

 New compounds purified by reverse phase chromatography and dried by centrifugal evaporation are usually amorphous. Crystallisation changes properties such dissolution rate and biologically active so ideally polymorphisms should be identified before the compound is taken forward. In reality initial screening is often performed on amorphous compound. Erroneous results derived from amorphous compound may mean a candidate API is needlessly rejected, or is further developed only for the crystalline form to be found unsuitable at a later stage. Opportunity to obtain crystals easily at an early stage of drug discovery, with minimal compound, provides the ability to identify polymorphisms, work up potential hits more efficiently, and provide seed crystals / solvent data for scale up.

References

1. Vrecer F, Vrbinc M, Moden A (2003). Characterization of Piroxicam Crystal Modifications. International Journal of Pharmaceutics, Vol.256(1-2), 3–15.

2. Darrington R (2014). New technology for pharma small-molecule crystallisation. Lab Product News, http://www.labcanada.com/features/pharmaceutical-small-molecule-crystallization/.