Polymorphism of Estramustine

Polymorphism of Estramustine TOMMYWADSTEN' AND NILS-OLOFLINDBERG*' Received April 26,1988,from 'Pharmacia LEO Therapeutics AB, Pharmaceutical Department, Box 941, S-257 09 Helsingborg, Sweden, and Accepted for publication December 21, 1988. *Development & Research WAfl, Surbrunnsgatan 58, S-17327 Stockholm, Sweden. ~.

~~~

~-

Abstract As the solubility in water of the cytotoxic drug estramustine is <1 mgiL, polymorphism can have an impact on the bioavailability of orally administered drug. Therefore, the solid state characteristics of estramustine samples, crystallized from different solvents, were investigated by means of X-ray crys'tallography, thermal analysis, and IR spectroscopy. The DSC data indicated the existence of several phases. Four forms-A, B, C, and Il-were confirmed. Phase A was obtained when crystallizing from solvents with a moderate or low dielectric constant (less than -24). This form was an anhydrate and found to be the stable one. When crystallizing from methanol, metastable solvates, with -0.5 mol for phase B or -1 mol for form C, were precipitated. Both types were transformed to phase A during storage. This desolvation was accelerated by bleating. Crys;tallizing from a mixture of acetone and water resulted in a monohydrate, farm D, which was converted to the anhydrous type A upon heating. As forms 8, C, and D were solvates which transformed to another crystal form upon desolvation, they were polymorphic solvates of the anhydrate, type A. Symmetry and unit cell dimensions of the stable form of estramustine (phase A) were determined by mean:; of a single-crys,talX-ray technique (orthorhombic, a = 23.90,b = 20.69,c = 8.76,space group P2,2,2,). In addition, the crystallographic parameters of form D were deduced from calculations based on powder diffraction data.

Estramustiine, estradiol3-[bis(2-chloroethyl)carbamatel,is one of the main metabolites of estramustine phosphate,' a drug used in the treatment of prostate cancer. Consequently, it is natural to examine the possibilities of using estramustine itself as the drug instead of. the phosphate ester. In water or gastrointestinal fluids, the solubility of estramustine is <1 mgiL. It is known that polymorphism can have an impact on the bioavailability of slightly soluble drugs.2 Preliminary differential scanning calorimetry (DSC) data on estramustine were complex and difficult to interpret; however, they indicated that polymorphism could not be excluded. Because of the low solubility and the indications of polymorphism, it was important to investigate the solid state characteristics of estramustine.

Experimental Section Materials-Estramustine was produced by Gaeleo, Cork, Ireland. The final step was crystallization from a methanolic solution by cooling. Drying of the centrifuge cake was performed at 30 "C in a 0022-3549/89/L~700-0563$0 1.0(7/0 0 7989, American Pharmaceutical Association

Table I-Dielectric Solvent

Constants of Solvents at 25 'Ce Dielectric Constant

Water Methanol Ethanol Acetone 2-Propanol Ethyl acetate lsopropyl ether mHexane a

78.5 32.6

24.3 20.7

18.3 6.0 3.9 1.9b

Data from refs 3 and 4. Determined at 20 "C.

drying oven. On one occasion, a vacuum dryer with horizontally rotating paddles was used. The drug was also crystallized on a laboratory scale from other solvents with different dielectric constants (see Table 11.334 In addition, recrystallization in methanol and from equal parts of acetone and water was performed on a laboratory scale. The crystals were collected and dried in a desiccator. X-ray Crystallography-A large crystal recrystallized from nhexane was chosen and studied by means of a single-crystal technique, employing a Weissenberg camera, in order to reveal the symmetry and cell dimensions. The powder samples were analyzed by means of X-raytransmission powder diffraction in a Guinier-Hagg camera with photographic recording, using KC1 (a = 6.2930 A) as a n internal standard and CuK,, radiation. Ry mathematical refinement of the observed data using a trial-and-error indexing program,5 the cell parameters were calculated. The film strips were measured with an optical film scanner in order to obtain accurate geometrical data and intensity distribution. The strongest relative line intensity for each sample has been adjusted to 100 in Table 111. In order to register low-angle diffraction lines, a standard reflecting Philips diffractometer with Cu radiation (Eindhoven, The Netherlands) was used. In some cases, a Rigaku Denki diffractometer (Tokyo, Japan) was employed as well. Thermal Analysis-Thermogravimetric analysis and its first derivative and DSC were recorded with standard set-ups, manufactured by Du Pont (Wilmington, DE), Perkin Elmer (Norwalk, CT), Setaram (Saint-Cloud, France), and Stanton Redcroft (London, UK). All measurements were run at the standard heating rate of 10 "Cimin in air. The sample sizes were on the order of 5-10 mg. The temperatures of the DSC peaks were taken a t the maximum heat flow. Infrared Spectroscopy-The IR spectra were recorded at 4000650 cm-l in a Perkin Elmer 157 instrument, applying the Nujol-mull technique with light grinding. Scanning Electron Microscopy-The samples were coated with gold in an ion sputter (Jeol JFC-1100, Tokyo, Japan) and observed in a scanning electron microscope (Jeol JSM-T 200) at magnifications ranging from 200 to 3500 times. Nuclear Magnetic Resonance Spectroscopy-The methanol content of some batches was checked by means of NMR in CDCl, in a Bruker instrument (AC 250, Karlsruhe, West Germany). In the sample crystallized from a mixture of acetone and water, the acetone content was also checked by means of NMR. Water Content-The water content of the sample crystallized from a mixture of acetone and water was determined by means of titration according to Karl Fischer. As the methanol or acetone content was specifically determined, thermogravimetry was carried out as a complementary water analysis. Journal of Pharmaceutical Sciences I 563 Vol. 78, No. 7, July 1989

Table 11-Crystallographic Estramustine

A Form D Form Weissenberg Single-crystal Powder Technique Diffractiona Diffraction

Data ~~

~

~~

Orlhorhombic Orthorhombic Orthorhombic

Symmetry a, A

13.977(9) 24.89(1) 6.645(4) 4

23.90(2) 20.69(1) 8.758( 7) 8

b, c, A Z

Density (calculated), g/cm3 Volume, A3 Space group a

Data for Polymorphs A and D of

1.27

1.35 4328 p212121

14.00(1) 24.95( 1) 6.655( 1) 4 1.26

2312 p212121

2324 p212121

From ref 8.

92.5 126.8'127.8

Results and Discussion Crystals of Form A-Symmetry and unit cell dimensions were determined by means of a single-crystal technique applied to large crystals recrystallized from n-hexane. These data are summarized in Table 11. The cell dimensions were further refined based on 39 observed data points. The results of the X-ray powder diffraction measurements with estramustine crystallized from ethanol, acetone, 2propanol, ethyl acetate, isopropyl ether, or n-hexane indicate crystals of type A. These solvents have dielectric constants from 24.3 for ethanol down to 1.9 for n-hexane (see Table I). This means that type A is obtained directly from solvents of moderate or low polarity. The characteristic X-ray powder diffraction patterns are presented in Table 111. The first three signals of type A in Table I11 were registered by means of a reflecting technique. The DSC thermograms of phase A, which is the anhydrate, are characterized by one endothermic peak only, with the melting point at -127 "C (Figure 1). The aim of our DSC measurements was not to make exact determinations of the desolvation (dehydration) temperature or melting point, but to obtain relevant DSC spectra. Four different brands of DSC equipment had to be used during the study. When a sample was tested in the different set-ups, there was an agreement of L 1 "C regarding the characteristic temperatures. However, the temperatures obtained with the Setaram equipment deviated from those of the other three because of a different construction; that is, the heating elements were placed in the walls of the calorimetric block and not in the sample holder. Therefore, the temperature scale from the Setaram thermograms was displaced so that the summits of these thermograms coincided with the corresponding summits of a thermogram from any one of the other three brands.

I1

I

20

40

I

I

60

80

I

I

1

100 120 140

Temperature, * C Figure I-The

DSC curves of estramustine types A and D. Form A: melting point; form D: the first peak corresponds to dehydration and the second peak is the melting point.

Crystals of Forms B and C-When estramustine is crystallized from methanol, solvates are obtained with -1 mol of methanol for phase C or -0.5 mol of methanol for phase B. Methanol measurements were made by means of the NMR technique, and the results were confirmed by thermogravimetry. According to Table I, methanol is a more polar solvent than the other solvents in the table which gave phase A. The crystals which are small plates similar to form A, -10 pm in the largest dimension, produce other X-ray powder diffraction patterns (see Table 111). Crystals of phase C are more irregular than those of form B. Due to the minute size of the crystallites, it was not possible to apply normal single-crystal methods to determine the proper symmetry and cell dimensions of type B. The microscopic exposures revealed thin plate-shaped crystals. By using a reflecting X-ray diffraction technique on a deliberately oriented sample and on another sample (where orientation effects were avoided) very sharp peaks were registered at d = 11.0 and d = 21.8 respectively. These signals may thus correspond to the layers in a two-dimensional lattice. As for type C, a reflecting technique similar to the one used with form B did not yield any conclusive evidence of layers. The first part of the characteristic and indexed X-ray powder patterns of types B and C are summarized in Table 111. The DSC thermogram of form B is characterized by two or three endotherms with temperatures at -50,70, and 127 "C,

A,

Table Ill-Characteristic X-ray Powder Patterns of the Four Polymorphs of Estramustine ~~

~

Type A d obsb d calc.b

Int.a

Type B hklC

Int.

d obs.

Type D

Type C

Int.

d obs.

Int.

~

14 5 45 91 25 31 22 53 15 11 9 a

d obs.

16.3 12.3 10.7 6.73 5.97 5.95 5.85 5.73 5.20 4.47 4.38

15.7 12.0 10.5 6.68 5.97 5.97 5.83 5.74 5.21 4.48 4.38

110 200 210 21 1 400 230 410 31 1 321 331 141

16 9 8 14 22 13 11 83 47 11 100

21.8 12.7 11.0 7.57 7.21 6.74 6.63 6.22 5.72 5.10 5.04

37 30 36 84 100 7 21 7 33 34 51

15.0 13.2 10.3 8.34 7.88 7.41 7.24 7.13 6.47 6.37 6.23

40 7 12 51 5 14 50 32 7 49 13

9.30 7.14 6.99 6.73 6.43 6.10 5.84 5.41 5.20 4.87 4.82

Relative intensity scale. The interplanar spacing, A, observed and calculated, respectively. The reflecting plane indices.

564 I

d calc.

hkl

9.31

120 130 200 210 011 220 111 121 031 131 201

~~

Journal of Pharmaceutical Sciences Vol. 78, No. 7,July 1989

7.15 7.00 6.74 6.43 6.10 5.84 5.41 5.20 4.87 4.82 -

r

\\8\\\,q-7 z i?.

UI

z s

67.8

\ \

\

h

\ \

I

\

I I

\ \ \

c

-------- - -- ..-.

-...

127.3

20

40

60

80

100 120 140 160 Temperature, "C

.... 180 200

Figure 2-The IISC curve (continuous line) and thermogravimetrical curve (dashed line) for estramustine type B. The first weight-dependent peak correspondls to desolvatiori and the second point is the melting point.

_ _ _ _ _ _ _ _ _ _ _-_

,_---

_--

2 rr c 0

m

E 5 P C

i 12 6.6

20

40

60 80 100 Temperature, 'C

120

140

Figure &The DSC curves (cointinuous line) for estramustine types A

and C, and the first derivative of the thermogravimetrical curve (dashed line) for estramustine phase C. Melting points of forms A and C are also shown. but the first endothemi is riot always present (Figure 2). An exothermic efrect was observed between the second and the third endotherm. Phase C has three endothermic peaks at -70, 86, and 127 "C (Figure 3). Simultaneous DSC and thermogravimetric measurements indicate that the first two endotherms on the DSC thermogram coincided with the changes on the thermogravimetric curves (see Figures 2 and 3 ) . Thus, the first two endotherms at -50 and 70 "C for form B and -70 and 86 "C for type C were caused by loss of methanol from the crystal lattice. After the loss of solvent, the sample was tested by X-ray powder diffractometry, and the X-ray pattern of form A was obtained. Phase C did not transform into B before the stable end product, phase A. The IR data indicate that forms B and C, which had identical IR slpectra except for the OH stretch region (-37003000 cm-'1, deviated from phase A with a peak at 3300 cm-' (Figure 4). This peak seems to be caused by hydrogen-bonded hydroxyl groups. The I!R spectra within the OH stretch region are not identical for types B and C. During storage, both forms B and C lost the crystal solvent and were converted to phase A. The time for this transfor-

4000

3000

2000

1800

1600

1400

1200

1000

800 650

Wave number, cm-l

Figure &The IR spectrum for estramustine type A. The spectra for forms 8, C, and D are similar to that of phase A. The most strikingly inconsistent parts have been marked by dotted lines and transposed above the curve of Type A.

mation depended above all on storage temperature and the size of the aggregates. Mixtures of A and B or A and C were registered during the beginning of storage until a complete transformation to type A had occurred. A rapid transformation to form A is obtained by heating at -90 "C. There are different views among authors regarding nomenclature. A review article states that solvated (hydrated) crystals do not meet the definition of polymorphism.6 On the other hand, different solvates and/or anhydrates are referred to as polymorphs.7 The latter definition has been used in this article. Further, solvates that transform to another crystal form upon desolvation are polymorphic solvates.7 Consequently, forms B and C are polymorphs or polymorphic solvates of type A. Crystals of Form D-When estramustine was crystallized from a mixture of two parts of acetone and one part of water (i.e., a medium with a high dielectric constant), a monohydrate was obtained.8 The crystallographic parameters deduced from this single-crystal investigation, are also summarized in Table 11. The sample recrystallized from equal parts of acetone and water was tested by X-ray powder methods and the results are given in Table 111. These results were refined (47 observed data points) according to the indexing program,K and the parameters obtained from these calculations are summarized in Table 11. No sign of acetone was observed by NMR. The water content was very close to 1 mol/mol of estramustine. The X-ray powder data confirmed the parameters from Punzi et al.8 Consequently, this sample was the monohydrate, phase

D. There are two endotherms at -93 and 127 "C on the DSC thermograms of form D (Figure 1). The first endotherm corresponds to the loss of crystal water, whereas the second endotherm is the melting point of phase A, which is formed when the crystal water is lost. This was verified with powder diffractometer testing. As can be seen on the DSC diagram, dehydration starts at 45-50 "C, indicating a rather unstable hydrate. The IR curve of form D deviates both from phases B and C and from form A. Thus, the carbonyl group peak at 1716 cm-' Journal of Pharmaceutical Sciences I 565 Vol. 78, No. 7, July 1989

has been shifted to 1690 cm-I (see Figure 4).However, the IR spectra within the OH stretch region (-3700-3000 cm-l) of both types C and D are very similar. Besides, the monohydrate has a peak at -1670 cm-I that is missing with forms B and C, which is characteristic of hydrates. A rapid transformation of type D to form A is obtained a t -100 "C (Figure 1). Consequently, this type is also a polymorphic solvate of the anhydrate. Both the solvates of methanol, phases B and C , were unsuitable as drugs because of the methanol content. However, the anhydrous form A might be used as the administered drug substance as well as the monohydrate.

4.

5. 6. 7. 8.

M. J.; Beyer, W. H., Eds.; CRC: Boca Raton, FL, 1984; pp E-49E-52. Martin, A.; Swarbrick, J.; Cammarata, A. In Physical Pharmacy, 3rd ed.; Lea & Febiger: Philadelphia, PA, 1983; p 126. Farkas, Laszlo; Werner, Per-Erik 2.Kristallogr. 1980, 151, 141152. Burger, Arthur Pharm. Int. 1982,3, 158-163. Byrn, Stephan R. Solid-state Chemistry ofDrugs; Academic: New York, 1982; pp 6 1 0 . Punzi, J. S.; Strong, P.D.; Pangborn, W.A.; Duax, W. L. Abstracts, National Meeting of the American Cr stallographic Association; Austin, TX, March, 1987; Americar Crystallographic Association: Buffalo, NY, 1987; Abstract PE 11.

References and Notes

Acknowledgments

1. Andersson, Sven B.; Gunnarsson, Per 0.;Nilsson, Torgny; Plym Forshell, Gustaf.Eur. J . Drug Metab. Pharmacokinet 1981,6,149154. 2. Fiese, Eugene F.; Hagen, Timothy A. In The Theory and Practice of Industrial Pharmacy; Lachman, L; Lieberman, H. A.; Kanig, J. L.; Eds.; Lea & Febiger: Philadelphia, PA, 1986; pp 176-178. 3. Handhook of Chemistry and Physics, 65th ed.; Weast, R. C.; Astle,

Many ersons at Pharmacia LEO Therapeutics AB have contributed to &is work. Their contributions are gratefully acknowledged. We are especially indebted to the following persons for their valuable contributions: Dr. Ann-Charlotte Eliasson, The University of Lund, for art of the DSC measurements, and Mr. Arne Olsson, Pharmacia L E 8 Therapeutics AB, for IR and NMR measurements and interpretations.

566 I Journal of Pharmaceutical Sciences Vd. 78,No. 7,Ju/y 1989