CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 4 441-444
Perspective How Many “New” Entities Can Be Created from One Active Substance? The Case of Cyclosporin A Alexandr Jegorov,*,§ Michal Husˇa´k,† Bohumil Kratochvı´l,† and Ivana Cı´sarˇova´# IVAX Pharmaceuticals, Research Unit, Branisˇ ovska´ 31, 370 05 C ˇ eske´ Budeˇ jovice, Czech Republic, Department of Solid State Chemistry, Prague Institute of Chemical Technology, Technicka´ 5, 166 28 Prague 6, Czech Republic, and Department of Inorganic Chemistry, Charles University, 128 43 Prague 2, Czech Republic Received March 17, 2003
ABSTRACT: The existence of various crystalline forms of active substances, which can potentially differ in their pharmacokinetic profile, bioavailability or stability, creates the possibility of extending the patent protection of “old” substances utilizing the structures of new crystalline forms. Using cyclosporin A as an example, this report points to the fact that even with relatively small molecules, having about 60 non-hydrogen atoms, the number of “new” entities that can be generated can be at times nearly infinite. It is fairly well documented1 that different polymorphs, hydrates, or solvates of one active substance can significantly differ in their density of packing, network of intermolecular hydrogen bonds, etc., which can affect their physical and chemical behavior. Hence, various forms may have different dissolution kinetics and thus also different pharmacokinetic profile and bioavailability. Accordingly, individual crystalline forms may be thought of as distinct solids having distinct advantageous and/or disadvantageous physical properties compared to other forms. This fact recently initiated a series of patents aimed to obtain an additional protection of sometimes very old, but commercially attractive, substances as, for example, in the case of paclitaxel.2,3 Although there is no doubt about certain advantages of particular crystalline forms, this paper is intended to show that due to the presence of various cavities in the structures, the increasing size of an organic molecule increases the number of possible combinations among the host molecule and their various guests. These combinations will always have “slightly” different physical properties, content of solvents, or residual solvents that can exhibit different possible risks to human health (ICH class), etc. Since the criteria for “novelty” and “difference” are not well established, it should be possible, based on this general feature of crystalline matter, to create a large number of “new” entities from one active substance. It is noteworthy to mention that using * To whom correspondence should be addressed. Tel: 420-385310331. Fax: 420-38-5310397. E-mail:
[email protected]. § IVAX Pharmaceuticals. † Prague Institute of Chemical Technology. # Charles University.
Figure 1. Structure of cyclosporin A.
this approach it also would be possible to prolong the patent protection of many already generic substances. On the other hand, in a number of cases, patent claims are mainly based on X-ray powder patterns, which refer to any possible isostructural series of compounds regardless of their real composition. Thus, this paper is aimed to open the discussion among scientists, lawyers, and registration agencies about the future rules in this relatively new field of pharmaceutical research. Cyclosporin A, Figure 1, is a cyclic undecapeptide widely used to prevent graft rejection in transplantations or for the treatment of various autoimmune diseases {CsA, ) cyclo(-MeBmt1-Abu2-Sar3-MeLeu4Val5-MeLeu6-Ala7-D-Ala8-MeLeu9-MeLeu10-MeVal11-), where MeBmt ) (4R)-4-[(E)-2-butenyl]-4,N-dimethyl-Lthreonine}. The X-ray structure of CsA was reported first by Loosli et al.4 (dihydrate, space group P41). The second crystalline form was originally considered to be anhydrous,5 then as ethanol solvate (0.2) hydrate (0.4),6
10.1021/cg0300127 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/10/2003
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Figure 2. Solvent accessible areas in structures of cyclosporin A on the 1 Å level (1 Å distance from the VdW sphere of the nearest atom). Top images: solvent accessible area in the 42 × 42 × 20 Å area of the crystal (map orientation parallel to the a,c lattice plane for structure of cyclosporin A bis-acetone hydrate (space group P41) on the left side and to the a,b lattice plane for structure of of cyclosporin A dibutyl ether clathrate (space group P21) on the right). Bottom images: solvent orientation in the cavity for above structures. Water molecule in cyclosporin A bis-acetone hydrate is involved in the hydrogen bond. Therefore, the oxygen atom is slightly outside the calculated cavity (011-0101 ) 2.716 Å).
and was finally refined as CsA monohydrate (space group P212121).7 This structure exhibit also acetyl derivatives of CsA.8 Another orthorombic form was mentioned only in the patent,5 but was never refined (diisopropyl ether solvate, space group P212121). Last structural type of CsA was reported with dimethylisosorbide,9 tetrahydrofuran, tetrahydropyran, dibutyl ether, and tert-butylmethyl ether10 and was denominated as the type of cyclosporin clathrates10 (space group P21). Here we report the new structure of cyclosporin A bisacetone hydrate (space group P41) crystallized from acetone-n-octane using (S)-1-amino-2-(methoxymethyl)pyrrolidone as the modifier. The choice of 1-amino-2(methoxymethyl)-pyrrolidone as the additive for crystallization originated from the idea to prove the theoretical possibility of chiral discrimination between enantiomers incorporated into the structure and calculation of free volume in the structures of P21 clathrates.10,11 Although 1-amino-2-(methoxymethyl)-pyrrolidone was not incorporated into the structure, only (S)1-amino-2-(methoxymethyl)-pyrrolidone facilitated crystallization of CsA bis-acetone hydrate, whereas the use of either (R)-1-amino-2-(methoxymethyl)-pyrrolidone or racemic 1-amino-2-(methoxymethyl)-pyrrolidone did not lead to crystallization. The structure is roughly isostructural with CsA dihydrate,4 but according to the overall composition it represents a “new” entity; moreover, the conditions employed
for crystallization would comply with the criteria for a new patentable process for crystallization. In contrast to CsA dihydrate, which creates small prisms from acetone, crystallization of CsA from n-octane/1-amino2-(methoxymethyl)-pyrrolidone/acetone yielded a CsA bis-acetone hydrate crystal morphology resembling sharp needles having several centimeters in length. CsA bis-acetone hydrate contains four intramolecular hydrogen bonds: D-Ala8NH‚‚‚COMeLeu6, Ala7NH‚‚‚ COMeVal11, Abu2NH‚‚‚COVal5, and Val5NH‚‚‚COAbu2. A large portion of the backbone spanning residues 1-5 adopts an antiparalel β-sheet conformation, the remaining residues form an open loop, with a cis-amide linkage between residues 9 and 10. Although CsA dihydrate and CsA bis-acetone hydrate are roughly isostructural, their physical properties are slightly different, for example, since acetone is not involved in any hydrogen bond, crystals of CsA bis-acetone hydrate are desolvatationprone. The origin of possible solvatomorphism of CsA stems from the presence of cavities12 in various structural types. Cyclosporin molecules are associated via van der Waals forces in crystal form giving rise to cavities that are occupied by solvent “guest” molecules. The shape and volume of the cavities was calculated12 by the program PLATON.13 The volume of a cavity is defined as the potential solvent area, i.e., the volume created by points having the distance larger than 1.2 Å from
Perspective
Crystal Growth & Design, Vol. 3, No. 4, 2003 443 Table 1. Parameters of Cavities in Structures of Cyclosporin A
cyclosporin A
monohydrate
dihydrate
bis-acetone, hydrate
dimethylisosorbide
di-n-butylether
di-n-butylether
(()-n-butyllactate
space group Z temp (K) unit cell V (Å3) all non-H atomsa atoms in cavitiesb
P212121 4 293 7216 372 (372 - 4 × 85) ) 32 32/4 ) 8 61
P41 4 293 7896 407 (407 - 4 × 85) ) 67 67/4 ) 17 332
P41 4 150 7751 400 (400 - 4 × 85) ) 60 60/4 ) 15 314
P21 2 293 4121 212 (212 - 2 × 85) ) 42 42/2 ) 21 438
P21 2 150 3961 204 (204 - 2 × 85) ) 34 34/2 ) 17 377
P21 2 293 4144 213 (213 - 2 × 85) ) 43 43/2 ) 21 463
P21 2 150 3987 205 (205 - 2 × 85) ) 35 35/2 ) 17 387
360
1326
1256
875
754
926
774
5%
17%
16%
21%
19%
22%
19%
1
2
9
12
9
9
10
one cavityc largest cavity V (Å3) Σ of cavities in unit cell V (Å3) volume of cavities (%) solvent atoms foundd packing coefficient cavity V (Å3)/ solvent atoms
7216/(4 × 85 + 7896/(4 × 85 + 7751/(4 × 85 + 4121/(2 × 85 + 3961/(2 × 85 + 4144/(2 × 85 + 3987/(2 × 85 + 4 × 1) 4 × 2) 4 × 9) 2 × 12) 2 × 9) 2 × 9) 2 × 10) ) 21.0 ) 23.0 ) 20.6 ) 21.2 ) 21.1 ) 22.0 ) 21.0 61.0 166.0 34.9 36.5 41.9 51.4 38.7
a Theoretical number of all non-H atoms in the unit cell ) V/19.4. b Theoretical number of non-H solvent’s atoms in the unit cell (CsA ) 85). c Theoretical number of non-H solvent’s atoms in one cavity (there is just one cavity per asymmetric unit cell in this case). d Number of the solvent’s non-H atoms found in one cavity.
the van der Waals sphere of any CsA atom. Furthermore, the shapes and volumes of the cavities can be also visualized, Figure 2. The basic parameters of cavities in the CsA structures are given in Table 1. The computed maximal continuous solvent suitable area in the range of 377-562 Å3 was localized in various cyclosporin clathrates of the P21 symmetry.9,10,14 Due to the thermal expansion, unit cell parameters and also the computed maximal continuous solvent suitable area increase with the increase of temperature used for the data collection. For example, in the case of CsA dibuthyl ether, the cavity volume increased by 4.6% from 150 to 293 K, Table 1. In each particular case, there is only one type of cavity in each of the cyclosporin structures. Under the assumption that the average packing coefficient for nonhydrogen atoms is 19.4 in such types of organic compounds,15 it can be calculated that about 15 or 17 nonhydrogen solvent atoms (in addition to 85 ones in CsA) could be incorporated into the asymmetric unit of the P41 or P21 forms of CsA, respectively, to achieve this value. However, as implied from the Table 1, the packing coefficient lies between 21.0 and 22.0 for all cyclosporins and is surprisingly independent of the crystalline form and conditions used. Consequently, this value can provide more reliable prediction within this series. It is clear from this calculation that the possible number and combination of molecules, which can be incorporated, are very high and for sure are not limited to the aforementioned series of ethers in the case of P21 structures or two acetone and one water molecule in the CsA P41 structure. In addition, both the packing coefficient and cavity volume/number of solvent atoms ratio (166 Å3, Table 1) calculated for CsA dihydrate indicate that the originally described composition, derived apparently from the crystal structure determination at ambient temperature, is unlikely. Such volume of cavity in CsA P41 indicates that solvents of similar size as in CsA P21 or several smaller solvents can be incorporated there. The occurrence of various cavities is connected not only with the particular example of CsA, but is fairly
common even with smaller organic compounds.1,16 Thus, with increasing molecular size there is an increase in the number of possible modes of molecular packing in crystalline form. This in turn gives rise to an increase in the number and diversity of various cavities and thus the number of potentially new crystalline forms. The example of cyclosporin A clearly demonstrates this phenomenon. Acknowledgment. This work was supported in part by Grants No. 203/99/M037, 203/00/1255, 203/01/0700, and 203/02/1417 by the Grant Agency of the Czech Republic. Supporting Information Available: Tables containing crystallization conditions (Table 1), X-ray crystallographic data (Table 2), final atomic parameters (Table 3), atomic thermal parameters (Table 4), bond distances and angles (Table 5), and ORTEP drawing of cyclosporin A bis-acetone hydrate are available free of charge via the Internet at http://pubs.acs.org. Crystallographic data for the structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number: CCDC 195079. Copies of the data can be obtained free of charge on application to CCDC, e-mail:
[email protected].
References (1) Byrn, S. R. Solid State Chemistry of Drugs; Academic Press: New York, 1982. (2) Perrone, R. K.; Stenberg, S. R.; Kaplan, M. A.; Saab, A.; Agharkar S. Crystalline Paclitaxel Hydrates, EP 0717041, 1995. (3) Authelin, J. R.; Didier, E.; Leveiller, F.; Taillepied, I. Method for preparing 4,10-diacetoxy-2R-benzoyloxy-5β,20-epoxy1,7β-dihydroxy-9-oxo-tax-11-en-13R-yl-(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate trihydrate. US Patent 6,002,022, 1996. (4) Loosli, H. R.; Kessler, H.; Oschkinat, H.; Weber, H. P.; Petcher, T. J.; Widmer A. Helv. Chim. Acta 1985, 68, 682704. (5) Giron, D.; List, M.; Richter, F.; Uike, Y.; Weber, H. P. Neue Kristallform von Cyclosporin, Verfahren zu ihrer Herstellung, pharmazeutische Pra¨parate enthaltend dies Kristallform sowie ihre Anwendung. Ger. Offen. DE 3843054, 1989.
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(6) Knott, R. B.; Schefer, J.; Schoenborn, B. P. Mater. Sci. Forum 1988, 27/28, 151-158. (7) Knott, R. B.; Schefer, J.; Schoenborn, B. P. Acta Crystallogr. 1990, C46, 1528-1533. (8) Kratochvı´l, B.; Jegorov, A.; Pakhomova, S.; Husˇa´k, M.; Bulej, P.; Cvak, L.; Sedmera, P.; Havlı´cˇek, V. Collect. Czech. Chem. Commun. 1999, 64, 89-98. (9) Husˇa´k, M.; Kratochvı´l, B.; Jegorov, A.; Mat’ha, V., Stuchlı´k, M., Andry´sek, T. Z. Kristal. 1996, 211, 313-318. (10) Jegorov, A.; Pakhomova, S.; Husˇa´k, M.; Kratochvı´l, B.; Zˇ a´k; Z., Cvak, L.; Buchta, M. J. Inclusion Phenom. Macrocyclic Chem. 2000, 37, 137-153. (11) Kratochvı´l, B.; Husˇa´k, M.; Jegorov, A. Chem. Listy 2001, 95, 9-17.
Perspective (12) Husˇa´k, M.; C ˇ ejka. J. MarchingCubeELD - electron density visualisation program for small molecules and its application on cyclosporins structures investigation. Book of Abstracts, European Crystallographic Meeting, ECM-19, Nancy, 2000, poster s3.m1p1. (13) Spek A. L. 2001 PLATON, A Multipurpose Crystallographic Tool; Utrecht University: Utrecht, The Netherlands. (14) Husˇa´k, M.; Kratochvı´l, B.; Jegorov, A.; Cı´sarˇova´, I. Collect. Czech. Chem. Commun. 2000, 65, 1950-1958. (15) Alkorta, I.; Rozas, I.; Elguero, J.; Foces-Foces, C.; Cano, F. H. J. Mol. Struct. 1996, 382, 205-213. (16) Brittain, H. G. Pharm. Technol. 2000, 116-125.
CG0300127
