- Email: [email protected]
Enhancement of water-solubility and bioactivity of paclitaxel using modified cyclodextrins
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 4, 369–371. 2006 DOI: 10.1263/jbb.102.369
© 2006, The Society for Biotechnology, Japan
Enhancement of Water-Solubility and Bioactivity of Paclitaxel Using Modified Cyclodextrins Hiroki Hamada,1* Kohji Ishihara,1 Noriyoshi Masuoka,1 Katsuhiko Mikuni,2 and Nobuyoshi Nakajima3 Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan,1 Carbohydrate Research Laboratory, Ensuiko Sugar Refining Co., Ltd., 1-1-1 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan,2 and Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization, Okayama Prefectural University, Soja, Okayama 719-1197, Japan 3 Received 8 June 2006/Accepted 26 July 2006
We investigated the aqueous solubility of paclitaxel using 11 kinds of cyclodextrins (CDs) and the bioactivity of the paclitaxel-CD inclusion complex. 2,6-Dimethyl β-cyclodextrin was the most effective and its solubility was 2.3 mM in a 0.1 M 2,6-dimethyl β-cyclodextrin aqueous solution. The inclusion complex of paclitaxel and 2,6-dimethyl β-cyclodextrin had a 1.23-fold polymerization activity as paclitaxel in a tubulin assay. [Key words: paclitaxel, cyclodextrin, inclusion complex, water solubility]
Paclitaxel (1), a diterpenoid ester, is an antitumor agent used in clinical trials for ovarian and breast cancers (2–4). Although paclitaxel has such a biological activity, its water solubility is very poor. Therefore, paclitaxel is solubilized in a mixture of ethanol and cremophore EL for clinical trials. Enhancing the solubility of paclitaxel has been paid significant attention because it is very important to decrease a patient’s burden. Cyclodextrin (CD) is a cyclic oligosaccharide with 6–8 glucose units bonded by an α-1,4-linkage, and it has a cavity in its molecule. Therefore, CD is well known to form inclusion complexes with many compounds, which prevents the oxidation of oils and involatile flavors, and solubilizes insoluble compounds (5–8). Hydroxypropyl β-CD forms an inclusion complex with paclitaxel through hydrophobic interactions (9). The conformation of paclitaxel dynamically changes upon switching from a polar solvent to a nonpolar one (10). We now report on the improvement of the solubility of paclitaxel in aqueous solutions through the use of cyclodextrins and the evaluation of the antitumor activity of the paclitaxel-CD inclusion complex. Furthermore, we discuss the possibility of developing the inclusion complex as a useful drug. α-Cyclodextrin (α-CD), β-CD, γ-CD, mono-6-O-maltosyl α-CD (G2-α-CD), mono-6-O-maltosyl β-CD (G2-β-CD), mono-6-O-maltosyl γ-CD (G2-γ-CD), heptakis-(2,6-di-Omethyl) α-CD (DM-α-CD), heptakis-(2,6-di-O-methyl) β-CD (DM-β-CD), and heptakis-(2,3,6-tri-O-methyl) β-CD (TM-β-CD) were obtained from Wako Pure Chemical Industries (Osaka). Random hydroxyethyl β-CD (HE-β-CD) and random hydroxypropyl β-CD (HP-β-CD) were purchased
from Cyclo Lab (Budapest, Hungary). Paclitaxel was obtained from Sumitomo Chemical (Osaka). An excess amount of paclitaxel was added to various CD aqueous solutions and mixed well for an appropriate time at 25°C. The mixture was centrifuged at 8000×g for 20 min and the supernatant was passed though a 0.45 µm membrane filter. The filtrate was analyzed by HPLC (column: CrestPak C18S (JASCO, Tokyo); mobile phase: methanol : water = 65 : 35; flow rate: 1.0 ml/min; column temperature: 35°C; detector: UV 230 nm). The amount of paclitaxel was calculated from the peak area of the paclitaxel standard curve under the same HPLC conditions (paclitaxel is excluded from CD-paclitaxel inclusion complex under the HPLC conditions described above). Paclitaxel (5 mg) and G2-β-CD, DM-β-DC or HP-β-CD (500 mg) were added to a 70 ml solution (organic solvent : water = 1 : 1). The organic solvents used were dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), methanol, acetone, acetonitrile, or dioxane. The mixture was agitated for 2 h at 25°C, and then freeze-dried. The obtained powder was dissolved in water to from a 6 mM CD solution. The solubility of paclitaxel was analyzed using the same method as described above. Tubulin was extracted from pork brain following the procedure previously reported by Shelanski et al. (11), and the microtuble assembly assay was performed according to the procedure described by Schiff et al. (12). The effects of 11 kinds of 6 mM CDs on the solubility of paclitaxel are shown in Table 1. For DM-β-CD, the solubility of paclitaxel was 49 ± 0.52 µM, which was the highest value in this work. The composition ratio of paclitaxel to DM-β-CD in the inclusion complex was estimated to be 1 : 2 moles by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses (data not shown). The chemically modified CDs, DM-α-CD, DM-β-CD, TM-β-CD, HE-
* Corresponding author. e-mail: [email protected] phone: +81-(0)86-256-9473 fax: +81-(0)86-256-8468 369
370
J. BIOSCI. BIOENG.,
HAMADA ET AL.
TABLE 1. Solubility of paclitaxel in aqueous solutions with 6 mM of various CDs Concn of paclitaxela (µM) None 0.41 ±0.05 α-CD 0.72 ±0.06 β-CD 1.1±0.08 γ-CD 0.93 ±0.05 0.74 ±0.04 G2-α-CD 1.2±0.08 G2-β-CD 0.83 ±0.07 G2-γ-CD DM-α-CD 8.1± 0.11 DM-β-CD 49±0.52 TM-β-CD 5.5± 0.11 HE-β-CD 4.1±0.14 HP-β-CD 7.8±0.12 a Values are means± SD from five indipendent experiments. Cyclodextrins
β-CD, and HP-β-CD, solubilized more of paclitaxel than did the nonchemically modified CDs, α-CD, β-CD, γ-CD, G2-α-CD, G2-β-CD, and G2-γ-CD. The solubility of paclitaxel increased with increasing concentration of DM-β-CD, as shown in Fig. 1. For the 100 mM DM-β-CD, paclitaxel was solubilized at 2.3 mM (2000 µg/ml). As a critical dose for humans, 60 µg/ml of paclitaxel is intravenously administrated (2). It is possible to use DMβ-CD, a solubilizer of paclitaxel, instead of the mixture of ethanol and cremophor EL, on the basis of solubility. With DM-β-CD, paclitaxel remained at approximately 100% after 50 h (see Fig. 2). Paclitaxel decreased by almost the same ratio in both G2-β-CD and HP-β-CD. Paclitaxel in the DMβ-CD solution was more stable than those in G2-β-CD and HP-β-CD. The effects of different solvent systems on the solubility of paclitaxel are summarized in Table 2. The solubility of paclitaxel was the highest in the combination of HP-β-CD and dioxane at 97 ± 0.47 µM. DMSO seemed to be a good solvent system because the highest solubility was obtained among all the three kinds of CDs. Both paclitaxel and the CDs were soluble in DMSO, and it was thought that they easily form an inclusion complex. Most of solubilies of paclitaxel in the organic solvent system were higher than those in water systems (see Table 1). Williams et al. reported that paclitaxel was less globular in nonpolar solvents than in polar solvents (9). It seems to be related to the conformation of paclitaxel in the solvent upon forming an inclusion complex with CD. Methew et al. reported that paclitaxel and its derivatives has a microtubule polymerization activity and that little or no depolymerization (induced by CaCl2) occurred in the presence of paclitaxel and its derivatives. (4). To examine such a bioactivity of the paclitaxcel-CD inclusion complex, the paclitaxel-DM-β-CD complex was subjected to the in vitro microtubule assembly assay. Microtubule assembly was monitored by the increase of turbidity (that is, by the apparent absorbance at 350 nm). The polymerization and depolymerization activity of the paclitaxel-DM-β-CD complex had paclitaxel values of 123 and 63, respectively (see Table 3). These results indicate that the bioactivity of paclitaxel remains in the paclitaxel-DM-β-CD inclusion complex.
FIG. 1. Effect of concentration of DM-β-CD on solubility of paclitaxel. Each data point shows a mean ± SD (n = 5).
FIG. 2. Stability of paclitaxel in CD solutions at 25°C. Closed triangles, DM-β-CD; closed circles, G2-β-CD; closed squares, HP-β-CD. Each data point shows a mean ± SD (n = 5).
TABLE 2. Effect of different solvent systems on solubility of paclitaxel G2-β-CDa DM-β-CDa HP-β-CDa (µM) (µM) (µM) Methanol 1.2 ± 0.13 74 ± 0.41 5.0 ± 0.11 DMSO 49± 0.44 89 ± 0.51 61 ± 0.39 Acetonitrile 2.0 ± 0.08 92 ± 0.47 21 ± 0.22 Dioxane 4.8 ± 0.11 93 ± 0.30 97 ± 0.47 Acetone 70 ± 0.28 51 ± 0.38 21 ± 0.53 THF 28± 0.19 12 ± 0.09 53 ± 0.23 a Values are means± SD from five indipendent experiments.
Solvent
VOL. 102, 2006
TECHNICAL NOTES
TABLE 3. Effect of paclitaxel and paclitaxel-CD Complex on in vitro tubulin assaya Substance Polymerizationb Depolymerizationb Paclitaxel 100 100 Paclitaxel-DM-β-CD 123 63 a Tubulin (10 µM) was polymerized at 30°C in the presence of 5 µM substance and 0.5 mM GTP in PEM (0.1 M PIPES, pH 6.9, 1 mM EGTA, and 1 mM MgSO4) buffer. The increase in turbidity was monitored by the apparent absorbance at 350 nm. b Polymerization or depolymerization rates were determined by setting the activity of paclitaxel to 100.
3.
4.
5.
We demonstrated an improvement in the solubility of paclitaxel using the modified CD and the evaluation of the antitumor activity of the paclitaxel-CD inclusion complex. Further detailed studies, including the clarification of the structure-bioactivity relationship of the paclitaxel-CD inclusion complex by NMR analysis, are now in progress in our laboratories. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Financial Assistance Program of the Social Cooperation Study (2006–2010). We wish to express our appreciation to Mr. Kiyoshi Nakajima, Tokyo Supply, Ltd., for his useful advice during the course of the work.
6. 7.
8.
9. 10.
REFERENCES 1. Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, A. T.: Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc., 93, 2325–2337 (1971). 2. McGuire, W. P., Rowinsky, E. K., Rosenshein, N. B., Grumbine, F. C., Ettinger, D. S., Armstrong, D. K., and
11. 12.
371
Donehower, R. C.: Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann. Intern. Med., 111, 273–279 (1989). Holmes, F. A., Walters, R. S., Theriault, R. L., Forman, A. D., Newton, L. K., Raber, M. N., Buzdar, A. U., Frye, D. K., and Hortobagyi, G. N.: Phase II traial of taxol, an active drug in the treatment of metastatic breast cancer. J. Natl. Cancer Inst. (Bethesda), 83, 1797–1805 (1991). Methew, A. E., Mejillano, M. R., Nath, J. P., Himes, R. H., and Stella, V. J.: Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity. J. Med. Chem., 35, 145–151 (1992). Szejtli, J.: Cyclodextrins and their inclusion complexes, p. 204–255. Academiai Kiadó, Budapest (1982). Szejtli, J.: Cyclodextrin technology, p. 186–334. In Davies, J. E. D. (ed.), Topics in inclusion science. Kluwer Academic Science, Dordrecht, Netherland (1988). Fromming, K-H. and Szejtli, J.: Cyclodextrins in pharmacy, p. 127–196. In Davies, J. E. D. (ed.), Topics in inclusion science, vol. 5. Kluwer Acadamic Publishers, Dordrecht, Netherland (1994). Loftsson, T., Baldvinsdottir, J., and Sigurgardottir, A. M.: The effect of cyclodextrins on the solubility and stability of medroxyprogesterone acetate and megestrol acetate in aqueous solution. Int. J. Pharm., 98, 225–230 (1983). Cserháti, T. and Holló, J.: Interaction of taxol and other anticancer drugs with hydroxypropyl-β-cyclodextrin. Int. J. Pharm., 108, 69–75 (1994). Williams, H. J., Scott, A. I., Dieden, R. A., Swindell, C. S., Chirlian, L. E., Francl, M. M., Heerding, J. M., and Krauss, N. E.: NMR and molecular modeling study of the conformations of taxol and of its side chain methylester in aqueous and non-aqueous solutions. Tetrahedron, 49, 6545– 6560 (1993). Shelanski, M. L., Gaskin, F., and Cantor, C. R.: Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. USA, 70, 765–768 (1973). Schiff, P. B., Fant, J., and Horwitz, S. B.: Promotion of microtubule assembly in vitro by taxol. Nature (London), 277, 665–667 (1979).
© 2006, The Society for Biotechnology, Japan
Enhancement of Water-Solubility and Bioactivity of Paclitaxel Using Modified Cyclodextrins Hiroki Hamada,1* Kohji Ishihara,1 Noriyoshi Masuoka,1 Katsuhiko Mikuni,2 and Nobuyoshi Nakajima3 Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan,1 Carbohydrate Research Laboratory, Ensuiko Sugar Refining Co., Ltd., 1-1-1 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan,2 and Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization, Okayama Prefectural University, Soja, Okayama 719-1197, Japan 3 Received 8 June 2006/Accepted 26 July 2006
We investigated the aqueous solubility of paclitaxel using 11 kinds of cyclodextrins (CDs) and the bioactivity of the paclitaxel-CD inclusion complex. 2,6-Dimethyl β-cyclodextrin was the most effective and its solubility was 2.3 mM in a 0.1 M 2,6-dimethyl β-cyclodextrin aqueous solution. The inclusion complex of paclitaxel and 2,6-dimethyl β-cyclodextrin had a 1.23-fold polymerization activity as paclitaxel in a tubulin assay. [Key words: paclitaxel, cyclodextrin, inclusion complex, water solubility]
Paclitaxel (1), a diterpenoid ester, is an antitumor agent used in clinical trials for ovarian and breast cancers (2–4). Although paclitaxel has such a biological activity, its water solubility is very poor. Therefore, paclitaxel is solubilized in a mixture of ethanol and cremophore EL for clinical trials. Enhancing the solubility of paclitaxel has been paid significant attention because it is very important to decrease a patient’s burden. Cyclodextrin (CD) is a cyclic oligosaccharide with 6–8 glucose units bonded by an α-1,4-linkage, and it has a cavity in its molecule. Therefore, CD is well known to form inclusion complexes with many compounds, which prevents the oxidation of oils and involatile flavors, and solubilizes insoluble compounds (5–8). Hydroxypropyl β-CD forms an inclusion complex with paclitaxel through hydrophobic interactions (9). The conformation of paclitaxel dynamically changes upon switching from a polar solvent to a nonpolar one (10). We now report on the improvement of the solubility of paclitaxel in aqueous solutions through the use of cyclodextrins and the evaluation of the antitumor activity of the paclitaxel-CD inclusion complex. Furthermore, we discuss the possibility of developing the inclusion complex as a useful drug. α-Cyclodextrin (α-CD), β-CD, γ-CD, mono-6-O-maltosyl α-CD (G2-α-CD), mono-6-O-maltosyl β-CD (G2-β-CD), mono-6-O-maltosyl γ-CD (G2-γ-CD), heptakis-(2,6-di-Omethyl) α-CD (DM-α-CD), heptakis-(2,6-di-O-methyl) β-CD (DM-β-CD), and heptakis-(2,3,6-tri-O-methyl) β-CD (TM-β-CD) were obtained from Wako Pure Chemical Industries (Osaka). Random hydroxyethyl β-CD (HE-β-CD) and random hydroxypropyl β-CD (HP-β-CD) were purchased
from Cyclo Lab (Budapest, Hungary). Paclitaxel was obtained from Sumitomo Chemical (Osaka). An excess amount of paclitaxel was added to various CD aqueous solutions and mixed well for an appropriate time at 25°C. The mixture was centrifuged at 8000×g for 20 min and the supernatant was passed though a 0.45 µm membrane filter. The filtrate was analyzed by HPLC (column: CrestPak C18S (JASCO, Tokyo); mobile phase: methanol : water = 65 : 35; flow rate: 1.0 ml/min; column temperature: 35°C; detector: UV 230 nm). The amount of paclitaxel was calculated from the peak area of the paclitaxel standard curve under the same HPLC conditions (paclitaxel is excluded from CD-paclitaxel inclusion complex under the HPLC conditions described above). Paclitaxel (5 mg) and G2-β-CD, DM-β-DC or HP-β-CD (500 mg) were added to a 70 ml solution (organic solvent : water = 1 : 1). The organic solvents used were dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), methanol, acetone, acetonitrile, or dioxane. The mixture was agitated for 2 h at 25°C, and then freeze-dried. The obtained powder was dissolved in water to from a 6 mM CD solution. The solubility of paclitaxel was analyzed using the same method as described above. Tubulin was extracted from pork brain following the procedure previously reported by Shelanski et al. (11), and the microtuble assembly assay was performed according to the procedure described by Schiff et al. (12). The effects of 11 kinds of 6 mM CDs on the solubility of paclitaxel are shown in Table 1. For DM-β-CD, the solubility of paclitaxel was 49 ± 0.52 µM, which was the highest value in this work. The composition ratio of paclitaxel to DM-β-CD in the inclusion complex was estimated to be 1 : 2 moles by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses (data not shown). The chemically modified CDs, DM-α-CD, DM-β-CD, TM-β-CD, HE-
* Corresponding author. e-mail: [email protected] phone: +81-(0)86-256-9473 fax: +81-(0)86-256-8468 369
370
J. BIOSCI. BIOENG.,
HAMADA ET AL.
TABLE 1. Solubility of paclitaxel in aqueous solutions with 6 mM of various CDs Concn of paclitaxela (µM) None 0.41 ±0.05 α-CD 0.72 ±0.06 β-CD 1.1±0.08 γ-CD 0.93 ±0.05 0.74 ±0.04 G2-α-CD 1.2±0.08 G2-β-CD 0.83 ±0.07 G2-γ-CD DM-α-CD 8.1± 0.11 DM-β-CD 49±0.52 TM-β-CD 5.5± 0.11 HE-β-CD 4.1±0.14 HP-β-CD 7.8±0.12 a Values are means± SD from five indipendent experiments. Cyclodextrins
β-CD, and HP-β-CD, solubilized more of paclitaxel than did the nonchemically modified CDs, α-CD, β-CD, γ-CD, G2-α-CD, G2-β-CD, and G2-γ-CD. The solubility of paclitaxel increased with increasing concentration of DM-β-CD, as shown in Fig. 1. For the 100 mM DM-β-CD, paclitaxel was solubilized at 2.3 mM (2000 µg/ml). As a critical dose for humans, 60 µg/ml of paclitaxel is intravenously administrated (2). It is possible to use DMβ-CD, a solubilizer of paclitaxel, instead of the mixture of ethanol and cremophor EL, on the basis of solubility. With DM-β-CD, paclitaxel remained at approximately 100% after 50 h (see Fig. 2). Paclitaxel decreased by almost the same ratio in both G2-β-CD and HP-β-CD. Paclitaxel in the DMβ-CD solution was more stable than those in G2-β-CD and HP-β-CD. The effects of different solvent systems on the solubility of paclitaxel are summarized in Table 2. The solubility of paclitaxel was the highest in the combination of HP-β-CD and dioxane at 97 ± 0.47 µM. DMSO seemed to be a good solvent system because the highest solubility was obtained among all the three kinds of CDs. Both paclitaxel and the CDs were soluble in DMSO, and it was thought that they easily form an inclusion complex. Most of solubilies of paclitaxel in the organic solvent system were higher than those in water systems (see Table 1). Williams et al. reported that paclitaxel was less globular in nonpolar solvents than in polar solvents (9). It seems to be related to the conformation of paclitaxel in the solvent upon forming an inclusion complex with CD. Methew et al. reported that paclitaxel and its derivatives has a microtubule polymerization activity and that little or no depolymerization (induced by CaCl2) occurred in the presence of paclitaxel and its derivatives. (4). To examine such a bioactivity of the paclitaxcel-CD inclusion complex, the paclitaxel-DM-β-CD complex was subjected to the in vitro microtubule assembly assay. Microtubule assembly was monitored by the increase of turbidity (that is, by the apparent absorbance at 350 nm). The polymerization and depolymerization activity of the paclitaxel-DM-β-CD complex had paclitaxel values of 123 and 63, respectively (see Table 3). These results indicate that the bioactivity of paclitaxel remains in the paclitaxel-DM-β-CD inclusion complex.
FIG. 1. Effect of concentration of DM-β-CD on solubility of paclitaxel. Each data point shows a mean ± SD (n = 5).
FIG. 2. Stability of paclitaxel in CD solutions at 25°C. Closed triangles, DM-β-CD; closed circles, G2-β-CD; closed squares, HP-β-CD. Each data point shows a mean ± SD (n = 5).
TABLE 2. Effect of different solvent systems on solubility of paclitaxel G2-β-CDa DM-β-CDa HP-β-CDa (µM) (µM) (µM) Methanol 1.2 ± 0.13 74 ± 0.41 5.0 ± 0.11 DMSO 49± 0.44 89 ± 0.51 61 ± 0.39 Acetonitrile 2.0 ± 0.08 92 ± 0.47 21 ± 0.22 Dioxane 4.8 ± 0.11 93 ± 0.30 97 ± 0.47 Acetone 70 ± 0.28 51 ± 0.38 21 ± 0.53 THF 28± 0.19 12 ± 0.09 53 ± 0.23 a Values are means± SD from five indipendent experiments.
Solvent
VOL. 102, 2006
TECHNICAL NOTES
TABLE 3. Effect of paclitaxel and paclitaxel-CD Complex on in vitro tubulin assaya Substance Polymerizationb Depolymerizationb Paclitaxel 100 100 Paclitaxel-DM-β-CD 123 63 a Tubulin (10 µM) was polymerized at 30°C in the presence of 5 µM substance and 0.5 mM GTP in PEM (0.1 M PIPES, pH 6.9, 1 mM EGTA, and 1 mM MgSO4) buffer. The increase in turbidity was monitored by the apparent absorbance at 350 nm. b Polymerization or depolymerization rates were determined by setting the activity of paclitaxel to 100.
3.
4.
5.
We demonstrated an improvement in the solubility of paclitaxel using the modified CD and the evaluation of the antitumor activity of the paclitaxel-CD inclusion complex. Further detailed studies, including the clarification of the structure-bioactivity relationship of the paclitaxel-CD inclusion complex by NMR analysis, are now in progress in our laboratories. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Financial Assistance Program of the Social Cooperation Study (2006–2010). We wish to express our appreciation to Mr. Kiyoshi Nakajima, Tokyo Supply, Ltd., for his useful advice during the course of the work.
6. 7.
8.
9. 10.
REFERENCES 1. Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, A. T.: Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc., 93, 2325–2337 (1971). 2. McGuire, W. P., Rowinsky, E. K., Rosenshein, N. B., Grumbine, F. C., Ettinger, D. S., Armstrong, D. K., and
11. 12.
371
Donehower, R. C.: Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann. Intern. Med., 111, 273–279 (1989). Holmes, F. A., Walters, R. S., Theriault, R. L., Forman, A. D., Newton, L. K., Raber, M. N., Buzdar, A. U., Frye, D. K., and Hortobagyi, G. N.: Phase II traial of taxol, an active drug in the treatment of metastatic breast cancer. J. Natl. Cancer Inst. (Bethesda), 83, 1797–1805 (1991). Methew, A. E., Mejillano, M. R., Nath, J. P., Himes, R. H., and Stella, V. J.: Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity. J. Med. Chem., 35, 145–151 (1992). Szejtli, J.: Cyclodextrins and their inclusion complexes, p. 204–255. Academiai Kiadó, Budapest (1982). Szejtli, J.: Cyclodextrin technology, p. 186–334. In Davies, J. E. D. (ed.), Topics in inclusion science. Kluwer Academic Science, Dordrecht, Netherland (1988). Fromming, K-H. and Szejtli, J.: Cyclodextrins in pharmacy, p. 127–196. In Davies, J. E. D. (ed.), Topics in inclusion science, vol. 5. Kluwer Acadamic Publishers, Dordrecht, Netherland (1994). Loftsson, T., Baldvinsdottir, J., and Sigurgardottir, A. M.: The effect of cyclodextrins on the solubility and stability of medroxyprogesterone acetate and megestrol acetate in aqueous solution. Int. J. Pharm., 98, 225–230 (1983). Cserháti, T. and Holló, J.: Interaction of taxol and other anticancer drugs with hydroxypropyl-β-cyclodextrin. Int. J. Pharm., 108, 69–75 (1994). Williams, H. J., Scott, A. I., Dieden, R. A., Swindell, C. S., Chirlian, L. E., Francl, M. M., Heerding, J. M., and Krauss, N. E.: NMR and molecular modeling study of the conformations of taxol and of its side chain methylester in aqueous and non-aqueous solutions. Tetrahedron, 49, 6545– 6560 (1993). Shelanski, M. L., Gaskin, F., and Cantor, C. R.: Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. USA, 70, 765–768 (1973). Schiff, P. B., Fant, J., and Horwitz, S. B.: Promotion of microtubule assembly in vitro by taxol. Nature (London), 277, 665–667 (1979).