Bioconjugate Chem. 2006, 17, 1624−1626
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A Solvent-Free Method for Isotopically or Radioactively Labeling Cyclodextrins and Cyclodextrin-Containing Polymers Ryan K. Zeidan,† Stacey A. Kalovidouris,§ Thomas Schluep,§ Robert Fazio,# Robert Andresini,# and Mark E. Davis*,† Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena California, 91125, Insert Therapeutics, 129 N. Hill Avenue, Suite 104, Pasadena, California 91106, and Vitrax, 660 S. Jefferson Street, Unit E, Placentia, California 92870. Received July 12, 2006; Revised Manuscript Received September 20, 2006
A method for installing a distinguishable label onto cyclodextrins or cyclodextrin-containing polymers is reported. Cyclodextrins (CD) and cyclodextrin-containing polymers are exposed to labeled (2H or 14C) ethylene oxide (EO) vapor and the alcohol groups on the CD ring open the EO to give ether-linked labeled methylenes and a terminal alcohol. This method provides for the incorporation of an easily tracked and quantified label without the use of solvents or purification steps. The method can be generalized for use with materials that contain nucleophiles other than alcohols, e.g., amines.
INTRODUCTION Cyclodextrin-containing compounds and materials are becoming more widespread in drug delivery applications due to commercial availability, ability to form inclusion complexes, biocompatibility, and other unique chemical properties that contribute to their ease of chemical functionalization (1). Cyclodextrins do not contain chromophores or fluorophores in the parent structure and thus create difficulties in monitoring by spectrophotometric methods. Herein, a robust and general method to incorporate labels (deuterium or 14C) to cyclodextrin compounds simply and without solvents or purification is described that utilizes the reactivity of sugars with ethylene oxide vapor. This labeling method is also applicable to other sugar-containing molecules as well as any material bearing a reactive nucleophile group. The ability to incorporate radioactive isotopes such as 14C provides for materials that can be used for pharmacokinetic and pharmacodynamic studies (tritium labels, for example, may be lost from material in vivo (2)). The addition of the EO to the CD does not change the total amount of alcohol groups and just inserts a two-methylene spacer onto the sugar. Thus, there is minimal differences in the physicochemical properties from the parent CD. LeMaistre and Seymour (3) reported that the reaction of sucrose in aqueous sodium hydroxide solution with ethylene oxide occurred in high yield. However, there are no reports of the reaction of EO with CD. Here, we react individual CDs and CD-containing polymers with EO.
EXPERIMENTAL SECTION General. Deuterated ethylene oxide was obtained as a gas from Sigma Aldrich. β-cyclodextrin was obtained from Wacker. Labeling with Ethylene Oxide-d4. β-Cyclodextrin (50 mg) was dried at 100 °C under vacuum for 12 h overnight in order to completely remove residual water (water reacts with ethylene oxide to form ethylene glycol if not removed). After drying, the solid β-cyclodextrin sample was then allowed to cool to RT. The lecture bottle with ethylene oxide-d4 was then attached * Corresponding author. E-mail:
[email protected]. † California Institute of Technology. § Insert Therapeutics. # Vitrax.
to the reaction flask, and gas was flowed over the material for 2 min at 17 psi. The reaction vessel was then placed under vacuum to remove unreacted ethylene oxide-d4. The resulting white solid was then analyzed by ESI-MS to test for EO incorporation. Deuterium NMR experiments were performed also to confirm the presence of the EO incorporation. Labeling with 14C Ethylene Oxide. The cyclodextrincontaining polymer conjugate of camptothecin, IT-101 (4-7) (1.6 g), was reacted with 1 mmol ethylene oxide [1,2-14C], 50 mCi/mmol, under anhydrous conditions. Ethylene oxide, [1,214C] was generated from bromoethanol, [1,2-14C] under basic conditions, dried, and vacuum transferred into a 50 mL glass pressure vessel containing IT-101. The resultant mixture, at approximately 0.5 atm, was allowed to incubate for 4 days. The excess ethylene oxide, [1,2-14C] was removed by vacuum transfer, and no further workup was performed. The specific activity was measured by gravimetric assay to be 1.01 µCi/mg. Yield 1440 µCi, remaining a light yellow solid.
RESULTS AND DISCUSSION The reaction of dried β-CD with ethylene oxide-d4 (EO-d4) was carried out with vapor EO (Figure 1). Ethylene oxide-d4 has a mass of 48 and for each EO-d4 that reacts with the β-CD a corresponding increase in mass should be detectable by ESI/ MS. The resulting solid product after exposure to EO-d4 was analyzed by ESI/MS to observe the amount of labeling that has occurred. When the β-CD is not dried in advance, there is only a trace of EO incorporation (see Supporting Information), as residual water reacts with EO to give ethylene glycol. The same experiment was then carried out with β-CD that was dried at 100 °C for 24 h prior to use and then treated with EO-d4 for 5 min. The resulting ESI/MS data are shown in Figure 2. It is clear that there are observable species in the mass spectrum that correspond to various levels of ethylene oxide incorporation into the β-CD. The parent peak at 1157.4 corresponds to [M + Na] for the parent β-CD. The corresponding peak for 1 addition of EO is at [M + Na] ) 1205.4 and is clearly present. The corresponding peaks for 2, 3, 4, and 5 EO additions are also observed, as well as trace peaks corresponding to 6 and 7 additions at m/z of 1445 and 1493. The distribution of labeled products is also very clearly an exponentially decreasing trend
10.1021/bc060211e CCC: $33.50 © 2006 American Chemical Society Published on Web 10/31/2006
Technical Notes
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Figure 1. Schematic of reaction of β-cyclodextrin with ethylene oxide-d4.
Figure 2. ESI/MS analysis of labeled β-CD.
as the m/z increases to values indicative of highly labeled species. By using shorter exposure time and lower reaction temperatures, less EO-d4 is incorporated (see Supporting Information). There is a need to incorporate radioactive labels into materials that can be used for in vivo studies. A major concern when labeling materials is whether or not the labeling significantly
alters the material properties from those of the parent compound. Incorporation of EO into CDs does alter the properties of the labeled compound from those of the parent CD (unlike placing the label in the sugar itself). However, the consequences of the slight differences in properties may be significantly outweighed by the ease in the labeling methodology with EO; especially for CD-containing materials.
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Figure 3. Schematic of radioactively labeled EO reaction with IT-101.
Radiolabeled 14C ethylene oxide would be expected to react with β-CD in a manner similar to the EO-d4. The radioactivity of the resulting material could be quantified to evaluate the level of incorporation of EO and provide for straightforward analysis if used in vivo. To illustrate the methodology on β-CD containing polymer, we used IT-101 (4-7), a linear, water soluble cyclodextrin-containing polymer that is conjugated with camptothecin (Figure 3) to react with 14C ethylene oxide. IT-101 was exposed to radiolabeled 14C ethylene oxide, and the resulting material was analyzed for radioactivity to quantify the extent of labeling. Note that in this case the drying step was carried out at room temperature to prevent polymerconjugate decomposition. This drying step is sufficient to show that no radioactively labeled ethylene glycol is formed (see Supporting Information). The labeled IT-101 contained 1.01 mCi/mg of radioactivity that corresponds to ca. 1.5 EO molecules added per polymer chain. The labeling was performed in the absence of solvents on the polymer conjugate, and when the “hot” IT-101 is dissolved in water it forms particles with average size of 43 nm (analysis was performed by The Southwest Research Institute). This size is consistent with published data of IT-101 that is not radiolabeled (4-7). These data suggest that substantial levels of radiolabel can be incorporated into IT-101 and its properties not significantly modified from the parent compound. Animal studies using radiolabeled IT-101 are in progress and will be reported at a later time. In summary, a simple and efficient method for labeling cyclodextrin-containing materials without the use of solvents or purification is described through reaction with ethylene oxide, and this methodology is applicable to any materials bearing
nucleophilic sites. When using radiolabeled ethylene oxide, an accurately quantifiable labeling method is achieved and it allows for the preparation of radiolabeled materials for in vivo studies. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) Davis, M. E., and Brewster, M. E. (2004) Cyclodextrin-based pharmaceutics: Past, present, future. Nat. ReV. Drug DiscoVery 3, 1023-1035. (2) Beumer, J. H., Beijnen, J. H., and Schellens, J. H. (2006) Mass balance studies, with a focus on anticancer drugs. Clin. Pharmacokinet. 45, 33-58. (3) LeMaistre, J. W., and Seymour, R. B. (1948) The Reaction of Sucose with Ethylene Oxide. J. Org. Chem. 13, 782-785. (4) Cheng, J. J., Khin, K. T., Jensen, G. S., Liu, A., and Davis, M. E. (2003) Synthesis of Linear, Beta-Cyclodextrin-Based Polymers and Their Camptothecin Conjugates. Bioconjugate Chem. 14, 10071017. (5) Cheng, J. J., Khin, K. T., and Davis, M. E. (2004) Antitumor Activity of β-Cyclodextrin Polymer-Camptothecin Conjugates Mol. Pharm. 1, 183-193. (6) Schluep, T., Cheng, J. J., Kim, K. T., and Davis, M. E. (2006) Pharmacokinetics and biodistribution of the camptothecin-polymer conjugate IT-101 in rats and tumor-bearing mice. Cancer Chemother. Pharmacol. 57, 654-662. (7) Schluep, T., Hwang, S. J., Cheng, J. J., Heidel, J. D., Bartlett, D. W., and Davis, M. E. (2006) Preclinical Efficacy of the Camptothecin-Polymer Conjugate IT-101 in Multiple Cancer Models. Clin. Cancer Res. 12, 1606-1614. BC060211E
