Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-05-28
2001-12-18
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
active
06331235
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of capillary electrophoresis separation, as well as laser-induced fluorescence to aid in detection, of benzoporphyrin derivative (BPD) stereoisomers. These separation methods permit all four enantiomers of BPD-MA and the two of BPD-DA to be completely separated with baseline separation with high resolution and efficiency. The use of laser-induced fluorescence detection greatly improves the analytical sensitivity and selectivity of CE of BPDs. The invention is well suited as a rapid and reliable method for the separation of BPD monoacid and diacid enantiomers in a preparative or analytical application. Of particular utility is the potential for use as an analytical method with clinical samples.
DESCRIPTION OF THE RELATED ART
Photodynamic therapy (PDT) is based on the observation that some photosensitizers accumulate somewhat selectivity in tumor tissue where they can be activated by light at wave-lengths in the visible region. The photosensitizers absorb photons of visible light and use the absorbed energy to generate singlet oxygen, which is considered responsible for most of the damage leading to tumor cell death and tumor ablation (Delaney et al. (1988)). A number of photosensitizers are currently under investigation including chlorins, purpurins, phthalocyanines, and benzoporphyrin derivatives, especially benzoporphyrin derivative monoacid ring A (BPD-MA) and Verteporfin (Sternberg et al. (1996)).
BPD-MA is a novel second-generation photosensitizer for PDT which exhibits lower skin phototoxicity than Photofrin (Richter et al. (1987)) which has already been approved by Health Boards around the world. BPD-MA has been shown to be effective for the treatment of cutaneous lesions; as a promising agent for bone marrow purging and for the treatment of leukemia (Jamieson et al. (1990)), as an antiviral agent for decontamination of blood and blood products (Neyndorff et al. (1990)) and most recently for the treatment of age related macular degeneration of the eye (Miller et al. (1993)).
The importance of determining the stereoisomeric or chiral composition of chemical compounds, especially those of pharmaceutical importance, cannot be overemphasized. Nearly 60% of the most frequently prescribed drugs in the United States possess one or more asymmetric centers in the drug molecule. The physiological effects of enantiomers of these racemic drugs have not always been examined. To ensure the safety and efficacy of currently used and newly developed drugs, it is important to isolate the enantiomers and to examine each one separately. Accurate assessment of enantiomeric purity of substances is critical to investigate their toxicological and pharmacological effects and to study pharmocokinetic profiles.
Traditionally, high-performance liquid chromatography (HPLC) has been the most commonly used method for the separation of porphyrins (Lim et al. (1988)), metalloporphyrins (Wan et al. (1994); Ho et al. (1994); Sato et al. (1994); and Beukeveld et al. (1994)), porphyrin dimers and trimers (Owens et al. (1996)), porphyrin isomers (Richter et al. (1992) and Udagawa et al. (1982)), and some porphyrin enantiomers (Kimmett et al. (1992) and Walker et al. (1988)). The large number of biological samples that need to be assayed during a typical pharmacokinetic study and the labor-intensive procedures in these HPLC techniques, required alternative methodologies for this kind of separation. Capillary electrophoresis (CE) is not new (Hjerten et al. (1967) and Jorgenson et al. (1981)) and there have been numerous subsequent studies (Wallingford and Ewing (1988); Pietta et al. (1994); Morin et al. (1993); Honda et al. (1990); Honda et al. (1989); and Hoffstetter-Kuhn et al. (1991)). The good reproducibility, sensitivity, rapidity, ease of automation and small sample requirements of CE have made it a promising bioanalytical technique (Kuhn et al. (1993)) for the separation of porphyrins and their analogues (Kuhn et al. (1993); Yao et al. (1996); Weinberger et al. (1990); Liu et al. (1995); Bowser (1996); and Bowser et al. (1997)). It has been especially useful in the separation of porphyrins because porphyrin fluorescence permits detection at low concentration.
Given the importance of BPDs in PDT and the desire to prepare specific stereoisomers for pharmaceutical, research and commercial/industrial applications, the present invention relates to a new CE method for the separation of BPD stereoisomers.
SUMMARY OF THE INVENTION
The invention is directed to the use of capillary electrophoresis (CE) methods for the rapid and reliable separation of benzoporphyrin derivative (BPD) stereoisomers, which can then be used in photodynamic therapy (PDT). In particular, laser-induced fluorescence mediated detection is used in combination with CE methods to improve the sensitivity and selectivity in the separation of stereoisomers of BPDs. Additionally, the methods of the invention can be used to completely separate with baseline separation enantiomers of BPDs (as presented in U.S. Pat. No. 5,171,749, which is hereby incorporated by reference as if fully set forth) as well as the derivatives of these compounds, especially the four enantiomers of BPD-monoacid (BPD-MA) and the two of BPD-diacid (BPD-DA), with high resolution and efficiency. Additional BPDs for separation are EA6 (as set forth in related application Ser. No. 08/852,494, which is hereby incorporated by reference as if fully set forth) and B3 (as set forth in related application Ser. No. 09/265,245, which is hereby incorporated by reference as if fully set forth).
The invention is also directed to methods of separating BPD stereoisomers as a preparative or analytical application. Because of superiority over known HPLC methods, the preparative or analytical applications of the invention include automated versions of the instant CE methods as well as versions utilizing a smaller sample size over traditional HPLC separation methods. Of particular preference is the use of the instant methods for the analysis of clinical samples of material containing BPDs.
Additionally, the invention includes the use of borate as a buffering system and cholate as a chiral selector for the separation of porphyrin compounds, including metalloporphyrins, porphyrin multimers and isomers, as well as the stereoisomers of such compounds. Borate and cholate can dynamically interact and complex with the BPD enantiomers and influence their electrophoretic properties and migration behavior. Particularly preferred conditions in such separations include the use of a capillary of 50 &mgr;m inner diameter and a 37 cm length (30 cm to detector), a field strength of +20 KV, a separation temperature of 20° C., a pH of 9.2, a borate concentration of 300 mM, 25 mM sodium cholate as a chiral selector, and 10% acetonitrile. A preferred baseline separation time is within 20 minutes.
Moreover, and beyond complete separation of enantiomers, the invention encompasses methods of separating the stereoisomers of different compounds, the regioisomers of a given compound, as well as the enantiomers of a given compound.
REFERENCES:
patent: 5171749 (1992-12-01), Levy et al.
Dixon et al. (“Capillary electrophoretic separation of cationic porphyrins”, Journal of Liquid Chromatography A, 802(Apr. 1998), 367-380.*
CAPLUS abstract of Wu et al. (“Separation of porphyrins using a .gamma.-cyclodextrin stationary phase”, J. Liq. Chromatogr. (Month unknown 1994), 17(5), 1111-24.*
CAPLUS abstract of Fanali et al. (“The utility of cyclodextrins in capillary electrophoresis”, J. Capillary Electrophor. (Month unknown 1994), 1(10, 72-8).*
CAPLUS abstract of Armstrong et al. (Derivatized cyclodextrins immobilized on fused-silica capillaries for eantiomeric separations via capillary electrophoresis, gas chromatograph, or supercritical fluid chromatography, Anal. Chem. (Month unknown 1993), 65(8), (114-17).*
Wu et al. (“Recent developments in Porphyrin Separations using Capillary Electrophoresis with Native Fluorescence Detection”, Journal of Liquid Chromatography,
Dolphin David
Peng Xuejun
Sternberg Ethan D.
Noguerola Alex
The University of British Columbia
Warden Jill
LandOfFree
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