Thomas G. Burke

An in vitro study of liposomal curcumin: Stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells

Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.

Simple and versatile high-performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of camptothecin anticancer drugs.

The well documented hydrolysis of the alpha-hydroxy-delta-lactone ring moiety in camptothecin and related analogues is routinely monitored using high-performance liquid chromatography (HPLC). Previous HPLC separations of the lactone and carboxylate forms of camptothecins have often required mobile phases containing three to four components; ion-pairing reagent to provide adequate retention of the carboxylate form of the drug; buffer to control the ionic strength and pH of the mobile phase; acetonitrile to control the retention of the lactone form and, in some instances, sodium dodecyl sulfate to reduce peak tailing. Because of the complexity of the mobile phases employed, development of these assays can be a laborious process, requiring re-optimization for each new analogue. In this study, we have developed a simple HPLC methodology for the simultaneous separation of the lactone and carboxylate forms of numerous camptothecin analogues. The mobile phase employed includes only triethylamine acetate (TEAA) buffer and acetonitrile. In this application, triethylamine serves multiple roles; as the ion-pairing reagent, as a masking agent for underivatized silanols and as the major buffer component. By altering only the composition of TEAA buffer with respect to acetonitrile, method development becomes a more streamlined and time efficient process. In this publication, we present the simultaneous separation of the lactone and carboxylate forms of camptothecin and four related analogues, namely, topotecan, GI147211, 10-aminocamptothecin and the CPT-11-SN-38 drug-metabolite pair. It is proposed that this new mobile phase, consisting of only triethylamine acetate buffer and acetonitrile, can be used for the analysis of the several camptothecin derivatives presently in clinical trials as well as the numerous other analogues in preclinical development.

The highly lipophilic DNA topoisomerase I inhibitor DB-67 displays elevated lactone levels in human blood and potent anticancer activity

The novel silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67) is 25- to 50-times more lipophilic than camptothecin and readily incorporates into lipid bilayers. Using the method of fluorescence anisotropy titration, we determined that DB-67 bound to small unilamellar vesicles composed of dilaurylphosphatidylcholine (DLPC) with an association constant (K value) of 5000 M(-1). This association constant is significantly higher than the K(DLPC) value observed for camptothecin (K(DLPC) value of 110 M(-1)). Using HPLC methods, we demonstrated that the presence of liposomal membranes readily stabilize the lactone form of DB-67. At drug and lipid concentrations of 10 microM and 0.3 mM, respectively, the lactone form of DB-67 persisted in liposome suspension after 3 h of incubation at 37 degrees C. Thus an advantage of a liposomal formulation of DB-67 is that the presence of lipid bilayers assists with stabilizing the key pharmacophore of the agent. The highly lipophilic character of DB-67, in combination with its 10-hydroxy moiety (which functions to enhance lactone stability in the presence of human serum albumin), results in DB-67 having superior stability in human blood with a percent lactone at equilibrium value of 30 [Cancer Res. 59 (1999) 4898; J. Med. Chem. 43 (2000) 3970]. Potent cytotoxicities against a broad range of cancer cells were observed for DB-67, indicating that DB-67 is of comparable potency to camptothecin. The impressive human blood stability and cytotoxicity profiles for DB-67 indicate it is an excellent candidate for comprehensive in vivo pharmacological and efficacy studies. Based on these promising attributes, DB-67 is currently being developed under the NCI RAID program. Due to its potent anti-topoisomerase I activity and its intrinsic blood stability, DB-67 appears as an attractive novel camptothecin for clinical development.

Campthotecin Design and Delivery Approaches for Elevating Anti‐Topoisomerase I Activities in Vivo

Abstract: The camptothecins as a class have exhibited unique dynamics and reactivity in vivo, with respect to both drug hydrolysis and blood protein interactions. These factors have confounded their pharmaceutical development and clinical implementation. Recent bench and clinical research alike indicates that the combination of medicinal chemical and drug delivery approaches has been and will continue to be highly valuable in improving the overall therapeutic indices of camptothecin‐based anti‐topoisomerase I therapies. In the future the development of camptothecin analogues that exhibit highly specific human albumin interactions will likely be avoided, and agents such as the highly lipophilic DB‐67 analogue with improved tissue stability will be evaluated. Drug delivery scientists will also devise better ways of targeting camptothecin therapies to solid tumors by using carriers such as tumor‐targeted long‐circulating liposomes.

Synthesis and evaluation of a novel E-ring modified α-hydroxy keto ether analogue of camptothecin

The synthesis of a novel E-ring modified keto ether analogue of camptothecin and homocamptothecin by the cascade radical annulation route is reported. The analogue, Du1441, is an isomer of homocamptothecin, but includes the alpha-hydroxy carbonyl functionality that camptothecin possesses and homocamptothecin lacks. Despite these similarities, the new keto ether analogue is inactive in cell assays, and implications for the structure/activity relationship are discussed.

Fluorescence Spectral Properties of the Anticancer Drug Topotecan by Steady‐State and Frequency Domain Fluorometry with One‐Photon and Multi‐Photon Excitation

Abstract— Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady‐state and time‐resolved fluorescence spectral properties of topote‐can with one‐and two‐photon excitation. Topotecan was found to display a high two‐photon cross section near 20 GM for wavelengths within the fundamental output of a Ti: sapphire laser, 800–880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one‐photon and two‐photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate‐buffered saline and propylene glycol. The steady‐state and time‐resolved data indicate that topotecan binds to a double‐helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA‐topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.

Comparison of Filter and Tunable Fluorescence Detection for the HPLC Simultaneous Quantitation of Lactone and Carboxylate Forms of Topotecan in Plasma

Abstract The hydrolysis of the α-hydroxy-δ-lactone ring moiety in topotecan is routinely monitored using high performance liquid chromatography (HPLC) with fluorescence detection. While both tunable and filter fluorescence detectors are commercially available, only the tunable detector has been studied for clinical and in vitro applications of topotecan. In the present study we have developed a simple HPLC method for the simultaneous separation of the lactone and carboxylate forms of topotecan in plasma, which, can be utilized for both clinical and in vitro studies. Limits of detection, percent relative standard deviation, and linear range for both the lactone and carboxylate forms of the drug in plasma are presented and compared using a tunable and filter fluorescence detector. Limits of detection in plasma of 0.10 ng/mL for carboxylate and 0.26 ng/mL for lactone have been obtained using a tunable fluorescence detector. A filter fluorescence detector produced limits of detection of 0.15 ng/mL for carboxy...

Recent Advances in Camptothecin Drug Design and Delivery Strategies

Camptothecin (CPT) (Fig. 1) and its related analogs are an expanding class of anticancer agents that have the potential to effect a broad and significant clinical impact (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Clinical interest in the CPTs is in large part based on their unique mode of action: these agents turn topoisomerase I (TOP-I), an enzyme that alleviates the torsional stress of supercoiled DNA, into an intracellular poison. The CPTs stabilize the covalent binding of TOP-I to its DNA substrate and the formation of these complexes leads to reversible, single-strand nicks. Initially, the nicks do not negatively affect the cellular viability; however, according to the fork collision model, the nicks are ultimately converted to irreversible and lethal double-strand breaks during DNA synthesis. As a result of this mechanism of action, the CPTs are regarded as being S-phase specific agents and are therefore toxic to cells that are actively replicating DNA (11,16,17). Because of their proliferating nature, cancerous cells spend more time in the S-phase relative to normal cells. Also, it has been shown that TOP-I is overexpressed in a variety of tumor lines (8,15). Logically, the accelerated rate of cell replication and the overexpression of TOP-I provide a limited basis for selectivity through which the CPTs can generate greater cytotoxicity against cancerous cells than against normal cells.

Determination of 9-nitrocamptothecin by precolumn derivatization and its metabolite 9-aminocamptothecin in a biological fluid using reversed-phase high-performance liquid chromatography with fluorescence detection

A novel insoluble topoisomerase I inhibitor, 9-nitrocamptothecin (9-NC), is in advanced stages of clinical development and has been used to treat a diverse array of tumor types, including breast, ovarian, pancreatic and haematological malignancies. We have established a sensitive high-performance liquid chromatography method using fluorescence detection for the quantitation of 9-NC. Non-fluorescent 9-NC is converted to fluorescent 9-aminocamptothecin (9-AC) via a one-step pre-column derivative reaction. The quantitative limit of 9-NC was 1 ng/ml and the method was reproducible with the respective intra- and inter-day variability falling below 5.0 and 9.0%. The determination of both 9-NC and its metabolite 9-AC in dog plasma was also achieved using the same chromatographic and detection conditions. In dog plasma, the quantitative limits of 9-AC and 9-NC were 0.25 and 1 ng/ml, respectively. The presence of 9-AC in the samples yielded no interference with the determination of 9-NC. However, individual matrices can affect the conversion efficiency of 9-NC, thus indicating that standard samples should be run for each matrix.

Fluorescence detection of camptothecin anticancer drugs by two-photon excitation

Hycamtin is a camptothecin anticancer analogue containing a dimethylaminomethyl substituent at position 9 and a hydroxy functionality at position 10. Using an excitation wavelength of 800 nm we have compared the two-photon cross sections and excited-state lifetimes from several camptothecins in phosphate buffered saline solution with and without the presence of human serum albumin (HSA). Drug and HSA concentrations of 10 (mu) M and 46 (mu) M were employed in our studies. In phosphate buffered saline solution containing HSA the following excited-state lifetimes (ns) and two- photon cross-sections (10-50 cm4 s/photon), respectively, were determined: hycamtin (4.3 nm, 36); camptothecin (1.3 ns, 1); 7-t-butyldimethylsilyl-10- hydroxycamptothecin (1.7 ns, 3.7); 7-t-butyldimethylsilyl- camptothecin (1.9 ns, 1.9); 7-trimethylsilyl-10- aminocamptothecin (6.3 ns; 35); and 7-trimethylsilyl-10- hydroxycamptothecin (1.8 ns; 2.2). Our results indicate that Hycamtin exhibits a high cross-section relative to the parent camptothecin molecule and represents one of the best camptothecin analogues to detect using two-photon excitation. Hycamtin was detected at concentrations as low as 0.05 (mu) M and 1 (mu) M in plasma and whole blood, respectively. The newly synthesized analogue 7- trimethylsilyl-10-aminocamptothecin was found to display similar lifetime and two-photon cross section values relative to Hycamtin. Thus, fluorescence detection with two- photon excitation may prove to be of advantage in the development of this promising new experimental therapeutic.