DNA-Binding Anticancer Drugs: One Target, Two Actions.

Amsacrine, an anticancer drug first synthesised in 1970 by Professor Cain and colleagues, showed excellent preclinical activity and underwent clinical trial in 1978 under the auspices of the US National Cancer Institute, showing activity against acute lymphoblastic leukaemia. In 1984, the enzyme DNA topoisomerase II was identified as a molecular target for amsacrine, acting to poison this enzyme and to induce DNA double-strand breaks. One of the main challenges in the 1980s was to determine whether amsacrine analogues could be developed with activity against solid tumours. A multidisciplinary team was assembled in Auckland, and Professor Denny played a leading role in this approach. Among a large number of drugs developed in the programme, N-[2-(dimethylamino)-ethyl]-acridine-4-carboxamide (DACA), first synthesised by Professor Denny, showed excellent activity against a mouse lung adenocarcinoma. It underwent clinical trial, but dose escalation was prevented by ion channel toxicity. Subsequent work led to the DACA derivative SN 28049, which had increased potency and reduced ion channel toxicity. Mode of action studies suggested that both amsacrine and DACA target the enzyme DNA topoisomerase II but with a different balance of cellular consequences. As primarily a topoisomerase II poison, amsacrine acts to turn the enzyme into a DNA-damaging agent. As primarily topoisomerase II catalytic inhibitors, DACA and SN 28049 act to inhibit the segregation of daughter chromatids during anaphase. The balance between these two actions, one cell cycle phase specific and the other nonspecific, together with pharmacokinetic, cytokinetic and immunogenic considerations, provides links between the actions of acridine derivatives and anthracyclines such as doxorubicin. They also provide insights into the action of cytotoxic DNA-binding drugs.

Optimization of Weight Ratio for DSPE-PEG/TPGS Hybrid Micelles to Improve Drug Retention and Tumor Penetration.

PURPOSE: To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration. METHODS: ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control. RESULTS: The ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5 nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL. CONCLUSIONS: The optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.

Quantitative determination of intracellular Asulacrine in MCF-7 breast cancer cells by liquid chromatography-mass spectrometry and its application to cellular pharmacokinetic studies of P188 modified liposomes.

Asulacrine (ASL), an analogue of amsacrine, has shown higher anti-breast and anti-lung cancer activity. Hereby, a new sensitive and selective liquid chromatography-mass spectrometry (LC/MS) method was developed to determine intracellular asulacrine. The chromatographic separation was performed on an Agilent Zorbax Extend-C18 column (2.1 mm i.d. × 50 mm, 5 µm) using gradient elution with water (2 mmol/L ammonium acetate and 0.1% acetic acid) and acetonitrile as the mobile phase. The detection was achieved with selected ion monitoring mode using electrospray ionization in positive mode with target ions at m/z 465.3 and m/z 326.1 for asulacrine and midazolam, respectively. The standard curve showed a good linearity with the lower limit of quantification of 1 ng/mL, as a result of which, the trace concentration of ASL in cell suspension could be quantified. The intra- and inter-day accuracy ranged from -5.28 to 6.5% and from -6.32 to 1.05%, and the intra- and inter-day precisions were no more than 7.65% and 11.71%, respectively. Additionally, no degradation of asulacrine was observed during stability evaluation. The method was proved to be powerful and practical to determine and compare the intracellular distribution and kinetics of ASL under different formulations in MCF-7 breast cancer cells.

Strategies to maximize liposomal drug loading for a poorly water-soluble anticancer drug.

PURPOSE: To develop a liposomal system with high drug loading (DL) for intravenous (i.v.) delivery of a poorly water-soluble basic drug, asulacrine (ASL). METHODS: A thin-film hydration and extrusion method was used to fabricate the PEGylated liposomal membranes followed by a freeze and thaw process. A novel active drug loading method was developed using ammonium sulphate gradient as an influx driving force of ASL solubilized with sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD). DL was maximized by optimizing liposomal preparation and loading conditions. Pharmacokinetics was evaluated following i.v. infusion in rabbits. RESULTS: Freeze-thaw resulted in unilamellar liposome formation (180 nm) free of micelles. Higher DL was obtained when dialysis was used to remove the untrapped ammonium sulphate compared to ultracentrifuge. The pH and SBE-ß-CD level in the loading solution played key roles in enhancing DL. High DL ASL-liposomes (8.9%w/w, drug-to-lipid mole ratio 26%) were obtained with some drug "bundles" in the liposomal cores and were stable in a 5% glucose solution for >80 days with minimal leakage (<2%). Surprisingly, following administration of ASL-liposomes prepared with or without SBE-ß-CD, the half-lives were similar to the drug solution despite an increased area under the curve, indicating drug leakage from the carriers. CONCLUSIONS: High liposomal DL was achieved with multiple strategies for a poorly-water soluble weak base. However, the liposomal permeability needed to be tailored to improve drug retention.

In-silico Design, ADMET Screening, MM-GBSA Binding Free Energy of Some Novel Isoxazole Substituted 9-Anilinoacridines as HER2 Inhibitors Targeting Breast Cancer.

BACKGROUND: Human Epidermal development factor Receptor-2 (HER2) is a membrane tyrosine kinase which is overexpressed and gene amplified in human breast cancers. HER2 amplification and overexpression have been linked to important tumor cell proliferation and survival pathways for 20% of instances of breast cancer. 9-aminoacridines are significant DNA-intercalating agents because of their antiproliferative properties. OBJECTIVE: Some novel isoxazole substituted 9-anilinoacridines(1a-z) were designed by in-silico technique for their HER2 inhibitory activity. Docking investigations of compounds 1a-z are performed against HER2 (PDB id-3PP0) by using Schrodinger suit 2016-2. METHODS: Molecular docking study for the designed molecules 1a-z are performed by Glide module, in-silico ADMET screening by QikProp module and binding free energy by Prime-MMGBSA module of Schrodinger suit. The binding affinity of designed molecules 1a-z towards HER2 was chosen based on GLIDE score. RESULTS: Many compounds showed good hydrophobic communications and hydrogen bonding associations to hinder HER2. The compounds 1a-z, aside from 1z have significant Glide scores in the scope of - 4.91 to - 10.59 when compared with the standard Ethacridine (- 4.23) and Tamoxifen (- 3.78). The in-silico ADMET properties are inside the suggested about drug likeness. MM-GBSA binding of the most intense inhibitor is positive. CONCLUSION: The outcomes reveal that this study provides evidence for the consideration of isoxazole substituted 9-aminoacridine derivatives as potential HER2 inhibitors. The compounds, 1s,x,v,a,j,r with significant Glide scores may produce significant anti breast cancer activity and further in vitro and in vivo investigations may prove their therapeutic potential.

Development and validation of bioanalytical method for the determination of asulacrine in plasma by liquid chromatography.

Asulacrine (9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-N,5-dimethyl-4-acridinecarboxamide), an analogue of the antileukaemia drug amsacrine, has high antitumour activity in mice and has also shown clinical activity. A simple method is described for the quantitation of asulacrine in plasma by liquid chromatography. Chromatographic separation was achieved on a reversed phase C 18 column (250 mm x 4.6mm, particle size 5 microm, Gemini) using isocratic elution (acetonitrile and 0.01 M sodium acetate buffer pH 4.0, 45/55, v/v) at a flow rate of 1 ml/min. Asulacrine and internal standard (the ethylsulphonanilide analogue) were measured using UV detection at 254 nm. The total chromatographic run-time was 8 min with asulacrine and internal standard eluting at approximately 4.7 and approximately 6.5 min, respectively. Limit of quantification was 0.1microg/ml. The linearity range of the method was 0.1-10 microg/ml (r2=0.9995). Mean recoveries from plasma were 100-105%. Intra-batch and inter-batch precision was 7.1 and 7.8%, respectively, and intra-batch and inter-batch accuracy (relative error) was 4.9 and 8.4%, respectively (n=8 in all cases). The bench top, freeze thaw, short-term storage and stock solution stability evaluation indicated no evidence of degradation of asulacrine. The validated method is simple, selective and rapid and can be used for pharmacokinetic studies in mice.

Multiseed liposomal drug delivery system using micelle gradient as driving force to improve amphiphilic drug retention and its anti-tumor efficacy.

To improve drug retention in carriers for amphiphilic asulacrine (ASL), a novel active loading method using micelle gradient was developed to fabricate the ASL-loaded multiseed liposomes (ASL-ML). The empty ML were prepared by hydrating a thin film with empty micelles. Then the micelles in liposomal compartment acting as 'micelle pool' drove the drug to be loaded after the outer micelles were removed. Some reasoning studies including critical micelle concentration (CMC) determination, influencing factors tests on entrapment efficiency (EE), structure visualization, and drug release were carried out to explore the mechanism of active loading, ASL location, and the structure of ASL-ML. Comparisons were made between pre-loading and active loading method. Finally, the extended drug retention capacity of ML was evaluated through pharmacokinetic, drug tissue irritancy, and in vivo anti-tumor activity studies. Comprehensive results from fluorescent and transmission electron microscope (TEM) observation, encapsulation efficiency (EE) comparison, and release studies demonstrated the formation of ML-shell structure for ASL-ML without inter-carrier fusion. The location of drug mainly in inner micelles as well as the superiority of post-loading to the pre-loading method , in which drug in micelles shifted onto the bilayer membrane was an additional positive of this delivery system. It was observed that the drug amphiphilicity and interaction of micelles with drug were the two prerequisites for this active loading method. The extended retention capacity of ML has been verified through the prolonged half-life, reduced paw-lick responses in rats, and enhanced tumor inhibition in model mice. In conclusion, ASL-ML prepared by active loading method can effectively load drug into micelles with expected structure and improve drug retention.

Metabolic interaction between methotrexate and 4'-(9-acridinylamino)methanesulfon-M-anisidide in the rabbit.

The interaction between methotrexate (MTX) and a new acridine antitumor agent and potent aldehyde oxidase inhibitor, 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA), was investigated both in vivo and in vitro. New Zealand White male rabbits were used for the former experiments under three pharmacokinetic designs: (a) a zero order infusion of mAMSA at 9 mg/h to steady state followed by a single i.v. bolus dose of MTX at 50 mg/kg while maintaining the infusion; (b) a zero order infusion of MTX at 7 mg/h to steady state followed by a single i.v. bolus dose of mAMSA at 5 mg/kg while maintaining the infusion, and (c) a zero order infusion of MTX at 3 mg/h to steady state followed by a zero order infusion of mAMSA at 3 mg/h while maintaining the MTX infusion. In (a) while the mean AUC for MTX (15815 +/- 1317 microMmin) with mAMSA (+mAMSA) remained essentially unchanged relative to that without mAMSA (-mAMSA) at the same dose (14832 +/- 5151 microMmin), the mean AUC of the metabolite 7-hydroxymethotrexate (7-OH MTX) decreased from 9338 +/- 3057 (n = 6, -mAMSA) to 5794 +/- 1371 microMmin (n = 6, +mAMSA). Urinary excretion of 7-OH MTX also decreased from 40.3 +/- 9.5% (n = 6) (-mAMSA) to 23.8 +/- 6.1% dose (n = 6) (P less than 0.01) (+mAMSA) in 8 h with essentially no change in MTX excretion. The fractional rate conversion of MTX to this metabolite (fmi) also decreased from 0.60 +/- 0.19 (n = 6) to 0.40 +/- 0.10 (n = 6) (P less than 0.05). No change in terminal half-lives of MTX and 7-OH MTX was apparent. In (b) MTX steady state levels increased with the concomitant decrease in 7-OH MTX levels in the presence of mAMSA such that their concentration ratios (7-OH MTX/MTX) decreased to 43, 54, 75, and 76% of the pre-mAMSA values, respectively, in four rabbits. In the presence of mAMSA, clearance of MTX at steady state decreased significantly relative to those without mAMSA. Similar results were also observed in (c) except that the perturbation of MTX metabolism was more profound consistent with the experimental setting. No change in protein binding of MTX or the metabolite was apparent in the presence of mAMSA. Rabbit liver homogenate was used in the in vitro experiments which yielded a classical competitive inhibition on the double-reciprocal plot when conversion of MTX to 7-OH MTX was monitored.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced, topoisomerase II-mediated DNA cleavage by gossypol.

Our earlier studies have shown that gossypol [1,1',6,6',7,7'-hexahydroxy-5,5-diisopropyl - 3,3'-dimethyl - (2,2'- binaphthalene)-8,8'-dicarboxyaldehyde], a male contraceptive, inhibits DNA synthesis by decreasing the activities of DNA polymerase alpha and beta, resulting in the arrest of cells in mid-S phase [L.J. Rosenberg, R.C. Adlakha, D.M. Desai, and P.N. Rao, Biochim. Biophys. Acta, 866: 258-267, 1986]. Now we have examined the effects of gossypol on another enzyme of importance to cellular functions, topoisomerase II (topo II). We have determined the consequences of gossypol treatment on 4'-(9-acridinylamino)methane-sulfon-m anisidide (m-AMSA)-induced topoisomerase II-mediated, protein-associated DNA cleavage using the alkaline elution technique. In HeLa cells pretreated with gossypol (3.4-17.5 microM) for 8-16 h we observed a dose- and time-dependent decrease (50-75%) in DNA cleavage compared to that quantified in cells treated with m-AMSA alone. Gossypol by itself did not induce more than 25 rad-equivalents of DNA single-strand breaks even at the highest dose tested (26 microM). [14C]m-AMSA uptake was identical in treated and untreated cells. Pretreatment of cells with another inhibitor of DNA synthesis, thymidine, which blocks cells at G1/S boundary increased the m-AMSA-induced DNA cleavage by 25%, suggesting that the effect of gossypol might be due to the arrest of cells in mid-S phase. In contrast to gossypol's effects on m-AMSA-induced DNA cleavage, m-AMSA-induced cytotoxicity was actually increased in gossypol pretreated cells. Gossypol blocked topo II strand passing activity (decatenation of kinetoplast DNA) of cellular extracts from HeLa cells. The inhibition of this activity by gossypol was synergistic with the inhibition produced by m-AMSA or etoposide. These data suggest that gossypol can both inhibit topo II catalytic activity and interfere with the stabilization of topo II-DNA complex formation by m-AMSA. These data indicate that the magnitude of m-AMSA-induced DNA cleavage may not necessarily parallel the magnitude of m-AMSA-induced cytotoxicity. The cytotoxicity data may rather be explained by an action of gossypol and m-AMSA to block topo II catalytic activity at a point in the enzyme's strand passing cycle prior to cleavage complex formation that might be particularly toxic to cells in S phase. Gossypol should therefore be useful in improving our understanding of the cellular role of topo II and the consequences of interference with topo II activity by active antineoplastic agents.

Phase I trial of the amsacrine analogue 9-[( 2-methoxy-4-[(methylsulfonyl)amino]-phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921).

CI-921, an analogue of amsacrine with superior activity against in vivo and in vitro experimental tumor models, has been studied in 16 patients with solid tumors refractory to chemotherapy or for which conventional therapy does not exist. Thirty-nine cycles were given and doses escalated from 39 to 810 mg/m2. This total dose was divided over 3 consecutive days and administered by 15-min infusion each day, repeated three times weekly. Neutropenia (Eastern Cooperative Oncology Group) Grade greater than or equal to 3 occurred at Day 8 (range, 7-13) in 10/13 courses at 648 mg/m2 and in 2/2 courses at 810 mg/m2 with recovery in 10 (range, 4-20) days. At 810 mg/m2 Grade 2 mucositis and phlebitis were noted. Mild nausea and venous irritation occurred in some patients at doses greater than or equal to 288 mg/m2. No objective response was seen. Pharmacokinetics were evaluated following 65 infusions on Days 1 and 3 with plasma concentrations of CI-921 measured by high performance liquid chromatography. Peak plasma concentrations ranged from 3.36 to 85.6 mumol/liter and were significantly correlated with dose. Mean (range) model-independent pharmacokinetic parameters were: distribution half-life, 0.46 h (0.24-1.08); elimination half-life, 2.63 h (1.08-4.98); mean residence time, 2.0 h (1.05-3.35); plasma clearance, 158 ml/h/kg (95-290); and steady-state volume of distribution, 319 ml/kg (219-614) with no significant difference between Day 1 and 3. Toxicity as defined by absolute granulocyte count nadir was significantly correlated with dose, area under concentration-time curve, and peak plasma concentration. The recommended dose for Phase II studies in this schedule is 648 mg/m2 (216 mg/m2 daily for 3 days) repeated every three weeks.

Mechanism of resistance of noncycling mammalian cells to 4'-(9-acridinylamino)methanesulfon-m-anisidide: comparison of uptake, metabolism, and DNA breakage in log- and plateau-phase Chinese hamster fibroblast cell cultures.

Resistance of noncycling cells to amsacrine (m-AMSA) has been widely reported and may limit the activity of this drug against solid tumors. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblasts (AA8 cells, a subline of Chinese hamster ovary-cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity induced by a 1-h exposure to m-AMSA or to its solid tumor-active analogue, CI-921. Studies with radiolabeled m-AMSA established that similar levels of drug were accumulated by log- and plateau-phase cells and that there was no significant drug metabolism in either of these cultures after 1 h. However, marked differences in sensitivity to m-AMSA-induced DNA breakage were observed using a fluorescence assay for DNA unwinding (Kanter P.M., and Schwartz, H.S., Mol. Pharmacol., 22: 145-151, 1982). Changes in sensitivity to DNA breakage occurred in parallel with changes in sensitivity to m-AMSA-induced cell killing. DNA breaks disappeared rapidly after drug removal (half-time approximately 4 min), suggesting that these lesions were probably mediated by DNA topoisomerase II. Resistance to m-AMSA may therefore be associated with changes in topoisomerase II activity in noncycling cells.

Pharmacokinetic and toxicity scaling of the antitumor agents amsacrine and CI-921, a new analogue, in mice, rats, rabbits, dogs, and humans.

The aim was to investigate interspecies relationships between body weight (W) (kg) and various pharmacokinetic parameters for the anti-tumor agents amsacrine and its 4-methyl-5-(N-methylcarboxamide) analogue, CI-921, and examine which pharmacokinetic parameter, if any, might be used to predict the toxicity of these agents. Pharmacokinetic, plasma protein binding, and toxicity data were available for CI-921 in mice, rats, rabbits, dogs, and humans. For amsacrine, similar interspecies pharmacokinetic data were available but toxicity and protein-binding data were available for only 3 species. Significant linear relationships were obtained for CI-921 between log W and log Vss (liters) (r = 0.971, P = 0.006), and log W and log Cl (liters/h) (r = 0.911, P = 0.031) resulting in the allometric equations Vss = 1.22W0.68 and Cl = 0.91W0.51. For amsacrine these corresponding equations were Vss = 3.37W0.81 (r = 0.996, P less than 0.001), and Cl = 2.28W0.46 (r = 0.952, P = 0.012). When interspecies differences in plasma protein binding were taken into account, the allometric relationships improved and the exponents of the power equations increased. For CI-921 the allometric equations for the kinetic parameters calculated from plasma "free" concentrations were: Vssfu (liters) = 247W0.93 (r = 0.984, P = 0.002) and Clu (liters/h) = 186W0.76 (r = 0.961, P = 0.009). The dog was a noticeable outlier in the relationship between the log maximum tolerated dose (MTD) (mg/kg) of CI-921 and log W. Omission of the latter resulted in a highly significant allometric relationship, MTD = 23.6W-0.14 (r = -0.988, P = 0.012). For amsacrine there was no significant allometric relationship between MTD and W. CI-921s prolonged t1/2 in the dog and the dog's increased susceptibility to CI-921 toxicity suggested a relationship between MTD and t1/2 (h). A significant linear relationship was observed between in MTD and t1/2 (r = -0.994, P less than 0.001), from which the following equation was developed MTD = 47.5e-0.51t1/2 Combining the amsacrine toxicity data in the latter relationship yielded a similar equation MTD = 44.7e-0.51t1/2 (r = -0.933, P less than 0.0001). It was concluded that allometric equations may be developed for CI-921 and amsacrine from animal pharmacokinetic data which allow a reasonable prediction of Cl and Vss in patients, despite these agents being eliminated mainly by biotransformation. However, similar relationships between toxicity and body weight were susceptible to variation between individual species. Species differences in the toxicity of these agents were predictable from the t1/2. This study emphasized the importance of pharmacokinetic data in preclinical toxicity and efficacy testing of antitumor agents.

Disposition of amsacrine and its analogue 9-([2-methoxy-4-[(methylsulfonyl)amino]phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921) in plasma, liver, and Lewis lung tumors in mice.

9-([2-Methoxy-4-[(methylsulfonyl)amino]phenyl]amino)-N,5-dimethyl-4- acridinecarboxamide (CI-921), an analogue of the clinical antileukemia drug amsacrine with improved solid tumor activity in mice, is currently being evaluated in patients. In order to determine whether CI-921 possesses any advantages over amsacrine in terms of tissue delivery, the pharmacokinetics of amsacrine and CI-921 were determined following i.v. injection in male B6D2F1 mice. Plasma kinetics in normal mice were measured following administration of 14.4, 28.9, and 57.7 mumol/kg. The kinetics in s.c. Lewis lung tumors, and in plasma and livers of normal and tumor-bearing mice were measured following administration of 57.7 mumol/kg. CI-921 and amsacrine were quantitated by high-performance liquid chromatography after extraction from plasma and from liver and tumor homogenates. In experiments with appropriate 3H-labeled compounds, both total and covalently bound radioactivity (determined after precipitation and washing with acetonitrile) were measured in plasma and in liver homogenates. Over this dose range, nonlinear kinetics were observed in plasma for unchanged CI-921 and amsacrine, and a reasonable fit was obtained with Michaelis-Menten kinetics to a one-compartment model for CI-921 (Km 3.7 mumol/liter; Vmax 18 mumol/h/kg; V ss 3.3 liter/kg) and a two-compartment model for amsacrine (Km 3.6 mumol/liter; Vmax 76 mumol/h/kg; Vss 4.8 liter/kg). The area under the concentration-time curve (AUC) for plasma following a dose of 57.7 mumol/kg was 31 mumol.h/liter for CI-921 and 6.3 mumol.h/liter for amsacrine. However, equilibrium dialysis measurements indicated high plasma protein binding with free drug fractions for CI-921 and amsacrine of 0.63 and 6.7%, respectively. In the liver, unchanged drug concentrations and total radioactivity for both compounds were approximately 10-fold those in plasma, and the tissue half-life of CI-921 was approximately 4-fold longer for CI-921 than for amsacrine. Plasma and liver kinetics in mice with s.c. Lewis lung tumors were similar to those in normal mice. Tumor half-lives of unchanged CI-921 and amsacrine were 3.9 and 2.7 h, respectively, considerably longer than those for plasma (1.2 and 0.30 h respectively) or liver (1.2 and 0.28 h, respectively). Tumor AUC values for CI-921 and amsacrine were 68 and 37 mumol.h/liter, respectively, as compared to the calculated AUC values for free drug in plasma of 0.19 and 0.42 mumol.h/liter, respectively. It is concluded that the uptake into tumors from the plasma free drug fraction is more efficient for CI-921 than for amsacrine.

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II.

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.

Amsacrine analog-loaded solid lipid nanoparticle to resolve insolubility for injection delivery: characterization and pharmacokinetics.

Amsacrine analog is a novel chemotherapeutic agent that provides potentially broad antitumor activity when compared to traditional amsacrine. However, the major limitation of amsacrine analog is that it is highly lipophilic, making it nonconductive to intravenous administration. The aim of this study was to utilize solid lipid nanoparticles (SLN) to resolve the delivery problem and to investigate the biodistribution of amsacrine analog-loaded SLN. Physicochemical characterizations of SLN, including particle size, zeta potential, entrapment efficiency, and stability, were evaluated. In vitro release behavior was also measured by the dialysis method. In vivo pharmacokinetics and biodistribution behavior of amsacrine analog were investigated and incorporated with a non invasion in vivo imaging system to confirm the localization of SLN. The results showed that amsacrine analog-loaded SLN was 36.7 nm in particle size, 0.37 in polydispersity index, and 34.5±0.047 mV in zeta potential. More than 99% of amsacrine analog was successfully entrapped in the SLN. There were no significant differences in the physicochemical properties after storage at room temperature (25°C) for 1 month. Amsacrine analog-loaded SLN maintained good stability. An in vitro release study showed that amsacrine analog-loaded SLN sustained a release pattern and followed the zero equation. An in vivo pharmacokinetics study showed that amsacrine analog was rapidly distributed from the central compartment to the tissue compartments after intravenous delivery of amsacrine analog-loaded SLN. The biodistribution behavior demonstrated that amsacrine analog mainly accumulated in the lungs. Noninvasion in vivo imaging system images also confirmed that the drug distribution was predominantly localized in the lungs when IR-780-loaded SLN was used.

Highly sensitive analysis of the anti-tumor agent 1-[4-(furo[2,3-b]-quinolin-4-ylamino)phenyl]ethanone in rat plasma by high-performance liquid chromatography using electrochemical detection.

A sensitive high-performance liquid chromatography method with electrochemical detection was developed for the purpose of determining the concentration of the new anti-tumor agent 1-[4-(furo[2,3-b]-quinolin-4-ylamino)phenyl]ethanone (FQPE) in rats. The plasma samples were spiked with the internal standard diclofenac and extracted using dichloromethane. A C(18) 250 mm x 4mm column was used for the separation of analyte with a mobile phase consisting of 50% acetonitrile and 50% pH 3.0 of sodium 1-pentansulfonate solution at a flow rate of 1.0 mL/min. FQPE was detected by electrochemical detector at 1.0 V and 20 nA. Intra-day and inter-day precision and accuracy were acceptable down to the limit of quantization of 1 ng/mL. The lower limit of detection (LOD) was 0.5 ng/mL. The pharmacokinetic parameters of FQPE in rats after intravenous administration of 2.1 and 4.2 mg/kg were determined. The apparent volume of distribution, half-life of elimination, and clearance showed no significant difference between the two dosages. The area under the plasma concentration time curve increased proportionally with dose. The half-life of FQPE was prolonged about 2.4-fold, compared with amsacrine.

Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation.

The ultimate aim of this study was to develop asulacrine (ASL)-loaded long-circulating liposomes to prevent phlebitis during intravenous (i.v.) infusion for chemotherapy. Poly(ethylene)glycol (PEG) and poloxamer 188-modified liposomes (ASL-PEGL and ASL-P188L) were developed, and ASL was loaded using a remote loading method facilitated with a low concentration of sulfobutyl ether-ß-cyclodextrin as a drug solubilizer. The liposomes were characterized in terms of morphology, size, release properties and stability. Pharmacokinetics and venous tissue tolerance of the formulations were simultaneously studied in rabbits following one-hour i.v. infusion via the ear vein. The irritancy was assessed using a rat paw-lift/lick model after subplantar injections. High drug loading 9.0% w/w was achieved with no drug leakage found from ASL-PEGL or ASL-P188L suspended in a 5% glucose solution at 30days. However, a rapid release (leakage) from ASL-PEGL was observed when PBS was used as release medium, partially related to the use of cyclodextrin in drug loading. Post-insertion of poloxamer 188 to the liposomes appeared to be able to restore the drug retention possibly by increasing the packing density of phospholipids in the membrane. In rabbits (n=5), ASL-P188L had a prolonged half-life with no drug precipitation or inflammation in the rabbit ear vein in contrast to ASL solution. Following subplantar (footpad) injections in rats ASL solution induced paw-lick/lift responses in all rats whereas ASL-P188L caused no response (n=8). PEGylation showed less benefit possibly due to the drug 'leakage'. In conclusion, drug precipitation in the vein and the drug mild irritancy may both contribute to the occurrence of phlebitis caused by the ASL solution, and could both be prevented by encapsulation of the drug in liposomes. Poloxamer 188 appeared to be able to 'seal' the liposomal membrane and enhance drug retention. The study also highlighted the importance of bio-relevant in vitro release study in formulation screening.

Properties of the nucleic acid photoaffinity labeling agent 3-azidoamsacrine.

This paper reports the study of the photochemical, physical, and biological properties of 3-azidoamsacrine. The binding of 3-azidoamsacrine to DNA was studied with UV spectroscopy. The UV spectral behavior is quite similar to that of the parent amsacrine and argues that 3-azidoamsacrine is a good photoaffinity labeling agent for amsacrine. The biological properties (cytotoxicity and mutagenicity) of 3-azidoamsacrine in the mammalian mutagenesis V79 and L5178Y assay systems were measured. Light-activated 3-azidoamsacrine is toxic, but not mutagenic, to V79 cells. 3-Azidoamsacrine with and without light activation, as well as amsacrine, are toxic and mutagenic to L5178Y cells. To probe the interactions of 3-azidoamsacrine with DNA, studies of the photoreactivity of this compound were conducted. 3-Azidoamsacrine was photolyzed in the presence of the plasmid pBR322, and the effect of the photoadducts on restriction endonuclease cleavage was investigated. Amsacrine and 3-azidoamsacrine, without light activation, did not block any of the restriction endonucleases. Light-activated 3-azidoamsacrine blocked cleavage by the restriction endonucleases AluI, HinfI, NciI, NaeI, DraI, Sau96I, HpaII, and HaeIII. Photolysis experiments with mononucleosides, blocked mononucleosides, dinucleotides, and DNA all indicated that 3-azidoamsacrine formed adducts with G and A. The structures of these adducts are discussed based upon mass spectral data. Thus, it appears that 3-azidoamsacrine covalently attaches to DNA and that this covalent binding results in the production of toxic and, in some cases, mutagenic lesions in mammalian cells and the inhibition of restriction endonuclease cleavage of DNA.

The clinical pharmacokinetics of N-5-dimethyl-9-[(2-methoxy-4-methyl-sulfonylamino)phenylamino]-4 -acridinecarboxamide (CI-921) in a phase 1 trial.

The pharmacokinetics of CI-921 were studied after 65 infusions over a 20-fold dose range (13-270 mg/m2 per day) in 16 patients during a phase 1 trial. CI-921 was given by a 15 min infusion on three consecutive days. Plasma samples were collected after the first and third infusions, and urine, at 6 h intervals throughout the 3 days. CI-921 concentrations were measured by an HPLC method. Maximum plasma concentrations ranged from 3-86 mumol/l. The plasma concentration-time disposition curves were mainly biphasic over the 24-h postinfusion period. There was no significant difference by the paired t-test between the Cmax, AUC, CL, Vss, MRT, t1/2 alpha, or t1/2 beta calculated for the first and third infusions. The means (range) of model-independent pharmacokinetic parameters were: CL, 158 (94-290) ml/h per kg; Vss, 319 (219-614) ml/kg; MRT, 2.1 (1.1-3.5) h; t1/2 alpha, 0.5 (0.2-1.1) h; and t1/2 beta, 2.6 (1.1-5.0) h. There was a strong linear correlation between the dose and the AUC and Cmax, suggesting linear kinetics over this dose range. A very small amount (less than 1%) of the total dose was excreted as unchanged CI-921 in the urine, mostly in the 12-h postinfusion period.

Pharmacokinetics of continuous-infusion amsacrine and teniposide for the treatment of relapsed childhood acute nonlymphocytic leukemia.

The systemic disposition of both amsacrine and teniposide was determined in children receiving treatment for resistant acute nonlymphocytic leukemia. As part of a phase I-II study, amsacrine and teniposide were given as continuous 72-h i.v. infusions at doses of 75-150 and 150-250 mg m-2 day-1, respectively. Plasma samples obtained during steady state were analyzed for drug concentrations by high-performance liquid chromatography assays specific for each compound. Clearance and systemic exposure values for both amsacrine and teniposide were calculated for 14 patients, and data were available for teniposide alone in an additional 14 subjects. Interpatient variability in clearance was substantial for each drug, producing overlapping systemic exposure across dose levels. No evidence of dose-dependent drug clearance was evident. Clearance values for teniposide given in combination with amsacrine were similar to previous values obtained when teniposide was given in an identical manner but as a single agent. In all, 80% of patients experienced some degree of mucositis after chemotherapy administration. Severe mucositis (Pediatric Oncology Group grades 3-4) occurred in 18% of cases, all of whom showed teniposide steady-state plasma concentrations above the median population value (11.9 micrograms/ml; P less than 0.0001). A comparison of the results of the present study on teniposide combined with amsacrine with those previously obtained for single-agent teniposide suggest that amsacrine produced little additive gastrointestinal toxicity. The evaluation of anti-cancer drug pharmacokinetics in individual patients during combination chemotherapy regimens helps to determine the relative importance of each agent when toxicity patterns are similar.