Dynamic assessment of mitoxantrone resistance and modulation of multidrug resistance by valspodar (PSC833) in multidrug resistance human cancer cells.

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, is one of the major causes for multidrug resistance (MDR). We report using confocal microscopy to study the roles of Pgp in mediating the efflux of the anticancer agent mitoxantrone and the reversal of MDR by the specific Pgp inhibitor valspodar (PSC833). The net uptake and efflux of mitoxantrone and the effect of PSC833 were quantified and compared in Pgp-expressing human cancer MDA-MB-435 (MDR) cells and in parental wild-type cells. The MDR cells, transduced with the human Pgp-encoding gene MDR1 construct, were approximately 8-fold more resistant to mitoxantrone than the wild-type cells. Mitoxantrone accumulation in the MDR cells was 3-fold lower than that in the wild-type cells. The net uptake of mitoxantrone in the nuclei and cytoplasm of MDR cells was only 58 and 67% of that in the same intracellular compartment of the wild-type cells. Pretreatment with PSC833 increased the accumulation of mitoxantrone in the MDR cells to 85% of that in the wild-type cells. In living animals, the accumulation of mitoxantrone in MDA-MB-435mdr xenograft tumors was 61% of that in the wild-type tumors. Administration of PSC833 to animals before mitoxantrone treatment increased the accumulation of mitoxantrone in the MDR tumors to 94% of that in the wild-type tumors. These studies have added direct in vitro and in vivo visual information on how Pgp processes anticancer compounds and how Pgp inhibitors modulate MDR in resistant cancer cells.

In vivo evaluation of diaminodiphenyls: anticonvulsant agents with minimal acute neurotoxicity.

Several diaminodiphenyl analogs were assessed in vivo for their capacity to inhibit seizure induction and propagation in rodents. Both 3,4'- and 4,4'-diaminodiphenyl compounds prevented seizures for as long as 4h after maximal electric shock induction. 4,4'-Diphenyl compounds bridged by a methylene, sulfide, or carbonyl linker also attenuated focal seizure acquisition in a kindling model. Of these analogs, based upon data generated in two rodent species, 4,4'-thiodianiline (1) was identified as the most active compound, significantly reducing seizure staging scores and after-discharge duration for several hours after systemic administration. All compounds were devoid of acute in vivo neurotoxicity at doses well above those required for anticonvulsant activity.

Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells.

P-glycoprotein (Pgp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells, and it plays a major role in causing multidrug resistance (MDR). Confocal microscopy was used to study in vitro and in vivo drug accumulation, net uptake and efflux, and MDR modulation by P-glycoprotein inhibitors in MDR1-transduced human MDA-MB-435mdr (MDR) cancer cells. The MDR cells were approximately 9-fold more resistant to the anticancer drug doxorubicin than their parental wild-type MDA-MB-435wt (WT) cells. Doxorubicin accumulation in the MDR cells was only 19% of that in the WT cells. The net uptake of doxorubicin in the nuclei of the MDR cells was 2-fold lower than that in the nuclei of the WT cells. Pgp inhibitors verapamil, cyclosporine A, or PSC833 increased doxorubicin accumulation in the MDR cells up to 79%, and it reversed drug resistance in these cells. In living animals, doxorubicin accumulation in MDA-MB-435mdr xenograft tumors was 68% of that in the wild-type tumors. Administration of verapamil, cyclosporine A, or PSC833 before doxorubicin treatment of the animals increased doxorubicin accumulation in the MDR tumors up to 94%. These studies have added direct in vitro and in vivo information on the capacity of the transporter protein Pgp to efflux doxorubicin and on the reversal of MDR by Pgp inhibitors in resistant cancer cells.

The effect of DB-67, a lipophilic camptothecin derivative, on topoisomerase I levels in non-small-cell lung cancer cells.

PURPOSE: To determine the in vitro drug sensitivity of two non-small-cell lung cancer cell lines after treatment with the novel lipophilic camptothecin derivative, 7- tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67), to determine if topoisomerase I protein levels decrease after treatment with DB-67, and to assess the duration and extent of topoisomerase I modulation after DB-67 exposure, in order to provide information about drug resistance that may be useful in determining an appropriate dosing schedule for DB-67. METHODS: The growth inhibition of the non-small-cell lung cancer cell lines A549 and H460 after exposure to DB-67 was evaluated with the MTS assay. A549 and H460 cells were treated for various times with DB-67 and topoisomerase I levels were determined by western blot analysis. In addition, A549 and H460 cells were treated with DB-67 for 24 h and topoisomerase I levels were determined by western blot analysis daily for 1 week after drug removal. RESULTS: DB-67 inhibited the growth of both A549 and H460 cells grown in culture; the A549 cells were more resistant to the cytotoxic effects of DB-67 than H460 cells. Notably, A549 cells had approximately one-half the baseline topoisomerase I than H460 cells. Topoisomerase I protein levels significantly decreased after 8-18 h of exposure to DB-67. Both A549 and H460 cells treated with DB-67 for 24 h had only negligible amounts of topoisomerase I at the end of treatment. However, within 24 h of drug removal topoisomerase I levels returned to near baseline levels in both cell lines. CONCLUSIONS: The decrease in topoisomerase I levels caused by DB-67 may represent a mechanism of resistance to this novel camptothecin derivative. Dosing DB-67 once every 48-72 h may maximize the interaction of the drug with topoisomerase I and should be considered as a potential dosing schedule in the preclinical and clinical development of this compound.

An in vivo evaluation of the antiseizure activity and acute neurotoxicity of agmatine.

Agmatine, an endogenous cationic amine, exerts a wide range of biological effects, including modulation of glutamate-activated N-methyl-D-aspartate (NMDA) receptor function in the central nervous system (CNS). Since glutamate and the NMDA receptor have been implicated in the initiation and spread of seizure activity, the capacity of agmatine to inhibit seizure spread was evaluated in vivo. Orally administered agmatine (30 mg/kg) protected against maximal electroshock seizure (MES)-induced seizure spread in rats as rapidly as 15 min and for as long as 6 h after administration. Inhibition of MES-induced seizure spread was also observed when agmatine was administered intraperitoneally. Agmatine's antiseizure activity did not appear to be dose-dependent. An in vivo neurotoxicity screen indicated that agmatine was devoid of any acute neurological toxicity at the doses tested. These preliminary data suggest that agmatine has promising anticonvulsant activity.

Phase I pharmacokinetic studies evaluating single and multiple doses of oral GW572016, a dual EGFR-ErbB2 inhibitor, in healthy subjects.

GW572016 is a dual EGFR-ErbB2 inhibitor that has promise as an anticancer agent. Two phase I studies were conducted to determine the safety, tolerability and pharmacokinetics of single and multiple doses given to healthy subjects. The single dose study evaluated two groups of eight subjects in an ascending dose, 4-way cross-over, while the multiple dose study evaluated twenty-seven healthy volunteers in an ascending dose, double-blind, randomized, placebo-controlled, staggered parallel design. No serious adverse events were seen in either study. The most common adverse events for subjects receiving GW572016 were headache, diarrhea, rash, cold symptoms, gastrointestinal symptoms, and elevated LFTs, which were similar between treatment and placebo groups. Absorption of single doses of GW572016 was slightly delayed, with median t(lag) of 15 minutes (range 0-90 minutes) and achieved peak serum concentrations at a median of three hours (range 1.5-6 hours) post-dose. Serum concentrations after multiple doses of GW572016 demonstrated no significant accumulation at the 25 mg dose, and approximately 50% accumulation at the 100 mg and 175 mg doses, achieving steady state in six to seven days. A modest time-dependent increase in serum concentrations also was detected with multiple doses of GW572016. Single and multiple oral doses of GW572016 were well tolerated in healthy subjects, and resulted in dose-related systemic exposure of GW572016.

The mechanism of L-canavanine cytotoxicity: arginyl tRNA synthetase as a novel target for anticancer drug discovery.

There is a clear need for agents with novel mechanisms of action to provide new therapeutic approaches for the treatment of pancreatic cancer. Owing to its structural similarity to L-arginine, L-canavanine, the beta-oxa-analog of L-arginine, is a substrate for arginyl tRNA synthetase and is incorporated into nascent proteins in place of L-arginine. Although L-arginine and L-canavanine are structurally similar, the oxyguanidino group of L-canavanine is significantly less basic than the guanidino group of L-arginine. Consequently, L-canavanyl proteins lack the capacity to form crucial ionic interactions, resulting in altered protein structure and function, which leads to cellular death. Since L-canavanine is selectively sequestered by the pancreas, it may be especially useful as an adjuvant therapy in the treatment of pancreatic cancer. This novel mechanism of cytotoxicity forms the basis for the anticancer activity of L-canavanine and thus, arginyl tRNA synthetase may represent a novel target for the development of such therapeutic agents.

L-Canavanine as a radiosensitization agent for human pancreatic cancer cells.

This study evaluated the in vitro effect of L-canavanine on cell cycle progression in the two human pancreatic cancer cells lines PANC-1 and MIA PaCa-2. After 72 h of exposure to L-canavanine, the percentage of cells in the radiosensitive G2/M phase of the cell cycle increased 6-fold in PANC-1 cells and 4-fold in MIA PaCa-2 cells, when compared to untreated cells. The capacity of L-canavanine to redistribute cells into the G2/M phase of the cell cycle was both concentration- and time-dependent. Since many drugs that cause cells to accumulate in the G2/M phase of the cell cycle are effective radiosensitization agents, the potential of L-canavanine to synergistically enhance the effects of ionizing radiation also was evaluated. The interaction between these treatment modalities was quantified using the median-effect equation and combination index analysis. L-Canavanine was found to be synergistic with radiation when either PANC-1 or MIA PaCa-2 cells were exposed to L-canavanine for 72 h prior to irradiation. These results suggest that L-canavanine in combination with radiation may have clinical potential in the treatment of pancreatic cancer.

The antiproliferative and immunotoxic effects of L-canavanine and L-canaline.

L-Canavanine and its arginase-catalyzed metabolite, L-canaline, are two novel anticancer agents in development. Since the immunotoxic evaluation of agents in development is a critical component of the drug development process, the antiproliferative effects of L-canavanine and L-canaline were evaluated in vitro. Both L-canavanine and L-canaline were cytotoxic to peripheral blood mononucleocytes (PBMCs) in culture. Additionally, the mononucleocytes were concurrently exposed to either L-canavanine or L-canaline and each one of a series of compounds that may act as metabolic inhibitors of the action of L-canavanine and L-canaline (L-arginine, L-ornithine, D-arginine, L-lysine, L-homoarginine, putrescine, L-omega-nitro arginine methyl ester and L-citrulline). The capacity of these compounds to overcome the cytotoxic effects of L-canavanine or L-canaline was assessed in order to provide insight into the biochemical mechanisms that may underlie the toxicity of these two novel anticancer agents. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. The elucidation of the biochemical mechanisms associated with the effects of L-canavanine and L-canaline on lymphoproliferation may be useful for maximizing the therapeutic effectiveness and minimizing the toxicity of these novel anticancer agents.