Effect of single and multiple administration of an O 6-benzylguanine/temozolomide combination: an evaluation in a human melanoma xenograft model

The purpose of the present study was to examine the effect of O ⁶-benzylguanine (O ⁶-BG) on the antitumour activity and toxicity of 8-carbamoyl-3-methylimidazo [5, 1-d ] -1,2,3,5-tetrazine-4(3H)-one (temo-zolomide) in a human malignant melanoma xenograft model following single and multiple administration of the combination. O ⁶-BG irreversibly inactivates the DNA-repair protein O ⁶-alkylguanine-DNA alkyltransferase (AGT), which confers resistance to temozolomide. Preadministration of O ⁶-BG (35 mg/kg, i.p.) 1 h prior to temozolomide (i.p.) was examined using single and daily × 5 dosing regimens in athymic mice bearing subcutaneous A375P xenografts. The AGT activity of A375P tumors was 95 ± 8 fmol/mg protein (mean ± SE, n = 4). O ⁶-BG alone completely suppressed xenograft AGT activity within 1 h of administration but had no effect upon tumor growth. O ⁶-BG did not significantly increase the tumor growth delay induced by a single 200-mg/kg dose of temozolomide (P>0.05, two-tailed Mann-Whitney test) but did increase the associated mean body weight loss (P<0.025). In contrast, when the same dose of temozolomide was divided into five equal fractions (40 mg/kg) and given with O ⁶-BG on 5 consecutive days, a comparable increase in toxicity was accompanied by a very significant increase in tumor growth delay (P<0.0025), equivalent to that produced by a 3-fold greater dose of temozolomide alone. O ⁶-BG with temozolomide also produced a greater antitumour effect than an equitoxic dose of temozolomide alone on this schedule (P<0.005). These data indicate that the enhancement of temozolomide antitumour activity by O ⁶-BG preadministration is dependent upon the schedule of drug administration, with multiple dosing of O ⁶-BG + temozolomide producing the greatest effect. The results also suggest that prolonged administration of the combination can lead to an increase in the therapeutic index of temozolomide. Received: 8 September 1996 / Accepted: 8 February 1997     

Dose intensity of uracil and tegafur in postoperative chemotherapy for patients with poorly differentiated gastric cancer

A retrospective analysis of postoperative chemotherapy had shown the continuous administration of UFT, an oral preparation of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil at a molar ratio of 1:4, to be effective for poorly differentiated gastric cancer. We therefore sought to determine prospectively the effective dose of postoperative chemotherapy with UFT for patients with poorly differentiated gastric cancer following a curative resection. We determined the effect of the combined intravenous administration of mitomycin C (MMC) and oral treatment with protein-bound polysaccharide Kreha (PSK), extracted from the basidiomycete Coriolus versicolor, and UFT at a dose of either 8 mg/kg or 12 mg/kg daily for 1 year. A total of 224 patients with poorly differentiated stage II–IV gastric cancer were entered into this study after undergoing a curative resection. No differences were observed between the two treatment groups in terms of prognostic factors, the toxicity rate or the doses of the drugs prescribed, other than UFT. The higher dose of UFT in maintenance therapy led to a decrease in the recurrence rate (P < 0.05), and increases in disease-free survival and cause-specific survival (P < 0.05). UFT at 12 mg/␣kg in postoperative chemotherapy was thus found- to improve the postoperative results with no increase in toxicity for poorly differentiated gastric cancer, and is also cost-effective for outpatients. Received: 8 February 1996 / Accepted: 27 November 1996     

Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel

Purpose: To determine, as we did for paclitaxel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drug's antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. Methods: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxel's antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. Results: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4 +/− 0.4% to 25.1 +/− 0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3 +/− 1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. Conclusions: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxel's antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors. Received: 3 March 1998 / Accepted: 14 May 1998     

Pharmacokinetics and pharmacodynamics of MX2 hydrochloride in patients with advanced malignant disease

The purpose of the present study was to investigate the pharmacokinetics and pharmacodynamics of the new morpholino anthracycline drug MX2. A total of 27 patients with advanced cancer participated in a dose-escalation study in the first cycle of treatment with drug given i.v. at doses of 10–50 mg/m² (total dose 16.8–107.5 mg). The mean total systemic plasma clearance (CL) of MX2 was 2.98 ± 1.68 l/min, the mean volume of distribution at steady state was 1460 ± 749 l and mean elimination half-life was 10.8 ± 5.1 h. The area under the plasma concentration-time curve (AUC) of MX2 was linearly related to the dose per kilogram and the dose per body surface area (r ² = 0.43, P < 0.01 and r ² = 0.44, P < 0.01, respectively). CL did not correlate with total body weight, lean body mass or body surface area. The mean elimination half-lives of the metabolites M1, M2, M3 and M4 were 11.8 ± 5.0, 21.9 ± 11.8, 19.0 ± 11.3 and 12.3 ± 6.3 h, respectively. The fractional E _max model produced a much better fit to the relative nadir neutrophil count versus dose data (r ² = 0.42) than to the relative nadir neutrophil count versus AUC or peak concentration (C _max) data (r ² = 0.15 and 0.09, respectively). There seemed to be a threshold dose of about 65 mg of MX2 at or above which a large proportion of patients had a nadir neutrophil count of less than 0.5 × 10⁹/l. This study shows that the pharmacokinetics of MX2 are similar to those of other anthracyclines. With other anthracyclines the degree of myelosuppression seems to depend more on the AUC and C _max than on the delivered dose; however, with MX2 the degree of myelosuppression depends more on the dose given than on drug exposure expressed as the AUC or C _max. Received: 18 February 1996 / Accepted: 20 December 1996     

Measurement of nitrobenzylthioinosine in plasma and erythrocytes: a pharmacokinetic study in mice

Purpose: Nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in many cell types, modulates the in vivo disposition of several cytotoxic nucleoside analogs. In this study, a radioligand binding assay was developed for measurement of the NBMPR content of plasma and erythrocytes. Methods: The assay was based on the competition between NBMPR and [³H]NBMPR for high-affinity sites on human erythrocyte membranes. With this assay, we followed in mice changes in the NBMPR content of blood plasma and erythrocytes, following the intraperitoneal injection of the disodium salt of NBMPR 5′-monophosphate (NBMPR-P), a prodrug form of NBMPR. Results: The radioligand binding assay was able to measure precisely as little as 2.5 pmol of NBMPR, allowing the direct determination of NBMPR concentrations in plasma as low as 16 nM. As few as 8 × 10³ molecules of NBMPR per cell could be determined in erythrocytes. The NBMPR content of plasma from mice injected with NBMPR-P was maximal at about 20 min after injection and declined to <0.2% of the peak value by 10 h. Erythrocyte-associated NBMPR was also maximal at 20 min, and declined to 11% of the peak value by 10 h after injection. Time courses for the disappearance of NBMPR from plasma and erythrocytes were monoexponential and yielded half-life values of 0.39 h and 0.68 h, respectively, an apparent volume of distribution of 0.61 l/kg, and a clearance of 1.1 l/h per kg. Conclusions: The radioligand binding assay is a sensitive and facile method for monitoring NBMPR concentrations in mammalian plasma and tissue extracts. Received: 20 August 1995 / Accepted: 17 December 1996     

In vitro antitumor activity of 3′-desamino-3′(2-methoxy-4-morpholinyl) doxorubicin on human melanoma cells sensitive or resistant to triazene compounds

A new methoxymorpholinyl derivative of Adriamycin (ADR), FCE 23762 (MRD), has recently been selected for phase I clinical trials for its reduced cardiotoxicity and for its cytotoxic activity against a broad spectrum of solid tumors and leukemias that are sensitive or resistant to ADR. The purpose of the present study was to compare the in vitro antitumor activity of MRD and ADR on human melanoma lines with different chemosensitivity to triazene compounds, among which dacarbazine remains a reference drug in the treatment of melanoma. Both MRD and ADR were tested in vitro on three melanoma lines, MI13443-MEL, SK-MEL-28, and M14, previously screened for their chemosensitivity to the triazene compound p-(3-methyl-1-triazeno) benzoic acid, potassium salt (MTBA). The three lines were also analyzed for P-170 expression, total glutathione (GSH) content, and GSH-related enzyme activity. All melanomas, whether sensitive or resistant to MTBA, were susceptible to anthracycline treatment. The cytotoxic activity of MRD was comparable with that of ADR, and no substantial difference was found in cell growth inhibition between the two drugs. When the relative chemosensitivity of the three lines was considered, SK-MEL-28 was found to be slightly less sensitive to MRD treatment than the other tumors. This finding seems to correlate with the higher GSH-peroxidase activity of this melanoma relative to that of the MI13443 and M14 lines. These results show a homogeneous response of melanoma lines to MRD treatment in vitro, suggesting that phase I clinical trials concerning this drug, which in vivo appears to be activated to a more cytotoxic metabolite, could be extended to metastatic melanomas, including those completely resistant to triazene compounds. Received: 11 August 1996 / Accepted: 20 January 1997     

Metabolism of 6-mercaptopurine in the erythrocytes, liver, and kidney of rats during multiple-dose regimens

Purpose: To describe the metabolism of 6-mercaptopurine (6-MP) in erythrocytes and tissues of rats after repeated administration of 6-MP at two dose levels and to provide evidence that in vivo modulation of 6-MP anabolism can be obtained by simultaneous treatment with ribavirin or hydroxyurea, two inhibitors of enzymes involved in the bioactivation of 6-MP to the active 6-thioguanine nucleotides (6-TGN). Methods: Rats were treated i.p. with 6-MP at 12.5 and 25 mg/kg daily for 12 days and erythrocyte, liver, and kidney levels of 6-mercaptopurine nucleotides (6-MPN) and 6-TGN were investigated during the accumulation phase and for 50 days after the end of treatment. In combination studies, ribavirin at 75 and 100 mg/kg per day (for 6-MP, 25 and 12.5 mg/kg per day) or hydroxyurea at 200 mg/kg per day were given i.p. for 12 days. The measurements of thionucleotide levels in rat samples were performed by high-pressure liquid chromatography (HPLC). Results: The maximal concentration (C_max) and the area under the concentration versus time curve (AUC) of 6-MPN and 6-TGN in erythrocytes and tissues increased significantly after the administration of 6-MP at 25 mg/kg per day as compared with 12.5 mg/kg per day. In particular, the C_max and AUC of 6-TGN in erythrocytes of rats treated with 6-MP at 25 mg/kg per day were approximately 5-fold higher than the 6-TGN values observed following treatment at 12.5 mg/kg per day. Moreover, 6-TGN levels in erythrocytes were significantly higher than those of 6-MPN (910.9 ± 53.1 and 286.8 ± 23.4 pmol/8 × 10⁸ cells for 6-TGN and 6-MPN, respectively, P < 0.05) after treatment with 6-MP at 25 mg/kg per day. The administration of ribavirin, an inhibitor of inosine monophosphate dehydrogenase, in association with 6-MP increased the amount of 6-MPN detected in erythrocytes and tissues while reducing 6-TGN levels in samples. The production and accumulation of 6-MPN and 6-TGN were increased in erythrocytes and tissues by hydroxyurea, an inhibitor of ribonucleotide reductase. Finally, a significant correlation between thionucleotide concentrations and erythrocyte counts was observed. Conclusion: The overall results demonstrate that 6-MP is actively metabolized in rats and that its biotransformation can be modulated by agents acting on enzymes of the purine metabolism, resulting in significant changes in erythrocyte and tissue levels of 6-MPN and 6-TGN. These findings provide evidence that the rat is a suitable model for investigation of the metabolism of 6-MP and its possible pharmacologic modulation. Received: 17 December 1997 / Accepted: 29 June 1998     

Activity of fenretinide plus chemotherapeutic agents in small-cell lung cancer cell lines

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide, 4HPR], a synthetic retinoid, is a potent inducer of apoptosis in small-cell lung cancer (SCLC) cell lines that may act through the generation of reactive oxygen species, suggesting that it may enhance the activity of other cytotoxic agents. In light of 4HPR's clinical potential and potent activity against SCLC cells, we evaluated the in vitro activity of 4HPR in combination with cisplatin, etoposide or paclitaxel. Methods: The growth-inhibitory activities of single-agent 4HPR, cisplatin, etoposide or paclitaxel, and combinations of 4HPR and individual chemotherapeutic agents, were evaluated using an MTT assay in two SCLC cell lines. Each two-drug combination was studied over a range of concentrations at a fixed ratio corresponding to the ratio of the IC₅₀ values of the individual agents. Data were analyzed by median-effect analysis as previously applied to drug combination studies. Results: All four agents inhibited growth in a dose-dependent manner in the NCI-H82 and NCI-H446 SCLC cell lines. At clinically reported drug concentrations that resulted in over 50% growth inhibition, the activities of the combinations 4HPR and cisplatin and 4HPR and etoposide were more than additive in both cell lines, and the activity of 4HPR plus paclitaxel was more than additive in NCI-H446 cells. Conclusion: 4HPR's potent single-agent activity, minimal toxicity, and potential synergy with standard cytotoxic drugs will allow for the development of promising investigational regimens for the treatment of patients with SCLC. Received: 17 February 1998 / Accepted: 20 May 1998     

Pharmacokinetics of dolasetron with coadministration of cimetidine or rifampin in healthy subjects

Purpose: Dolasetron is a selective 5-HT₃ receptor antagonist. The purpose of this study was to determine the effect of cimetidine and rifampin on the steady-state pharmacokinetics of orally administered dolasetron and its active reduced metabolite, hydrodolasetron. Methods: A group of 18 healthy men (22 to 44 years old) were randomized to receive each of the following three treatments in a three-period crossover design: 200 mg dolasetron daily (treatment A); 200 mg dolasetron daily plus 300 mg cimetidine four times daily (treatment B); or 200 mg dolasetron daily plus 600 mg rifampin daily (treatment C). Each study period was separated by a 14-day washout period. Serial blood samples were collected before the first dose (baseline) on day 1 and at frequent intervals up to 48 h after the morning dose on day 7 for quantification of dolasetron and its metabolites, hydrodolasetron (both isomers), 5′OH hydrodolasetron, and 6′OH hydrodolasetron. Serial urine samples were also collected at baseline and during the periods 0–24 and 24–48 h following the morning dose on day 7, and analyzed for dolasetron and its metabolites. Results: Plasma and urine dolasetron concentrations were below quantifiable concentrations for all three treatments. Mean steady-state area under the plasma concentration-time curve (AUC_ss(0–24)) of hydrodolasetron increased by 24%, mean apparent clearance (CL_app,po) decreased by 19%, and maximum plasma hydrodolasetron concentration (C_max,ss) increased by 15% when dolasetron was coadministered with cimetidine. When dolasetron was given with rifampin, mean hydrodolasetron AUC_ss(0–24) decreased by 28%, CL_app,po increased by 39%, and hydrodolasetron C_max,ss decreased by 17%. Small differences were found in mean t_max (0.7 to 0.8 h), CL_r (2.0 to 2.6 ml/min per kg), and t_1/2 (7.4 to 8.8 h) for hydrodolasetron between treatment periods. Approximately 20% and 2% of the dolasetron dose were excreted in urine as the R(+) isomer and S(−) isomer of hydrodolasetron, respectively, across all three treatments. Dolasetron mesylate was well tolerated in this study during all three treatment periods, with the highest incidence of adverse events reported during the control period when dolasetron mesylate was given alone. Conclusion: Based on the small changes in the pharmacokinetic parameters of dolasetron and its active metabolites, as well as the favorable safety results, no dosage adjustments for dolasetron mesylate are recommended with concomitant administration of cimetidine or rifampin. Received: 10 February 1998 / Accepted: 1 June 1998     

Inter- and intraindividual variation in dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells

Purpose: The activity of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in fluorouracil catabolism, has been reported to vary according to time of day. We wished to determine whether peak and trough DPD activities occurred at uniform times in six subjects, whether individual patterns fit a discernible profile, and whether such patterns were consistent and reproducible over time. Methods: Mononuclear cells were isolated from peripheral blood at 3-h intervals over a 24-h period on three different dates over a 6-month period. DPD activity was determined by incubating cellular lysates with [³H]FUra and measuring [³H]dihydrofluorouracil formation over time. Results: When the data were averaged by study date for each subject, the median value for the average DPD activity (11.0 pmol/min per 10⁶ cells) was significantly different from both the median peak (21.1 pmol/min per 10⁶ cells, P = 0.004) and median trough activities (4.0 pmol/min per 10⁶ cells, P = 0.002). Within the six␣subjects, the average DPD activity for the three study dates differed by a median of 2.4-fold (range 1.2- to 4.8-fold). The time at which peak and trough DPD activities occurred varied between subjects: 8 of the 17 peaks (47%) occurred between 10:00 p.m. and 6:00 a.m., 6 (35%) occurred between 8:00 a.m. and 3:00 p.m., and 3 (18%) occurred between 5:00 p.m. and 8:15 p.m. Thus, the time of day when the peak occurred was essentially randomly distributed over the 24-h period of observation (P = 0.68). Ten (59%) of the trough DPD activities occurred between 7:00 a.m. and 3:00 p.m. The median interval between the peak and trough was 6.5 h. The data were also expressed as percent of the mean for each individual's 24-h sampling period, and reordered as time from peak rather than as the actual time of day. When the combined data for all cycles was considered, the trough occurred 6–9 h after the peak, and the DPD levels subsequent to the peak did not display merely random variation (P = 0.0055). Conclusions: DPD activity levels and the times at which peak and trough DPD activities occurred varied both between and within subjects. If fluctuations in DPD activity influence the tolerability of fixed-rate infusions of FUra, these data suggest that a single variable-rate infusion regimen may not be suitable for all patients nor for a given individual treated over several months. Received: 1 April 1996 / Accepted: 7 October 1996     

A phase I study of recombinant human soluble interleukin-1 receptor (rhu IL-1R) in patients with relapsed and refractory acute myeloid leukemia

Purpose: The recombinant human interleukin-1 receptor (rhu IL-1R) is a soluble truncated form of the type 1 full-length membrane-bound receptor that binds IL-1 with identical affinity to that of the membrane form. As such, it may have clinical potential by sequestering IL-1, thereby preventing it from binding to its membrane-bound receptor and eliciting a biological effect. As IL-1 has been shown to regulate leukemic cell proliferation in an autocrine fashion, a phase I trial of rhu IL-1R was conducted in patients with relapsed and refractory acute myeloid leukemia (AML). Methods: The study group comprised 11 patients who were sequentially treated on one of three dose levels, receiving a single intravenous (i.v.) bolus dose on day 1 followed by 13 days of daily subcutaneous (s.c.) injections with the option of an additional 14 days of treatment if a response of stable disease or better was achieved. Dose level 1 i.v. bolus 500 g/m², s.c. dose 250 g/m² per day (five patients); dose level 2 i.v. bolus 1000 g/m², s.c. dose 500 g/m² per day (three patients); dose level 3 i.v. bolus 2000 g/m², s.c. dose 1000 g/m² per day (three patients). Owing to limited drug availability, the study was designed to only examine these three dose levels. Results: rhu IL-1R was well tolerated. There was no grade 3 or 4 non-hematological toxicity related to the study drug and the maximum tolerated dose was not reached. No IL-1R-blocking antibodies developed during the course of the study. Serum levels of IL-1, IL-6 and TNF were undetectable before, during and after rhu IL-1R administration. The terminal half-life after i.v. dosing was at least 7–12 h, and after s.c. dosing 2–4 days. Serum levels of rhu IL-1R up to 360- and 25-fold those of pretreatment levels were achieved after i.v. and s.c. dosing respectively. No patient had a complete, partial or minor response to treatment; four had stable disease and seven had progressive disease. Conclusions: rhu IL-1R therapy was safe but did not have any apparent antileukemic effect at the doses administered. Received: 6 October 1997 / Accepted: 1 April 1998     

Disposition of conjugate-bound and free doxorubicin in tumor-bearing mice following administration of a BR96-doxorubicin immunoconjugate (BMS 182248)

Purpose: The chimeric BR96–doxorubicin (DOX) immunoconjugate, BMS 182248, has induced remissions and cures of human lung adenocarcinoma (L2987) implanted in athymic mice. The purpose of this study was to evaluate the biodistribution of DOX after BMS 182248 administration to tumor-bearing mice and to evaluate the ability of BMS 182248 to target DOX to tumors. Methods: For this evaluation, L2987-implanted mice were given BMS 182248 (5 mg DOX/kg; three doses 4 days apart) and the levels of both conjugate-bound and free DOX in plasma, tumor, liver and heart were determined. Results: Conjugate-bound DOX comprised the majority of plasma DOX, with relatively low levels of free DOX present. From plasma, conjugate-bound DOX distributed to the tissues examined with the order of concentration (per gram of tissue) being tumor > liver > heart. Free DOX was also detected in liver and heart, but at concentrations lower than those present after an equivalent DOX dose (5 mg/kg; three doses 4 days apart). The total exposure of heart to free DOX after BMS 182248 administration was about one- quarter of that found after the administration of DOX alone. The elimination kinetics of both conjugate-bound and free DOX from heart and liver after BMS 182248 administration paralleled those observed from plasma, indicating that equilibrium had been attained between these nontumor tissues and plasma. The elimination kinetics of both entities from tumors, however, were different from those from plasma, liver and heart. BMS 182248 produced sustained levels of both conjugate-bound and free DOX which were present throughout the experiment. This suggested that, in contrast to normal tissues, tumor tissue retention of BMS 182248 by antigen-promoted binding had occurred and that the kinetics of free DOX in the tumors were controlled by the rate of release of DOX from tumor-associated BMS 182248. As a result of this retention, the tumor concentrations of free DOX after BMS 182248 administration exceeded those produced by i.v. administration of DOX at the same dose, a finding consistent with the greater antitumor activity of BMS 182248 relative to DOX. BMS 182248 also liberated DOX upon incubation with rat liver lysosomes and was accumulated by L2987 cells in culture, with the subsequent intracellular release of DOX. Conclusions: BMS 182248 effectively delivered DOX to L2987 xenografts implanted in athymic mice and produced higher and more prolonged tumor concentrations of free DOX than the administration of DOX alone. Following BMS 182248 administration, normal tissues (liver and heart) were exposed to lower overall concentrations of free DOX than were produced by administration of an equivalent DOX dose. Received: 18 June 1996 / Accepted: 27 November 1996     

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity

Purpose: Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185^erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. Methods: We performed Western blot analysis of Raf-1, p185^erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. Results: We found that downregulation of Raf-1, p185^erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 μM). These inactive drugs also had no or minimal antiproliferative activity (IC₅₀ > 3 μM). All other analogs (at a concentration of 2 μM) downregulated p53, p185^erbB2, and Raf-1, and also displayed cytotoxicity (IC₅₀ in the range 6–600␣nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC₅₀ for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185^erbB2 and Raf-1 were observed at doses that were 4–5 times greater than the cytotoxic IC₅₀. Conclusions: These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185^erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism. Received: 6 June 1996 / Accepted: 28 September 1996     

Pharmacokinetics of intrapericardial administration of 5-fluorouracil

A 30-year-old patient with metastatic breast adenocarcinoma was diagnosed as having a malignant pericardial effusion. Methods: The patient was treated with two courses of 200 mg 5-fluorouracil (5-FU) followed by 20 mg cisplatin 5 h later directly infused into the pericardial space through a catheter. The drug levels of the 5-FU were monitored during the second treatment. The half-life of 5-FU in the pericardial space was 168.6 min with a concentration of 0.113 mg/ml still detected at 5 h. The area under the curve (AUC) was estimated to be 4.739 mg h/ml. The plasma concentrations of 5-FU ranged from 0.022 to 0.04 mg/ml throughout the infusion. Results: There was no significant change in the patient's blood counts or chemistry profile. She did not experience any side effects during the treatment. A pericardial window was performed 2 days later when balloon pericardiectomy was unsuccessful. The patient eventually succumbed to her disease 4 months later, but without evidence of pericardial effusion. Conclusions: We conclude that pericardial infusion of 5-FU allowed a high concentration of 5-FU to be achieved within the pericardial sac with a greatly increased half-life over that of systemic 5-FU treatment (168 min vs 6–20 min), and with little systemic toxicity. Received: 12 September 1996 / Accepted: 12 December 1996     

Biodistribution of an antibody-enzyme conjugate for antibody-directed enzyme prodrug therapy in nude mice bearing a human colon adenocarcinoma xenograft

The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5–0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1–0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg. Received: 21 July 1996 / Accepted: 22 December 1996     

Antiestrogenic activities of alternate-substituted polychlorinated dibenzofurans in MCF-7 human breast cancer cells

Purpose: 1,3,6,8-Substituted alkyl polychlorinated dibenzofurans (PCDFs), typified by 6-methyl-1,3,8-triCDF (MCDF), inhibit 17β-estradiol (E2)-induced responses in the rodent uterus and human breast cancer cells. The major purpose of the experiments reported here was to determine the structure-dependent antiestrogenic activities of several alternate-substituted (1,3,6,8- and 2,4,6,8-) PCDFs. Methods: The antiestrogenic activities were determined in MCF-7 human breast cancer cells using two assays, that is E2-induced cell proliferation and induction of chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with the E2-responsive Vit-CAT plasmid. Results: MCDF (10⁻⁵ M ), 6-isopropyl-1,3,8-triCDF, 6-ethyl-1,3,8-triCDF, 3-isopropyl-6-methyl-1,8-diCDF, and 6-methyl-2,4,8-triCDF, inhibited both E2-induced cell proliferation and CAT activity in MCF-7 cells. All of the remaining ten congeners inhibited either E2-induced cell proliferation or CAT activity, but not both responses. Conclusions: The antiestrogenic activity of the alternate-substituted PCDFs involves interactions between the aryl hydrocarbon and estrogen receptor signaling pathways. Although these compounds exhibited antiestrogenic activity in MCF-7 cells, the effects of individual congeners were response-specific, and there were no apparent structure-activity relationships. Received: 26 July 1996 / Accepted: 16 November 1996     

A comparison of a CB17 scid mouse model and the tetrazolium-dye assay using human haematological tumour cell lines

 Haematological tumours in the CB17scid mouse produce a disseminated blood-borne disease analogous to that seen in humans. The CB17scid mouse model has been applied to study the efficacy of chemotherapeutic agents on tumours. Using three human tumour-cell lines of haemopoietic origin (CCRF-CEM, Raji, HS-Sultan), we established disseminated tumours in scid mice and studied the in vivo response of these tumours to four chemotherapeutic agents (daunorubicin, idarubicin, ifosfamide, etoposide). The in vitro drug-resistance profiles of the same cell lines to these drugs were also determined by the tetrazolium-dye (MTT) assay. Differences were found in the patterns of resistance and sensitivity of the cell lines in the in vivo and in vitro systems tested. Since the scid mouse model determines the in vivo response of both host and tumour to cytotoxic agents, it may be more valid than the other models in determining drug resistance of haematological malignancies. Received: 5 November 1995/Accepted: 20 March 1996     

Protection against doxorubicin-induced cardiotoxicity in weanling rats by dexrazoxane

Purpose: Dexrazoxane (DZR) protects against anthracycline-induced cardiotoxicity in several laboratory animal species and in patients with breast cancer. Encouraging results have also been obtained in a limited number of pediatric oncology patients. We conducted studies to determine the safety and cardioprotective activity of DZR in the doxorubicin (DOX)-treated weanling rat simulating the rapidly growing immature child. Methods: Male weanling rats and young adult rats, 20␣days old and 7 weeks old, respectively, were given 1 mg/kg DOX i.v., either alone or with 20 mg/kg DZR, once weekly for 7 weeks. Rats were sacrificed at weeks 8, 12 or 26 following blood collection for hematology and serum chemistry. Hearts were weighed and examined histologically. Results: DOX, either alone or with DZR, inhibited growth, and body weight remained below that of controls throughout the 26 weeks of study. There were no biologically significant hematologic changes in either the DOX- or DZR + DOX-treated young rats. DOX caused a slight increase in liver and kidney weights relative to body weight and a slight increase in serum cholesterol and triglycerides in the young rats. These effects were ameliorated or delayed by DZR. DOX, either alone or with DZR, caused a marked atrophy of the testes in the young rats which had recovered by week 26. In the mature rats, DOX caused a significant decrease in the WBC 1 week after the last treatment, and the WBC was significantly lower in the rats given DZR + DOX compared to those given DOX alone. There were marked increases in liver and kidney weight, serum cholesterol and triglycerides in the mature rats given DOX alone but not in those given DZR + DOX. There was also a marked testicular atrophy in the mature rats given either DOX or DZR + DOX but, unlike that observed in the young rats, this had not returned to normal by week 26. DOX-induced cardiotoxicity was less severe in the younger rats than in the mature rats but in both age groups, the lesion progressed rapidly until week 12, 5 weeks after the last dose, and remained relatively stable or progressed slightly thereafter. DZR provided significant cardioprotection in both age groups at all time points examined. Moreover, in both age groups, the severity of the cardiomyopathy in the DZR-treated rats was somewhat less at week 26 than it was at week 12. Conclusions: The results indicate that the pharmacologic effects of DZR, including its ability to protect against cardiotoxicity, are similar in immature and adult male animals treated with DOX. Received: 3 March 1998 / Accepted: 13 May 1998     

Protective role of metallothionein in renal toxicity of cisplatinum

To elucidate the protective role of metallothionein (MT) in the prevention of cisplatinum (cis-DDP) toxicity, we investigated the lethal and renal toxicities caused by cis-DDP in MT-null transgenic mice in comparison with wild-type control mice, and examined the effect of pretreatment with bismuth nitrate or zinc sulfate on the cis-DDP nephrotoxicity. The MT-null mice were of mixed 129 Ola and C57BL/6 genetic background. Since no differences in cis-DDP nephrotoxicity were observed between these strains, C57BL/6J mice were used as the wild-type control. The basal MT levels in the kidneys were negligible in the MT-null mice and 2.93 ± 0.77 μg/g tissue in the C57BL/6J mice. In terms of both the lethal and renal toxicities of cis-DDP, MT-null mice were far more sensitive than C57BL/6J mice. Preinduction of renal MT synthesis by administration of bismuth nitrate or zinc sulfate protected C57BL/6J mice from cis-DDP nephrotoxicity. In the case of MT-null mice, however, renal MT could not be induced by pretreatment with these metal compounds, and renal toxicity of cis-DDP was not prevented by this pretreatment. These results suggest that MT is an important factor with the potential to suppress the development of cis-DDP toxicity. Received: 17 June 1996 / Accepted: 27 November 1996     

A pilot phase I trial of continuous hyperthermic peritoneal perfusion with high-dose carboplatin as primary treatment of patients with small-volume residual ovarian cancer

Purpose: Because intraperitoneal (i.p.) therapy may provide a therapeutic advantage and because hyperthermia enhances carboplatin (CBDCA) cytotoxicity, we evaluated the feasibility, toxicity, and pharmacokinetics of CBDCA given via continuous hyperthermic peritoneal perfusion (CHPP) in patients with small-volume residual ovarian cancer. Patients and Methods: Six patients underwent optimal cytoreductive procedures (residual disease ≤5 mm) as initial treatment of stages II and III epithelial ovarian adenocarcinoma. All patients received a 90-min CHPP at a CBDCA dose of 800–1200 mg/m², with the perfusate being recirculated rapidly from a reservoir through a heat exchanger, resulting in i.p. temperatures of 41–43 °C. Plasma, perfusate, and urine samples were collected and platinum was quantified by flameless atomic absorption spectrophotometry. Results: At no time did any patient's core temperature exceed 40 °C. Peak perfusate platinum concentrations were 8- to 15-fold higher than peak ultrafilterable plasma concentrations. The permeability-area product was extremely high and variable (14–90 ml/min), resulting in a regional advantage of 1.9–5.3. The percentage of the dose absorbed ranged widely from 27% to 77%. Dose-limiting hematologic toxicity was observed at a dose of 1200 mg/m² and this was associated with a CBDCA AUC in plasma of 11 mg min ml⁻¹. Conclusions: CHPP with CBDCA was safely given to three patients at a dose of 800 mg/m², and dose-limiting hematologic toxicities observed at 1200 mg/m², correlated with the plasma CBDCA exposure established when lower doses of CBDCA are given systemically. The pharmacokinetic data are consistent with the expected effect of vigorous mixing on the exposed peritoneal surface area. Variable drug absorption and clearance make the prediction of systemic exposure highly uncertain. These findings may have important implications for novel therapies given i.p. Received: 9 March 1998 / Accepted: 11 June 1998