Histamine H1 Receptor Activation Stimulates Mitogenesis in Human Astrocytoma U373 MG Cells

In human astrocytoma U373 MG cells that express histamine H₁ receptors (180 ± 6fmol/mg protein) but not H₂ or H₃ receptors, histamine stimulated mitogenesis as assessed by [³H]-thymidine incorporation (173 ± 2% of basal; EC₅₀, 2.5 ± 0.4M). The effect of 100M histamine was fully blocked by the selective H₁ antagonist mepyramine (1M) and was markedly reduced (93 ± 4% inhibition) by the phospholipase C inhibitor U73122 (10M).The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100nM) stimulated [³H]-thymidine incorporation (270 ± 8% of basal), and this response was not additive with that to 100M histamine. The incorporation of [³H]-thymidine induced by 100M histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 ± 7% inhibition at 300nM) and by the compound PD 098,059 (30M, 62 ± 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2.These results show that histamine H₁receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.     

Metallothionein-like proteins and cell resistance tocis-dichlorodiammineplatinum(II) in L1210 cells

Summary Our studies on the mechanism of resistance of the murine leukemia L1210-PDD line tocis-dichlorodiammineplatinum(II) (cis-DDP) have not shown why it is 10-fold more resistant to the drug than the L1210 line. For this reason we investigated metallothionein-like proteins (MTs) in these cells. Soluble protein extracts from cultures treated for 24 h withcis-DDP, zinc sulphate or saline were anaerobically eluted from columns of chemically reduced Sephadex G-75, and the profiles of zinc, copper and platinum were determined along with those for incorporated radioactive cyst(e)ien and tyrosine. Both salinetreated cell lines contained similar levels of MTs, which were induced by exposure to a minimally toxic level of zinc (100 M). Zinc induction of MTs was nearly 4-fold greater in L1210 than in L1210-PDD cells. The levels of mRNA for metallothionein I (MTI) and (MTII) in uninduced cells were measured by dot-blotting with a cDNA probe. The L1210-PDD cells contained 80% of the MTI and 41% of the MTII compared with L1210 cells, confirming the similar levels in uninduced cells. L1210-PDD cells were 2-fold more sensitive than L1210 cells to cadmium and equally sensitive to zinc. Thus, the resistance of L1210-PDD cells tocis-DDP was not associated with cross-resistance to group II_b metals, whereas their sensitivity to cadmium did reflect the relative inability of the cells to synthesize MTs. The L1210 cells produced MTs when treated with 0.5 and 5.0 M cis-DDP, but the L1210-DDP cells did not when treated with 5.0–40 M cis-DDP. Small amounts of platinum (<21% of the total eluted) were bound to MTs in both cell lines, but platinum provided a minor portion of the MT-bound metals, with zinc and copper contributing the bulk. The basis for the resistance of L1210-PDD cell tocis-DDP is neither an increased level of MTs in the resistant cells nor an enhanced ability to increase the synthesis of MTs after drug exposure.This work was funded from a Peter Crimmins Research Fellowship held by P. G. F.     

Measurement of in vivo glucose transport from blood to tissue of experimentally-induced glioma in rat brain

Summary In experimentally-induced F98 glioma of rat brain, regional blood flow and glucose transfer were assessed by means of double tracer autoradiography to measure Michaelis-Menten constants for the determination of unidirectional glucose transport across the blood-tumor and blood-brain barrier. In brain regions opposite the tumor hemisphere, the maximal glucose transport rate constant, T_m, ranged from 1.41 ± 0.12 to 3.22 ± 0.29 mol/g/min and the half saturation transport constant of glucose, K_t, varied from 2.78 ± 0.83 to 5.6 ± 1.94 mol/ml (estimate ± standard error of the estimate) yielding a normoglycemic unidirectional glucose inward transport which ranged from 1.24 ± 0.24 to 1.97 ± 0.13 mol/g/min (mean ± standard deviation). In the tumor periphery, the T_m and the Kt values were 3.64 ± 0.56 mol/g/ml and 7.32 ± 2.12 mol/min, and in the tumor center, 1.77 ± 0.25 mol/ml and 2.76 ± 1.13 mol/min, respectively. The unidirectional glucose influx of tumor periphery and center in normoglycemia was 1.98 ± 0.22 and 1.34 ± 0.16 mol/g/min, respectively. Despite comparable unidirectional glucose influxes, however, glucose metabolism of tumor tissue located in the periphery (0.83 ± 0.12 mol/g/min) and the center (0.41 ± 0.10 mol/g/min) of the tumor mass exceeded that of normal gray matter by about 68% and 100% which indicates uncoupling between glucose transport and phosphorylation in experimentally-induced F98 glioma of rat brain.     

Evaluation of plasma 5-fluorouracil nucleoside levels in patients with metastatic breast cancer: relationships with toxicities

This paper describes the relationship between 5-fluorouracil (FUra)-derived toxicities and plasma levels of the FUra anabolites 5-fluorouridine (FUrd) and 5-fluoro-2-deoxyuridine (FdUrd) monitored in patients receiving continuous infusions of FUra (1000 mg/m² per 24 h) over 5 days preceded by the administration of cisplatin (100 mg/m²). A total of 63 courses of this treatment were given as second-line chemotherapy to 17 patients with metastatic breast cancer. The active FUra anabolites FUrd and FdUrd were monitored twice daily in the plasma by highperformance liquid chromatography. Data were analyzed using multiple analysis of variance (ANOVA). Only a low proportion of patients exhibited measurable plasmatic levels of FUrd (43%) and FdUrd (70%). The areas under the plasma concentration-time curves (AUC) determined over 120 h for FUrd (AUC_FUrd) and for FdUrd (AUC_FdUrd) were found to be statistically significantly different for chemotherapy cycles with and those without myelosuppression. Chemotherapy cycles without neutropenia were associated with low AUC_FUrd values (mean±SEM, 2.9±0.7 g ml^–1 h) and high AUC_FdUrd values (14.1±2.7 g ml^–1 h), respectively, whereas courses with myelosuppression (WHO grades 2–4) showed inverse profiles with high AUC_FUrd values (16.3±2.3 g ml^–1 h) and low AUC_FdUrd values (3.1±1.0 g ml^–1 h), respectively. A statistically significant difference in AUC_FdUrd values was also observed between cycles with and those without mucositis (P=0.0027), with AUC_FdUrd values being 22.6±5.6 and 7.8±1.9 g ml^–1 h, respectively. Whereas hematotoxicity could be correlated with both AUC_FUrd and AUC_FdUrd values, mucositis was associated with high AUC_FdUrd levels. Moreover, a negative correlation was found between the AUCs determined for FUrd and FdUrd (P=0.002), indicating that activation of FUra via FUrd or via FdUrd may involve competitive processes. Therefore, to follow the development of the major FUra-derived toxicities, measurement of FUrd and FdUrd plasma levels appeared very attractive.     

Bioavailability and pharmacokinetics of the cardioprotecting flavonoid 7-monohydroxyethylrutoside in mice

Purpose The pharmacokinetics and bioavailability of monoHER, a promising protector against doxorubicin-induced cardiotoxicity, were determined after different routes of administration.Methods Mice were treated with 500 mg.kg^–1 monoHER intraperitoneally (i.p.), subcutaneously (s.c.) or intravenously (i.v.) or with 1000 mg.kg^–1 orally. Heart tissue and plasma were collected 24 h after administration. In addition liver and kidney tissues were collected after s.c. administration. The levels of monoHER were measured by HPLC with electrochemical detection.Results After i.v. administration the AUC_{0–120 min} values of monoHER in plasma and heart tissue were 20.5±5.3 mol.min.ml^–1 and 4.9±1.3 mol.min.g^–1 wet tissue, respectively. After i.p. administration, a mean peak plasma concentration of about 130 M monoHER was maintained from 5 to 15 min after administration. The AUC_{0–120 min} values of monoHER were 6.1±1.1 mol.min.ml^–1 and 1.6±0.4 mol.min.g^–1 wet tissue in plasma and heart tissue, respectively. After s.c. administration, monoHER levels in plasma reached a maximum (about 230 M) between 10 and 20 min after administration. The AUC_{0–120 min} values of monoHER in plasma, heart, liver and kidney tissues were 8.0±0.6 mol.min.ml^–1, 2.0±0.1, 22.4±2.0 and 20.5±5.7 mol.min.g^–1, respectively. The i.p. and s.c. bioavailabilities were about 30% and 40%, respectively. After oral administration, monoHER could not be detected in plasma, indicating that monoHER had a very poor oral bioavailability.Conclusions MonoHER was amply taken up by the drug elimination organs liver and kidney and less by the target organ heart. Under cardioprotective conditions (500 mg/kg, i.p.), the C_max was 131 M and the AUC was 6.3 M.min. These values will be considered endpoints for the clinical phase I study of monoHER.     

Biochemical modulation of 5-fluorouracil with or without leucovorin by a low dose of brequinar in MGH-U1 cells

Summary Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical antitumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU±leucovorin (LV)±brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC₅₀) for brequinar, FU, and FU+LV (100 m) were 0.4, 20, and 10 m, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC₁₀ (0.1 m) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 m brequinar and FU±LV did not increase the cytotoxicity of FU±LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 m brequinar for 24 h alone or followed by a 2-h exposure to FU (25 m)±LV (100 m). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU + brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 m hypoxanthine and 25 m FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).Supported by the National Cancer Institute, Canada, and Du Pont Pharma, Mississauga, Canada     

Binding of vinblastine to recrystallized human α1-acid glycoprotein

Summary The binding of vinblastine (VLB) to recrystallized human ₁-acid glycoprotein (₁-AGP) dissolved in phosphate-buffered saline (pH 7.4) was determined at different drug concentrations using the technique of continuous ultrafiltration. Vinblastine was extremely highly bound (>99.0%) at a drug concentration of 4.0 mol·l^-1, dropping to under 60% at 65.0 mol·l^-1. Binding was best described by a two-class model with higher-(9.4 M^-1) and lower-(0.1 M^-1) affinity sites but with a similar number of binding sites (1.5 as against 1.1 lower-affinity sites). These results strongly suggest that ₁-AGP would be a major binding protein for VLB in plasma.     

Pharmacokinetics and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide after i.v. administration in the mouse

Summary The pharmacokinetics, tissue distribution and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide(AC) were studied after i.v. administration to mice. Over the dose range of 9–121 mol/kg (3–40 mg/kg), AC displayed linear kinetics with the following model-independent parameters: clearance (C), 21.0±1.9 l h^–1 kg^–1; steady-state volume of distribution (V_ss), 11.8±1.4 l/kg; and mean residence time (MRT), 0.56±0.02 h. The plasma concentration-time profiles for AC fitted a two-compartment model with the following parameters:C _c, 19.4±2.3 l h^–1 kg^–1; V_c, 7.08±1.06 l/kg;t _1/2 13.1±3.5 min; andt _1/2Z, 1.60±0.65 h. AC displayed moderately high binding in healthy mouse plasma, giving a free fraction of 15.9%–25.3% over the drug concentration range of 1–561 M. After the i.v. administration of 30 mol/kg [³H]-AC, high radioactivity concentrations were observed in all tissues (especially the brain and kidney), showing a hight _1/2c value (37–59 h). At 2 min (first blood collection), the AC concentration as measured by high-performance liquid chromatography (HPLC) comprised 61% of the plasma radioactivity concentration (expressed as AC equivalents/l). By 48 h, 73% of the dose had been eliminated, with 26% and 47% of the delivered drug being excreted by the urinary and faecal routes, respectively; <1% of the total dose was excreted as unchanged AC in the urine. At least five distinct radiochemical peaks were distinguishable by HPLC analysis of plasma extracts, with some similar peaks appearing in urine. The 121-mol/kg dose was well tolerated by mice, with sedation being the only obvious side effect and no significant alterations in blood biochemistry or haematological parameters being recorded. After receiving a dose of 152 mol/kg, all mice experienced clonic seizures for 2 min (with one death occuring) followed by a period of sedation that lasted for up to 2h. No leucopenia occurred, but some mild anaemia was noted. There was no significant change in blood biochemistry. A further 20% increase in the i.v. dose (to 182 mol/kg) resulted in mortality, with death occurring within 2 min of AC administration.Supported by the Auckland Medical Research Foundation and the Cancer Society of New Zealand     

In vitro evaluation of a novel chemotherapeutic agent,adozelesin, in gynecologic-cancer cell lines

Summary Adozelesin is a derivative of an extremely cytotoxic compound, CC1065. This entirely new class of drug binds preferentially to DNA and facilitates alkylation reaction. In the present study, we used the adenosine triphosphate (ATP) chemosensitivity assay to compare the cytotoxic potency of Adozelesin with that of common chemotherapeutic agents in ten gynecologic-cancer cell lines. Flow cytometry was also used to study its effects on cell-cycle kinetics. The mean drug concentrations required to produce a 50% reduction in ATP levels as compared with controls [IC₅₀] were: Adriamycin, 0.17±0.06 m; 4OH-Cytoxan, 18±3 m; cisplatin, 17±7 m; 5-fluorouracil, 183±116 m; and Adozelesin, 11.0±5.4pm. Thus, Adozelesin was 10⁴–10⁷ times more potent than Adriamycin, cisplatin, 5-fluorouracil, and Cytoxan. Cell kinetics studies revealed significant S and G2 blocks such as those previously reported for other alkylating agents.Supported in part by an American Cancer Society Clinical Oncology Fellowship and an American Cancer Society/Florida Division Startup Grant (both awarded to H. N. N.)     

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 M VBL and 500 M VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 M VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (K _i values) of 55 M for VBL and 110 M for VCR. Since theK _i values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.This study was supported financially by the Norwegian Cancer Society     

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma

Summary Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. Thein vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-/ (MulFN-/) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [³H]-thymidine incorporation, respectively. Incubation (24 h) of G-26 cells with As-S, As-P or MulFN-/, resulted in a dose dependent decrease in cell viability (IC₅₀=125M As-S; 175M As-P and 3.6×10⁴ U/ml MulFN-/) and proliferation (IC₅₀=157M As-S; 185M As-P and 3.6×10⁴ U/ml MulFN-/). A combined exposure to 175 M As-S and 800 U/ml of MulFN-/ resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-/, but not with human interferon- lymphoblastoid or human interferon-. Ascorbyl esters inhibited cytosolic GST activity (1–50=15.0 M As-S and 28.5 M As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 M and 2.0 M for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 M As-S or 800 U/ml MulFN-/.     

Dosing of thioTEPA for myeloablative therapy

High-dose thioTEPA is used frequently in myeloablative regimens for marrow transplantation, but the need for dose adjustments in obese patients has not been explored. We determined the pharmacokinetics of thioTEPA and its metabolite TEPA during first-dose infusion of thioTEPA 150–250 mg/m² given daily for 3 days in combination with busulfan and cyclophosphamide, and evaluated the results for correlations with toxicity and dosing strategies. The study included 15 adults undergoing marrow transplantation for hematologic malignancies. Plasma samples were obtained at various times over a 24-h period, and concentrations of thio TEPA and TEPA were measured by gas chromatography. At 22–24 h after initiation of a 4-h infusion, the mean ±SE plasma concentration of thioTEPA was 124±63 ng/ml, while that of TEPA was 235±69 ng/ml. For CFU-GM and BFU-E growth in vitro, the IC₅₀s of thioTEPA were 83 ng/ml and 16 ng/ml, respectively, and the IC₅₀s of TEPA were 141 ng/ml and 47 ng/ml, respectively. Using a twocompartment model, the mean thioTEPA Vc was 47.4±4.7 l/m², t_1/2 19±5 min,t _1/2 3.7±0.5 h, and plasma clearance 302±21 ml/min per m². The mean AUCs were 6.9–16.2 mg h/l for thioTEPA and 8.9–21.2 mg h/l for TEPA, while the mean peak concentrations were 0.95–2.08 g/ml for thioTEPA and 0.88–1.90 g/ml for TEPA. There was a significant association of grades 2–4 maximum regimen-related toxicity (RRT) with TEPA peak >1.75 g/ml and with combined thioTEPA and TEPA AUC >30 mgh/l (5/6 vs 0/9,P=0.01 for both comparisons), suggesting that drug exposure was an important determinant of toxicity and, potentially, efficacy. ThioTEPA Vc correlated best with adjusted body weight (r=0.74,P=0.0015). In an evaluation of 74 adults receiving thioTEPA 750 mg/m² in combination with busulfan and cyclophosphamide, the maximum RRT for patients at ideal weight was significantly greater than that for obese patients dosed on ideal weight (mean RRT grade 1.7 vs 1.0,P=0.004) but did not differ from the maximum RRT for obese adults dosed on actual or adjusted weights. We recommend that for obese patients thioTEPA be dosed on adjusted body weight. Measurements at time-points after 24 h are needed to determine when thioTEPA and TEPA concentrations are below myelosuppressive levels and safe for marrow infusion.Supported in part by a grant from the American Cyanamid Corporation     

Disposition and metabolism of bruceantin in the mouse

Summary A microbiologic assay of the agar diffusion type, employing a strain of yeast tentatively identified as Candida macedoniensis, was developed to study the disposition, excretion, and metabolism of the antitumor agent bruceantin (BN; NSC 165563) in the mouse. Bioautographic studies showed the assay to be specific for BN. Normal and tumor-bearing male BDFl mice were studied. Tumor-bearing mice were implanted sc with 10 ⁶ L1210 ascites cells and held for 7 days prior to dosing. Average tumor weight was 307 mg and advanced generalized disease was evident. Three groups of 5 or 10 mice were injected iv with BN (1.5 mg/kg; approx. LD₁₀). At various times after injection, blood, tissues, urine, and feces were obtained and extracted with chloroform to recover BN. Recovery of BN was in the range of 91 to 121%. Disposition and excretion of BN were similar for normal and tumor-bearing mice. Decay of BN in blood was biphasic with -phase half-lives of 5 to 6 min. Estimated half-lives for the -phase were possibly >0.5 day. Average zero time intercepts for -and -phases were 550 and 51 ng/ml respectively. Higher levels of BN were found in lung, pancreas, intestine, and spleen (1–6 g/g) than in liver, kidney, and tumor (0.3–0.5 g/g) after 15 min. Concentrations of BN in brain and peritoneal fat were below detectable limits (0.1 g/g tissue). Urine and fecal excretion of BN accounted for 2% of the dose after 24 h. In vitro metabolism studies using a postmitochondrial microsome fraction of liver, lung, and kidney suggest that bruceantin is inactivated by an NADPH-dependent enzyme present in liver but not in lung or kidney.Supported by contract NO1-CM-87162 from the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Department of Health, Education, and Welfare     

18b-甘草次酸诱导人乳腺癌细胞凋亡及与细胞内Ca2+释放的关系

Objective: To study the effects of 18-glycyrrhetinic acid (GA) on proliferation inhibition, apop- totic induction, and the relationship between GA-induced apoptosis and intracellular Ca²⁺ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 mol/L to 250 mol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 mol/L, 150 mol/L, and 200 mol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 mol/L GA for 24 h, intracellular Ca²⁺ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 mol/L to 250 mol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose- dependent fashion. IC50 of the proliferation inhibition was 234.33 mol/L. Treated with 100 mol/L, 150 mol/L, and 200 mol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca²⁺ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca²⁺ level may be depended on apoptosis induced by GA in MCF-7 cells.     

The effect of cimetidine on cyclophosphamide metabolism in rabbits

Summary Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 Ci [³H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3±7.3 to 33.5±9.5 min (mean ± SD;P=0.036) but did not significantly alter the AUC_{0–8 h} for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC_{0–8 h} for 4-hydroxycyclophosphamide (189.4±77 vs 364.6±126.7 mol min/l^–1;P=0.016) as compared with control values. A higher AUC_{0–8 h} value for phosphoramide mustard (53.7±69.2 vs 95.7±34.7 mol min/l^–1) was also observed after cimetidine dosing but the difference was not significant (P=0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.Supported in part by the Department of Veteran Affairs and grant CA-49186 from the National Institutes of Health (NIH)Department of Clinical Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China     

Pharamacokinetic Modeling for Boronophenylalanine-fructose Mediated Neutron Capture Therapy: 10B Concentration Predictions and Dosimetric Consequences

A two-compartment open model has been developed for predicting ¹⁰B concentrations in blood following intravenous infusion of the L-p-boronophenylalanine-fructose complex in humans and derived from pharmacokinetic studies of 24 patients in Phase I clinical trials of boron neutron capture therapy. The ¹⁰B concentration profile in blood exhibits a characteristic rise during the infusion to a peak of 32g/g (for infusion of 350mg/kg over 90min) followed by a biexponential disposition profile with harmonic mean half-lives of 0.32±0.08 and 8.2±2.7h, most likely due to redistribution and primarily renal elimination, respectively. The mean model rate constants k ₁₂, k ₂₁, and k ₁₀ are (mean ± SD) 0.0227±0.0064min^–1, 0.0099±0.0027min^–1, 0.0052±0.0016min^–1, respectively, and the central compartment volume of distribution V ₁ is 0.235±0.042L/kg. In anticipation of the initiation of clinical trials using an intense neutron beam with concomitantly short irradiations, the ability of this model to predict, in advance, the average blood ¹⁰B concentration during brief irradiations was simulated in a retrospective analysis of the pharmacokinetic data from these patients. The prediction error for blood boron concentration and its effect on simulated dose delivered for each irradiation field are reported for three different prediction strategies. In this simulation, error in delivered dose (or, equivalently, neutron fluence) for a given single irradiation field resulting from error in predicted blood ¹⁰B concentration was limited to less than 10%. In practice, lower dose errors can be achieved by delivering each field in two fractions (on two separate days) and by adjusting the second fraction's dose to offset error in the first.     

Systemic treatment with murine recombinant interleukin-1β inhibits the growth and progression of malignant glioma in the rat

We investigated the effects of daily subcutaneous (SC) injections of 100, 200, or 400 g/kg murine recombinant interleukin-1 (rIL-1) or its excipient on normal Fischer 344 rats and ones harboring a malignant RT-2 glioma. The tumor model has a predictable course with animals dying on days 14–17 following an intracerebral inoculation of 10⁴ RT-2 glioma cells. Treatments with (rIL-1) or excipient began on day seven post-tumor inoculation and continued for 7 days. We observed no significant effect on core body temperatures although there was a significant (p < 0.05) decrease in body weight in all (rIL-1) treated animals. When tumor-bearing animals became moribund, they received an intraperitoneal injection of bromodeoxyuridine (BUdr) and were sacrificed two hours later. Blood samples were obtained prior to their sacrifice by transcardiac perfusion with a buffered aldehyde solution. Recombinant IL-4ß affected blood differentials; causing neutrophilia, lymphopenia, and slight thrombocythemia. The BUdr labeling index of glioma cells did not significantly differ between treatment groups, although tumors differed histologically at the time of necropsy. Tumors of rIL-1 treated animals had more extensive necrosis and a greater degree of leukocyte infiltration. Survival studies were conducted in which rats were given continuous daily SC injections of (rIL-1) until day of death. Overall survival between the two groups differed significantly in studies using 100 g/kg/d (p < 0.05); (rIL-1ß) treated rats had a mean survival time of 22 (± 3.0) days while excipient controls had a mean survival time of 17 (± 0.5) days. Similarly, at a dose of 200 g rIL-1(3/kg/d), mean survival was significantly (p < 0.05) increased as compared to excipient controls (18.75 ± 1.5 vs. 15.25 ± 1.7 days, respectively). Daily injections of 400 g/kg did not significantly increase the survival of glioma bearing animals, possibly as a consequence of fIL-1ß toxicity at this dose.     

Modulation of the in vitro cardiotoxicity of doxorubicin by flavonoids

Cancer therapy with the anthracycline doxorubicin (Dox) is limited by cardiomyopathy, which develops in animals and patients after cumulative dosing. Generation of free radicals by Dox may be involved in this cardiotoxicity. Dox binds strongly to metal ions, especially iron(III). This Dox-metal complex stimulates the generation of free radicals through self-reduction of the complex. We investigated the possibility of inhibiting Dox-induced cardiotoxicity by scavenging of free radicals and/or chelating metal ions. The effects of Dox, both alone and in combination with iron-chelating agents, were studied on inotropy of the isolated mouse left atrium, lipid peroxidation (LPO) in cardiac microsomal membranes, ferricytochrome c (cyt.c³⁺) reduction, and oxygen consumption. The flavonoids 7-monohydroxyethylrutoside (mono-HER) and 7,3,4-trihydroxyethylrutoside (tri-HER) and the ethylenediaminetetraacetic acid (EDTA) analogue ICRF-198 and its precursor ICRF-187 were used as iron-chelating agents. The latter were used for comparison since ICRF-187 has been reported to inhibit the cardiotoxic effects of Dox both in vitro and in vivo. Only the flavonoids could inhibit the negative inotropic effect of Dox (35M) on the mouse left atrium; in the presence of tri-HER (500 M) the beating force decreased by 18% instead of 50%, whereas mono-HER completely prevented the Dox-induced negative inotropic effect. ICRF-198 and both flavonoids (500 M) completely inhibited Dox (35M)-induced LPO, whereas ICRF-187 provided 65% inhibition. The observation that both cyt.c³⁺ reduction and oxygen consumption induced by the Dox-iron(III) complex (50/16.6 M Dox₃Fr³⁺) could be inhibited by superoxide dismutase proved the involvement of superoxide anions (O ₂ ^– ). The iron-chelating agents (50 M) inhibited cyt.c³⁺ reduction by 91% (mono-HER), 43% (tri-HER), and 100% (ICRF-198). Only mono-HER and ICRF-198 (50 M) were capable of inhibiting the oxygen consumption by 70% and 43%, respectively. It is concluded that flavonoids offer a good perspective for further studies on the prevention of Dox-induced cardiomyopathy.     

Tamoxifen-Induced Increases in Cytoplasmic Free Ca2+ Levels in Human Breast Cancer Cells

Tamoxifen has been shown to increase cytoplasmic free Ca²⁺ levels [Ca²⁺]_i in renal tubular cells and bladder cancer cells, and to alter Ca²⁺ signaling in MCF-7 breast cancer cells. The present study examined the effect of tamoxifen on [Ca²⁺]_i in ZR-75-1 human breast cancer cells using fura-2 as an indicator. Tamoxifen increased [Ca²⁺]_i at a concentration above 2M with an EC₅₀ of 5M. Removing extracellular Ca²⁺ reduced the response by 48±2%. In Ca²⁺-free medium, after tamoxifen-induced [Ca²⁺]_i increased had returned to baseline, adding 3mM Ca²⁺ increased [Ca²⁺]_i in a concentration-dependent manner. Further, pretreatment with 10M tamoxifen abolished the [Ca²⁺]_i increase induced by 1M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor); and conversely, pretreatment with thapsigargin prevented tamoxifen from releasing more Ca²⁺. Tamoxifen (10M)-induced Ca²⁺ release was not changed by inhibiting phospholipase C activity with 2M U73122. Trypan blue exclusion assay revealed that tamoxifen (1–10M) did not alter viability after 1min of incubation, but killed 10% of cells after 3–10min of incubation. Together, this study shows that tamoxifen (>2M) induced a significant, immediate increase in [Ca²⁺]_i in ZR-75-1 breast cancer cells. Tamoxifen acted by releasing Ca²⁺ from the endoplasmic reticulum Ca²⁺ stores in a manner independent of phospholipase C activity, and by inducing Ca²⁺ entry from extracellular medium. Tamoxifen may be of mild cytotoxicity after acute exposure.     

Prevention of development of dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat by the new nonsteroidal antiestrogen EM-800 (SCH57050)

The effect of EM-800, a new non-steroidal antiestrogen having pure antiestrogenic activity, was studied on chemical carcinogenesis induced by dimethylbenz(a)anthracene (DMBA) as well as on serum lipids and bone mass in the rat. Treatment with EM-800 orally, once daily, for 282 days (9 months), starting 3 days before DMBA administration, decreased the incidence of tumors from 95% in control animals to 60% (p < 0.01), 38% (p < 0.01), and 28% (p < 0.01) at the daily doses of 25 g, 75 g, and 250 g, respectively. The average number of tumors per animal decreased from 4.5 ± 0.5 tumors in the control group to 0.9 ± 0.2 (p < 0.01), 0.5 ± 0.2 (p < 0.01), and 0.3 ± 0.1 (p < 0.01) tumors in the rats treated with the above-indicated doses of the antiestrogen. In addition, treatment with the increasing doses of EM-800 reduced serum cholesterol levels to 64%, 56%, and 48% of control, while serum triglycerides decreased to 31%, 28%, and 30% of control. Bone mineral content (BMC) and bone mineral density (BMD) of total skeleton, femur, and lumbar spine were not significantly affected following 282 days of treatment with EM-800. However, treatment with EM-800 inhibited the urinary ratio of hydroxyproline to creatinine (HP/Cr) from 14.0 ± 3.90 mol/mmol in controls to 7.6 ± 0.8 (p < 0.05), 6.8 ± 0.8 (p < 0.01), and 6.8 ± 1.1 (p < 0.01) mol/mmol, respectively, while the same treatment had no effect on serum total alkaline phosphatase (tALP) activity or urinary calcium and phosphorus excretion. The 25 g, 75 g, and 250 g daily doses of EM-800 inhibited uterine weight by 35% (p < 0.01), 62% (p < 0.01), and 66% (p < 0.01), while vaginal weight was reduced by 8% (p < 0.05), 30% (p < 0.01), and 38% (p < 0.01), respectively. In agreement with the 27% increment (p < 0.05) in ovarian weight at the highest antiestrogen dose used, serum androstenedione (p < 0.05), androst-5-ene-3,17-diol (p < 0.01), testosterone (p < 0.05), and estradiol (p < 0.01) levels were increased. The present data show that EM-800 prevents the development of DMBA-induced mammary tumors while simultaneously inhibiting uterine and vaginal weight, reducing serum cholesterol and triglyceride levels, and having no adverse effect on bone mass following 9 months of treatment in the rat.This revised version was published online in October 2005 with corrections to the Cover Date.