Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil

Summary Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 M LCV concurrently with or after 10 M FUra following MTX (1 M) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV schedulling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-³H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 M LCV with 10 M FUra after 1 M MTX resulted in significantly more ternary complex formation than did 1 M MTX before 10 M FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-³H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.Abbreviations MTX methotrexate - FUra 5-fluorouracil - LCV d, 1-N ⁵-formyltetrahydrofolic acid, calcium salt (folinic acid, leucovorin) - F dUMP 5-fluoro-2-deoxyuridylate - FUTP 5-fluorouidine-5-triphosphate - PRPP 5-phosphoribosyl-1-pyrophosphate - CH₂FAH₄ N ^5,10-methylenetetrahydrofolate - dUMP 2-deoxyuridylate - dTMP thymidylate - TS thymidylate synthetase - PBS phosphate-buffered saline - NaCl 8.0 g - KCl 0.2 g - Na₂HPO₄ 1.15 g - KH₂PO₄ 0.2 g in 1 1 H₂O, pH 7.4 - FdUrd 5-fluoro-2-deoxyuridine This work was supported in part by grant CH-145 from the American Cancer Society and by grants CA-27130 and CA-08341 from the National Cancer Institute. LLD was supported by Postdoctoral Training Grant S-T32-CA09085-08 from the National Institutes of Health and EC was the recipient of a Cancer Research Award from the American Cancer Society     

Effect of lonidamine on the cytotoxicity of four alkylating agents in vitro

Summary We examined the ability of lonidamine, which has been described as an inhibitor of cellular respiration and glycolysis, to enhance the cytotoxicity of alkylating agents to MCF-7 human breast-carcinoma cells. Lonidamine was increasingly cytotoxic to MCF-7 cells with increasing time of exposure. With a 12-h exposure, the IC₅₀ for lonidamine was about 365 M, and with a 24-h exposure it was about 170 M. A drug concentration of 250 M was chosen for use in the drug combination studies. Lonidamine appeared to have a dose-modifying effect on cisplatin (CDDP), producing increasingly supraadditive cell kill with increasing CDDP concentration. When simultaneously incubated with lonidamine for 1 h, 500 M CDDP yielded a cell kill that was 2 log greater than additive cytotoxicity. Extending the exposure to lonidamine for 12 h after CDDP treatment led to a small, additional aliquot of cell kill of about 2.5-fold over the CDDP concentration range. Lonidamine also appeared to have a dose-modifying effect on melphalan cytotoxicity in the melphalan concentration range of 100–500 M. Between concentrations of 10 and 100 M melphalan, the drug combination survival after 1 h exposure fell within the envelope of additivity for the two agents. However, maintaining the presence of lonidamine for an additional 12 h increased the effect such that the combination was supraadditive over the entire concentration range of melphalan. Simultaneous exposure to 4-hydroperoxycyclophosphamide (4-HC) and lonidamine for 1 h resulted in greater than additive cell kill, and extending the lonidamine exposure period such that lonidamine was present during and 12 h after 4-HC treatment further increased this effect. Lonidamine had a moderate effect on the cytotoxicity of carmustine (BCNU) with a 1 h simultaneous exposure; however, this treatment combination reached greater than additive cytotoxicity only at the highest concentration of BCNU tested. Extending the lonidamine exposure time for an additional 12 h resulted in supraadditive cell kill over the BCNU concentration range. Therefore, when lonidamine was present during exposure to the alkylating agent and its presence was then extended for an additional 12 h, a synergistic cell kill was produced with all four alkylating agents tested.This work was supported by a grant from DeSanctis Consultants, Montreal, Canada and National Cancer Institute Grant IPOI-CA38493     

Biochemical and antitumor activity of trimidox,a new inhibitor of ribonucleotide reductase

Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC₅₀) at 5 M, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC₅₀ of 500 M. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 M, a concentration that inhibits cell proliferation by 50% (IC₅₀) or at 100 M for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC₅₀ values of 7.5 and 50 M, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC₅₀ values of 12 and 87 M, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82 in male mice and 112% in female mice). The antitumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans.Abbreviations Didox N,3,4-trihydroxybenzamide - MTT {3-[4,5-dimethyl-thiazo-2yl]}-2,5-diphenyl tetrazolium bromide - PBS phosphate-buffered saline - trimidox 3,4,5-trihydroxybenzamidoxime HCl     

Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells

Summary Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72%±20% survival of cells for epirubicin vs 45%±13% for doxorubicin at a concentration of 0.2 m;P0.005). As analyzed by the DiSC assay, 0.2 m epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival,P0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 m, the mean intracellular uptake of epirubicin in leukemic cells was 0.43±0.26 nmol/mg protein as compared with 0.33±0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 m was 0.47±0.25 nmol/mg protein as compared with 0.31±0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.     

Studies on the myelosuppressive activity of doxorubicin entrapped in liposomes

Summary The myelosuppressive activity of doxorubicin encapsulated in liposomes of differing lipid composition and size was quantified in mice by measurement of changes in spleen weight, peripheral white blood cells (WBC), and bone marrow nucleated cells. Following i. v. administration of free doxorubicin at a dose of 20 mg/kg, a 90% reduction in marrow cellularity was observed on day 3. The marrow nucleated cell count was similar to control values by day 7. Administration of an equivalent dose of doxorubicin that was encapsulated in large (diameter, 1.0 m) egg phosphatidylcholine/cholesterol (EPC/Chol)(molar ratio, 5545) liposomes induced an 80% reduction in bone marrow cellularity that lasted for periods of >7 days. Similar results were obtained following administration of large (1.0 m) liposomal doxorubicin systems formulated with distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (molar ratio 5545). In contrast, liposomal doxorubicin prepared using small (diameter, 0.1 m) DSPC/Chol liposomes induced only a 40% reduction (day 3) in bone marrow cellularity, which returned to control values by day 7. Other indicators of doxorubicin-mediated myelosuppressive activity (spleen weight loss and peripheral leukopenia) correlated well with changes observed in marrow cellularity. An exception to this, however, was observed in animals treated with small (0.1-m) DSPC/Chol liposomal doxorubicin, which displayed peripheral leukopenia for periods of >14 days. This extended leukopenia was not observed following administration of small (0.1-m) EPC/Chol liposomal doxorubicin. Marrow-associated liposomal lipid and doxorubicin were quantified to determine if the extent of doxorubicin-mediated myeloid toxicity could be correlated to changes in biodistribution of the entrapped drug. It was demonstrated that 10–20 times more doxorubicin is delivered to the bone marrow when the drug is given encapsulated in largeliposomes than when it is associated with small liposomes. These data are useful in defining characteristics of liposomal preparations that modulate the myelosupressive behaviour of entrapped antineoplastic agents.Abbreviations MLV multilamellar vesicle - LuV large unilamellar vesicle - SUV small unilamellar vesicle - EPC egg phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DSPC distearoylphosphatidylcholine - Chol cholesterol - RES reticuloendothelial system - QELS quasielastic light scattering - FACS fluorescence-activated cell sorting - WBC white blood cell - HBSS HEPES buffered saline solution     

Pharmacokinetics of PEG-l-asparaginase and plasma and cerebrospinal fluidl-asparagine concentrations in the rhesus monkey

The pharmacokinetics of the polyethylene glycol-conjugated form of the enzymel-asparaginase and the depletion ofl-asparagine from the plasma and cerebrospinal fluid (CSF) following an i.m. dose of 2500 IU/m² PEG-l-asparaginase was studied in rhesus monkeys. PEG-l-asparaginase activity in plasma was detectable by 1 h after injection and maintained a plateau of approximately 4 IU/ml for more than 5 days. Subsequent elimination from plasma was monoexponential with a half-life of 6±1 days. Plasmal-asparagine concentrations fell from pretreatment levels of 14–47 M to <2 M by 24 h after injection in all animals and remained undetectable for the duration of the 25-day observation period in four of six animals. In two animals, plasmal-asparagine became detectable when the PEG-l-asparaginase plasma concentration dropped below 0.1 IU/ml. Pretreatment CSFl-asparagine levels ranged from 4.7 to 13.6 M and fell to <0.25 M by 48 h in five of six animals. CSFl-asparagine concentrations remained below 0.25 M for 10–14 days in four animals. One animal had detectable CSFl-asparagine concentrations within 24 h and another had detectable concentrations within 1 week of drug administration despite a plasma PEG-l-asparaginase activity profile that did not differ from that of the other animals. These observations may be useful in the design of clinical trials with PEG-l-asparaginase in which correlations among PEG-l-asparaginase pharmacokinetics, depletion ofl-asparagine, and clinical outcome should be sought.     

Loss of viability and induction of DNA damage in human leukemic myeloblasts and lymphocytes by m-AMSA

Summary The effects of m-AMSA on in vitro viability and on the induction of DNA damage were examined in low-growth-fraction cell populations of human leukemic myeloblasts and normal lymphocytes. A significant individual variation in the drug-induced reduction of in vitro viability was observed in studies with five selected leukemic patients. The concentration of m-AMSA required to reduce viability by 50% within 48 h ranged from 0.25 M to in excess of 5.0 M for the leukemic myeloblasts as against about 2.0 M for the samples of normal lymphocytes. Alkaline elution studies showed that m-AMSA induced protein-associated DNA strand breaks (PADB) in both myeloblasts and lymphocytes. Depending upon the m-AMSA concentration, there was a 4- to 9-fold difference in the level of PADBs induced by a given drug concentration in the myeloblasts of eight patients studied. The level of PADBs was saturable with respect to both drug concentration (5–10 M) and exposure time (45–10 M). The PADBs were repaired rapidly in all the lymphocyte and myeloblast samples studied, with over 90% of this DNA damage being repaired within 45 min after resuspension of the cells in drug-free medium. These studies of m-AMSA in low-growth-fraction samples of human lymphocytes and myeloblasts show both similarities and differences in the action of this drug compared with previously published studies using the high-growth-fraction mouse L1210 system.This work was supported by the NCI (Canada), MRC (Canada), and the Alberta Heritage Fund: Applied Cancer Program     

Differential effects of estrogen,tamoxifen and the pure antiestrogen ICI 182,780 in human drug-resistant leukemia cell lines

ICI 182,780, a potent, new steroidal antiestrogen without apparent agonist activity, appears to be a potent modulator of the classic multidrug resistance (MDR) phenotype in the CEM/A7, CEM/VLB₁₀₀ and K562/VIN100 MDR cell lines. This reagent had no effect on the respective parental CCRF-CEM and K562 cell lines. The use of 1.25 M ICI 182,780 resulted in a 6- to 7-fold decrease in doxorubicin resistance in the CEM/A7 and CEM/VLB₁₀₀ cell lines. A dose-response effect was observed at ICI 182,780 concentrations of up to 5 M. As compared with tamoxifen (TAM), ICI 182,780 was 2 and 4 times more effective in the K562/VIN100 and CEM/A7 cell lines, respectively. ICI 182,780 at 0.625 M increased [³H]-daunomycin uptake (P<0.0001) as effectively as 5 M TAM in the resistant CEM/A7 line. Drug-efflux studies showed that 5 M ICI 182,780 significantly decreased drug efflux as compared with 5 M TAM (P<0.0001). Estradiol (EST) at 10 M increased doxorubicin resistance by 1.2–1.3 times in the CEM/A7 and CEM/VLB₁₀₀ cell lines and significantly decreased drug accumulation (P=0.002) and retention (P<0.001) in the CEM/A7 cell line. However, the addition of 10 M EST to 1–2 M ICI 182,780 did not inhibit the ability of ICI 182,780 to modulate doxorubicin resistance in the two resistant cell lines. Using reverse-phase high-performance liquid chromatography (HPLC) to measure lipophilicity, we found no apparent association between the ability of ICI 182,780, TAM or EST to modulate resistance and their relative hydrophobicity.This work was supported in part by grants from the Department of Veterans Affairs, Canberra, ICI Pharmaceuticals and the AntiCancer Council of Victoria, Australia     

Metabolism of taxol by human and rat liver in vitro: A screen for drug interactions and interspecies differences

Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of³H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein,³H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 M, and diphenhydramine 200 M, had little or no effect on 6-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6-hydroxytaxol formation with IC₅₀ values of 36 M, 37 M, 16 M and 1 l/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 l/ml it had little or no effect on 6-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.Part of this work was presented in poster form at the 84th Annual Meeting of the American Association for Cancer Research, Orlando, Fl., USA, 1993     

Inhibitory effect of bile salts on the enterohepatic circulation of methotrexate in the unanesthetized rat: Inhibition of methotrexate intestinal absorption

Summary The effect of conjugated and unconjugated bile salts on the intestinal absorption of methotrexate (MTX) in the unanesthetized rat was investigated using a recycling perfusion technique. We initially determined the general characteristics of MTX absorption in vivo. Absorption of low (0.5 M) and high (6 M) concentrations of MTX was linear with time for 60 min perfusion and occurred at rates of 0.2 and 1.65 nmol/100 cm dry length/min, respectively. Absorption of 0.5 M MTX was pH-dependent and increased with decreasing perfusate pH. Absorption of MTX involves two processes: (1) a saturable process with a K_t of 0.98 M, and (2) a nonsaturable diffusion process. The unconjugated deoxycholate and the conjugated taurocholate inhibited the intestinal absorption of 1 M MTX in a concentration-dependent manner. The inhibitory effect of bile salts was reversible, and was not due to damage to the intestinal mucosa. The structural analogues folic acid and 5-methyltetrahydrofolate and the organic anions rose bengal and sulfobormophthalein were also inhibitory to MTX absorption. This study demonstrates that a variety of organic anions inhibit MTX intestinal absorption. The possible therapeutic importance of this observation is discussed.An abstract of this work was presented at the American Gastroenterology Annual Meeting, New Orleans, LA, May, 1984 and appeared in Gastroenterology 86: 1228, 1984Supported by the U.S. Public Health Service Grant AG 2767     

β-Cyclodextrin tetradecasulfate/tetrahydrocortisol±minocycline as modulators of cancer therapies in vitro and in vivo against primary and metastatic lewis lung carcinoma

Tetrahydrocortisol, -cyclodextrin tetradecasulfate, and minocycline used alone or in combination are not very cytotoxic toward EMT-6 mouse mammary tumor cells growing in monolayer. Tetrahydrocortisol (100 M, 24 h) and -cyclodextrin tetradecasulfate (100 M, 24 h) protected EMT-6 cells from the cytotoxicity of CDDP, melphalan, 4-hydroperoxycyclophosphamide, BCNU, and X-rays under various conditions of oxygenation and pH. Minocycline (100 M, 24 h) either had no effect upon or was additive with the antitumor alkylating agents or X-rays in cytotoxic activity toward the EMT-6 cells in culture. The combination of the three modulators either had no effect upon or was to a small degree protective against the cytotoxicity of the antitumor alkylating agents or X-rays. The Lewis lung carcinoma was chosen for primary tumor growth-delay studies and tumor lung-metastases studies. Tetrahydrocortisol and -cyclodextrin tetradecasulfate were given in a 1:1 molar ratio by continuous infusion over 14 days, and minocycline was given i.p. over 14 days, from day 4 to day 18 post tumor implantation. The combination of tetrahydrocortisol/-cyclodextrin tetradecasulfate diminished the tumor growth delay induced by CDDP and melphalan and produced modest increases in the tumor growth delay produced by cyclophosphamide and radiation. Minocycline co-treatment increased the tumor growth delay produced by CDDP, melphalan, radiation, bleomycin, and, especially cyclophosphamide, where 4 of 12 animals receiving minocycline (14×5mg/kg, days 4–18) and cyclophosphamide (3×150 mg/kg, days, 7, 9, 11) were long-term survivors. The 3 modulators given in combination produced further increases in tumor growth delay with all of the cytotoxic therapies, and 5 of 12 of the animals treated with the 3-modulator combination and cyclophosphamide were long-term survivors. Although neither tetrahydrocortisol/-cyclodextrin tetradecasulfate, minocycline, nor the three modulator combination impacted the number of lung metastases, there was a decrease in the number of large lung metastases. Treatment with the cytotoxic therapies alone reduced the number of lung metastases. Addition of the modulators to treatment with the cytotoxic therapies resulted in a further reduction in the number of lung metastases. These results indicate that agents that inhibit the breakdown of the extracellular matrix can be useful additions to the treatment of solid tumors.Abbreviations 14(SO₄)ßCD -cyclodextrin tetradecasulfate - THC tetrahydrocortisol - CDDP cis-diamminedichloroplatinum(II) - 4-HC 4-hydroperoxycyclophosphamide - BCNU N,N-bis(2-chloroethyl)-N-nitrosourea - CAM chick embryo chorioallantoic membrane; IC₅₀, concentration of a drug required to kill 50% of the cells This work was supported by NIH grant P01-CA38493 and a grant from Bristol-Myers-Squibb, Inc., Wallingford, Connecticut     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC₅₀s of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC₅₀s of 452 M, 56 M and 32 M, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC₅₀ of 290 M. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.     

Intracellular concentrations of anti cancer drugs in leukemic cells in vitro vs in vivo

Summary A comparison of intracellular concentrations of daunorubicin, doxorubicin and ara-C in myeloid blast cells was carried out in vivo and in vitro. In vivo, blood samples were obtained from 27 patients with acute nonlymphoblastic leukemia during and up to 4 days after drug infusion. Leukemic cells were isolated and drug concentrations were determined by HPLC. Before treatment, leukemic cells from 21 patients were isolated from blood and bone marrow, and in vitro incubations were done with anthracyclines for 1–3 h at concentrations of 0.1–1.0 M and with ara-C for 1 h to 5 days at concentrations of 0.5–5.0 M. The cells were cultured for 5 days, during which cell samples were taken for drug determination. The results showed that incubation with 0.2 M daunorubicin for 1 h and 0.2 M doxorubicin for 3 h and continuous exposure to 0.5 M ara-C gave intracellular concentration curves similar to those obtained in vivo. After 5 days' culture, the cytotoxic effect was determined by vital dye staining with fast green, the addition of an internal standard of fixed goose erythrocytes, cytospin centrifugation and counter-staining of living cells with haematoxylin/eosin (DiSC). Incubations at the above-mentioned concentrations exerted a cytotoxic effect of approximately 50%. We conclude that in mimicking the in vivo situation, it is important to consider differences in intracellular pharmacokinetics.     

Cytotoxicity of ketoconazole in malignant cell lines

Summary The cytotoxic effects of ketoconazole, an antifungal agent known to have some activity against human prostate cancer, adrenal cancer, and male metastatic breast cancer, were evaluated using colony-growth and clonogenic assays in eight malignant cell lines. The cytotoxicity of ketoconazole showed a dose-and time-dependent pattern, with the following concentrations, inhibiting 90% of the growing colonies (IC₉₀): MCF 7 (human breast cancer) 7.25 g/ml, T 47 D (human breast cancer) 9.0 g/ml, MiaPaCa (human pancreatic carcinoma) 10.0 g/ml, COLO 357 (human pancreatic carcinoma) 9.5 g/ml, HCT 8 (human colonic adenocarcinoma) 27.1 g/ml, DU 145 (human prostatic cancer) 40.0 g/ml, AR 42 J (rat pancreatic carcinoma) 9.0 g/ml, and L1210 (murine leukemia) 8.6 g/ml. Since a concentration of 10 g/ml can be achieved in humans, the use of ketoconazole in human malignancies might be worthy of clinical evaluation.This investigation was supported in part by a grant from the German Volkswagen Foundation, Hannover, Federal Republic of Germany and by a gift from Dr Virgil Gianelli, Stockton, California     

Immune adjuvants for chemotherapy or radiotherapy in the 9L rat brain tumor model

Summary The therapeutic efficacy and toxicity of three biological response modifiers,Corynebacterium parvum (Cp), Chinese blister beetle extract (CBBE), recombinant human IL-1 (rhIL-1), used alone or in combination with chemotherapy or radiotherapy, were investigated in the intracerebral (ic) rat 9L brain tumor model. Used alone, Cp (2mg/rat, ip plus 70g/rat, ic), CBBE (5l of an ethanol extract, ic), or IL-1 (lg/rat, ic or 1g/rat × 3, q 3 d, ic), had no effect on animal survival compared to the untreated or saline treated controls. When combined with chemotherapy or radiotherapy, the three immunotherapeutic agents did not show any additive effects on survival compared to that observed with systemic BCNU (12mg/kg), local ic bleomycin (0.25 unit), or local radiotherapy (16 Gy). While ic IL-1 did not produce evident toxicity, there was fatal toxicity caused by ic Cp or CBBE treatment in a few animals. The combination of Cp and bleomycin produced severe neurotoxicity, resulting in the early death of animals. This study demonstrates a lack of efficacy of the nonspecific immune adjuvants IL-1, Cp or CBBE, used either alone or combined with cytotoxic chemotherapy or radiotherapy, in this rat brain tumor model.     

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.     

Glutathione depletion increases the cytotoxicity of melphalan to PC-3, an androgen-insensitive prostate cancer cell line

Summary Prostate cancer that is androgen-insensitive is unresponsive to a wide spectrum of cytotoxic agents, including all of the alkylating agents. Since a major pathway for the detoxification of the alkylating agents is conjugation with glutathione (GSH), GSH depletion has proved to be effective as a technique to restore melphalan sensitivity in melphalan-resistant cancer cell lines. However, the effect of GSH depletion has not been widely studied in tumor cell lines that have not developed resistance due to previous exposure to alkylating agents. Thus, we decided to investigate GSH depletion as a technique to increase melphalan cytotoxicity to PC-3 cells, an androgen-insensitive prostate cancer line. After 2 and 6 h incubation with 0.25–5 m melphalan, virtually no effect was observed on either clonogenic lethality or MTT viability until 5 m exposures. A 24-h incubation of the cells with 100 m buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, reduced the GSH content by 70%–75%. Following GSH depletion, an increase in clonogenic lethality and a decrease in MTT viability occurred after exposure to concentrations as low as 0.25 m. The dose modification factor ranged from 2.9 after 2 h incubation to 4.5 at 6 h. These results provide support for additional studies in prostate cancer for further investigation of GSH depletion as a technique to induce sensitivity to alkylating agents in this chemotherapy-resistant tumor.     

Effects of Combination Chemotherapy with Biscoclaurine-derived Alkaloid (Cepharanthine) and Nimustine Hydrochloride on Malignant Glioma Cell Lines

In the treatment of malignant glioma, chemotherapy plays a critical role as do surgical resection and irradiation. Cepharanthine (CEP), a biscoclaurine-derived alkaloid, reportedly potentiates the effects of antitumor agents and induces apoptosis in some cancer cells. Here, we examined the effects of CEP, alone and in combination with nimustine hydrochloride (ACNU), on the in vitro proliferation of malignant glioma cells. The cell lines used were U87MG, U251MG, and T98G. At concentrations from 1 to 10g/ml, CEP-promoted cell proliferation somewhat; growth inhibition was noted at concentrations of 15g/ml and higher. Phase-contrast microscopy showed that cells tended to detach from the culture dishes and that cell density became sparse at the higher concentrations. DAPI fluorescence nuclear staining revealed condensation and fragmentation of nuclei, indicating the induction of apoptosis. To examine the cascade of apoptosis, the caspase inhibitors YVAD and DEVD were added. They inhibited CEP-induced apoptosis in U251MG cells (a p53-mutant cell line), but not in U87MG cells (a p53 wild-type cell line), suggesting that in CEP-induced apoptosis two possible cascades are in play. In combination with ACNU, the effects of the higher concentrations of CEP were enhanced.     

The growth-inhibitory effect of 4-hydroperoxycyclophosphamide against human non-small cell lung carcinoma is enhanced by low-dose difluoromethylornithine

Summary Surgically unresectable buman non-small cell lung carcinoma (NSCC) is highly resistant to present chemotherpy and radiation therapy regimens. Cyclophosphamide, a potent alkylating agent, has shown some efficacy, especially in combination chemotherapy. Difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC) which produces minimal toxicity in animals and humans, has shown antiproliferative effect against human SCC in culture but a much smaller effect (cytostatic) against NSCC. We therefore investigated 4-hydroperoxycyclophosphamide (4HC) and DFMO alone and in combination against a human NSCC line (NCI-H157). Cells were treated with DFMO at graded concentrations of 0 to 800 M from day 0 to day 7. On day 3, cells were exposed for 1 h to 4HC at graded concentrations of 0 to 80 M, washed, and refed with media containing DFMO at initial concentrations. On day 7, cells were counted by hemacytometer. Cells treated with DFMO or 4HC alone exhibited dose-dependent growth inhibition. Growth inhibition by 4HC was enhanced through combination with DFMO. On day 7, 50 M (5×10^-5 M) DFMO effected a 37% inhibition, 8 M 4HC 47% inhibition, and the combination of 50 M DFMO and 8 M 4HC yielded an elevated 71% inhibition. The growth inhibitory effect and potentiating effect of DFMO were reversible upon addition of putrescine (PU) to the culture medium. The combination of DFMO and 4HC, two agents with different toxicity spectra, may represent an effective chemotherapeutic regimen for the treatment of lung cancer.Abbreviations DFMO difluoromethylornithine - FBS fetal bovine serum - 4HC 4-hydroperoxycyclophosphamide - NSCC non-small celllung carcinoma - ODC ornithine decarboxylase - PBS phosphate-buffered saline - PU putrescine - RPMI 1640 Roswell Park Memorial Institute 1640 medium - SCC small cell lung carcinoma The studies were supported in part by grants RO1 CA-43280 and VO1 CA-37606 from the National Institutes of Health. Gordon D. Luk is a recipient of a Faculty Research Award from the American Cancer Society