Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in plasma and bone marrow of children receiving low-dose oral methotrexate

Summary The absorption, distribution, and elimination kinetics of low-dose p.o. methotrexate (MTX) were repeatedly studied in 19 children during maintenance treatment of childhood acute lymphoblastic leukemia. Plasma concentrations, urinary elimination, and bone marrow concentrations of MTX and 7-hydroxymethotrexate (7-OH-MTX) were monitored during 24 h following a routime p.o. dose (30 mg/m²) using high-pressure liquid chromatography. Significant interindividual variability was found in time to peak concentration (30–180 min), peak concentration (0.41–2.77 M), and to a lesser extent the half-lives (t_1/2: 32.8–86.1 min; t_1/2: 43.6–350.0 min; t_1/2 absorption: 25.2–60.3 min) and plasma area under the concentration-time curve from zero to infinity (195.6–818.5 M.min). Significant amounts of 7-OH-MTX were detected in plasma, with a mean area under the concentration-time curve from zero to infinity of 208 M.min compared with 365.6 M.min for MTX. High concentrations of 7-OH-MTX were present in bone marrow 24 h after oral MTX (15/19 patients) and were at least five fold those in plasma and three fold the concentration of MTX in bone marrow. In four patients occasionally neither MTX nor metabolite could be detected. Repeated examination of these pharmacokinetic parameters in plasma and bone marrow showed that the intraindividual variability was small.This study was supported by the Netherlands Cancer Foundation Koningin Wilhelmina Fonds     

Inhibition of 7-hydroxymethotrexate formation by amsacrine

Summary The inhibition of methotrexate (MTX) biotransformation to 7-hydroxymethotrexate (7-OH-MTX) by 4-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA) was studied in bile-drained rats in vivo and in incubates of isolated rat hepatocytes and rat-liver homogenate in vitro. In vivo, i.v. administration of 10 mg/kg mAMSA prior to [³H]-MTX infusion (50 mg/kg) led to a significant alteration in 7-OH-MTX kinetics. 7-OH-MTX peak concentrations and AUC in bile and serum were reduced by 75% and the recovery of MTX as 7-OH-MTX in bile and urine decreased by 70%, whereas MTX pharmacokinetics remained unaltered. In suspensions of isolated hepatocytes. 10 m mAMSA led to a 54% decrease in 7-OH-MTX formation. However, the hepatocellular influx and efflux of MTX was not perturbed by mAMSA. Preincubation of rat-liver homogenates with 1.25–10 m mAMSA reduced the formation of 7-OH-MTX by up to 73%. mAMSA appeared to inhibit MTX hydroxylation competitively, exhibiting aK _iof 3 m. Due to its inhibition of the MTX-oxidizing system, mAMSA may be beneficial in combination chemotherapy with MTX by reducing 7-OH-MTX-associated toxicity and, possibly, enhancing the cytotoxic effects of MTX.This study was financially supported by the Norwegian Cancer Society and the Erna and Olav Aakre Foundation for Cancer Research     

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     

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 M, and concentrations of 7-OH-MTX declined triphasically, showing at1/2 value of 2.45 min, at1/2 value of 30.5 min, and a terminal half-life (t1/2) of 240 min. The total clearance value was 14.5 ml min^–1 kg, and the postdistributional volume of distribution (V) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.This study was supported financially by the Norwegian Cancer Society     

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     

Relationship between in vivo drug exposure of the tumor interstitium and inhibition of tumor cell growth in vitro: a study in breast cancer patients

A novel approach is described to simulate effect site pharmacodynamics of anticancer drugs. This approach is based on (i) the in vivo measurement of unbound, interstitial drug pharmacokinetics (PK) in solid tumor lesions in patients and (ii) a subsequent pharmacodynamic (PD) simulation of the time versus drug concentration profile in an in vitro setting. For this purpose, breast cancer cells (MCF-7) were exposed in vitro to the time versus interstitial tumor concentration profiles of 5-fluorouracil (5-FU) and methotrexate (MTX) from primary breast cancer lesions in patients. This led to a maximal reduction in the viable cell count of 69 on day 4, and of 71 on day 7 for 5-FU and MTX, respectively. This effect was dependent on the initial cell count and was characterized by a high interindividual variability. For 5-FU there was a significant correlation between the maximum antitumor effect and the intratumoral AUC (r=0.82, p=0.0005), whereas no correlation could be shown for MTX (r=0.05, p=0.88). We conclude, that the in-vivo-PK / in-vitro-PD model presented in this study may provide a rational approach for describing and predicting pharmacodynamics of cytotoxic drugs at the target site. Data derived from this approach support the concept that tumor penetration of 5-FU may be a response-limiting event, while the response to MTX may be determined by events beyond interstitial fluid kinetics.     

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma

Summary Fluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5-deoxy-5-fluorouridine (5dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 °C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH₂FH₄), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH₂FH₄, but our results were not always reproducible.This study was supported by grants from Université Paul Sabatier, Conseil Régional Midi-Pyrénées-CNRS and Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés (INSERM)     

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     

Pharmacogénétique des fluoropyrimidines. La méthylènetétrahydrofolate réductase

Résumé: Lefficacité optimale des fluoropyrimidines nécessite des concentrations élevées en 5-10 méthylènetétrahydrofolate (CH₂FH₄), contrôlées par la méthylènetétrahydrofolate réductase (MTHFR) qui convertit irréversiblement le CH₂FH₄ en 5-méthyltétrahydrofolate (CH₃FH₄). Les polymorphismes 677CT et 1298AC du gène MTHFR sont associés à une baisse dactivité de lenzyme. Les tumeurs «MTHFR mutées» devraient donc être plus sensibles aux fluoropyrimidines que les tumeurs «MTHFR sauvages». Les études expérimentales et cliniques publiées à ce jour tendent à montrer une plus grande sensibilité au 5-FU (associé ou non à lacide folinique) dans les tumeurs MTHFR mutées par rapport aux tumeurs sauvages. Ces résultats montrent la nécessité de poursuivre ces recherches afin de confirmer limpact de ces polymorphismes sur lefficacité des fluoropyrimidines.     

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     

Involvement of nuclear steroid/thyroid/retinoid receptors and of protein kinases in the regulation of growth and of c‐erbB and retinoic acid receptor expression in MCF‐7 breast cancer cells

Nuclear steroid/thyroid/retinoid receptors and cerbB membrane receptor tyrosine kinases control epithelial growth and differentiation. Retinoid receptors can dimerize with the vitamin D receptor, the glucocorticoid receptor or the thyroid receptor. Furthermore, multiple cerbB receptor dimers have been identified. It has been shown that some of these receptor pathways communicate with each other via crossconnected regulatory networks. Molecular interactions between retinoid receptors or estrogen receptors (ER) and cerbB2, and between ER and retinoic acid receptor(RAR) have been reported. Here, we demonstrate the effects of steroids/thyroids/retinoids and of activators of protein kinase A (forskolin, Forsk) and C (12Otetradecanoylphorbol13acetate, TPA), on growth and expression of cerbB and RARs in MCF7 breast cancer cells, which contain high levels of RAR and , and which express significant amounts of cerbB2 and 3. All transretinoic acid (tRA), the antiestrogen ICI 182 780 (ICI), Forsk and TPA reduced, whereas triiodothyronine and 17estradiol (E₂) stimulated cell growth. Flow cytometry revealed that tRA and E₂ reduced cerbB2 and 3, whereas tamoxifen, Forsk and TPA upregulatedcerbB2. cerbB3 was coregulated with cerbB2. Northern analysis demonstrated that RAR was downregulated by dexamethasone, ICI, and TPA, whereas vitamin D₃ and E₂ upregulated RAR. RAR expression was less responsive to such treatment, being reduced only by ICI and Forsk. These data indicate that nuclear receptor and protein kinase signaling communicate with each other and control the expression of RARs and cerbB receptors. Efficient growth control requires the coordinated interplay of both receptor systems.     

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.     

Cytokine‐regulated urokinase‐type‐plasminogen‐activator (uPA) production by human breast fibroblasts in vitro

It has been shown that, in breast stroma, urokinasetype plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPAproducing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumorderived human breast fibroblasts to produce uPA protein and the myofibroblast marker smoothmuscleactin (SMA) in response to various cytokines implicated in the process of tissueremodeling during malignant transformation.We found that fibroblasts produced increased amounts of uPA protein after exposure to aFGF, bFGF, EGF, PDGFBB, and IFN, were unaffected in this respect by IL6, MCSF, GMCSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL1, TNF, IGFI, and IGFII. None of these cytokines were able to induce a striking increase in the fraction of SMApositive fibroblasts. On the other hand, 25pM TGF₁ increased the fraction of SMApositive fibroblasts 5fold in both normal and tumortissuederived fibroblasts. Nonetheless, the normalderived fibroblasts were unaffected in their uPAproducing capacity by TGF₁, and the tumorderived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basaluPAproduction of both normal and tumorderived fibroblasts was increased by autocrinely produced bFGFlike activity, and that the basaluPAproduction of at least the normalderived fibroblasts was decreased by autocrinely produced IGFlike activity.Altogether, our data suggest an active role for fibroblasts in the process of uPAdirected breast tumor proteolysis.     

Selective Decrease of Serum Immunoglobulin G1 as Marker for Early Stages of Invasive Breast Cancer

The diagnostic value of the decrease in percentage of immunoglobulin G1 (%IgG1) in breast cancer was analyzed with special emphasis on early tumor stages. IgG1 and total IgG were preoperatively measured in the sera of a total of 801 individuals using a modified quantitative affinity chromatography. Group A consisted of 174 healthy individuals of both sexes, group B of 324 female patients with benign breast disease, and group C of 303 patients with invasive and non-invasive breast cancer. Within group C, 13 patients presented with intraductal carcinoma, and 22 patients with a pT1a-tumour (diameter less than 0.5cm). The %IgG1 values were compared among groups A, B and C. In addition, correlations were sought between %IgG1 values of group C and tumor size, stage (UICC), histopathological grade and oestrogen (ER) and progesteron receptor (PR) expression. The mean value of %IgG1 in group A was 63.3±0.5 s.e.m., in group B 57.75±0.4 s.e.m. and in group C 52.37±0.5 s.e.m. The differences of mean values were highly significant between all three groups. Sensitivity and specificity of %IgG1 to discriminate between group A and C were 75% and 87%, and between group B and C 62% and 63%, respectively. The significant decrease of %IgG1 in total serum IgG is able to distinguish patients with breast cancer of more than 5mm in diameter from healthy controls and patients with benign breast diseases. Finally, calculated posterior probabilities revealed that within certain concentration limits %IgG1 may provide predictive information with high xprobabilities.     

Induction of insulin‐like growth factor binding protein expression by ICI 182,780 in a tamoxifen‐resistant human breast cancer cell line

Earlier studies in our laboratory demonstrated that the steroidal antiestrogen ICI 182,780 is very effective in abolishing the tamoxifenresistant proliferation of MCF 7/523 cells [1]. In addition, preliminary binding studies showed that ICI 182,780 increased the binding of insulinlike growth factor (IGF)I to the MCF 7/523 cells, although this finding was not the result of an increase in the expression of the insulinlike growth factorI receptor (IGFIR). Hence, we reasoned that the inhibition of tamoxifenresistant cell growth by ICI 182,780 might have been due to increased expression of insulinlike growth factor binding proteins (IGFBPs).We observed the upregulation of noninsulinsuppressible IGFI binding in both the tamoxifensensitive MCF 7/521 cell line (1.5fold) and the tamoxifenresistant MCF 7/523 cell line (2.5fold) after 5 days of treatment with ICI 182,780 (10^–7 M) in serumfree medium, suggesting a role for cellassociated IGFBPs. Affinity crosslinking experiments confirmed the presence of an IGFI:IGFBP complex of approximately 38kDa in tamoxifen or ICI 182,780treated cells. Western ligand blots showed higher levels of a soluble 30kDa IGFBP in media conditioned by either of the subclones that had been treated with ICI 182,780, an effect consistently opposed by estrogen (E₂:10^–9 M). RTPCR showed higher levels of IGFBP5 mRNA than any of the other known IGFBPs, suggesting that this was the major IGFBP subtype. The protein was subsequently identified by Western immunoblotting as IGFBP5. In conclusion, we postulate that this may be a mechanism contributing to the greater potency of ICI 182,780 in the growth inhibition of the MCF 7/523, tamoxifenresistant cell line.     

Pharmacokinetics and metabolism of β-2′-deoxythioguanosine and 6-thioguanine in man

Summary Resistance to the antileukemic agent 6-thioguanine (TG) inevitably develops in animal tumors. However, a new agent, -2-deoxythioguanosine (-TGdR) can overcome TG resistance in animal tumor models and is therefore of potential clinical use. The pharmacokinetics of radiolabeled TG were compared with those of -TGdR in patients with cancer after intravenous administration. [³⁵S]--TGdR (5.4 mg/kg, 200 mg/m², 200 Ci total) was administered to five patients; the radiolabel in the plasma declined with an initial half-life (t_1/2) of 14 min and a terminal t_1/2 of 19.3 h. Within 24 h, 65% of the radiolabel was excreted in the urine. In contrast, after administration of [³⁵S]-6-TG (3.4 mg/kg, 125 mg/m², 200 Ci total) the average initial t_1/2 was 40 min while the terminal phase t_1/2 was 28.9 h. Urinary excretion of the radiolabel was 75% of the dose 24 h after administration. Both thiopurines were rapidly and extensively degraded and excreted as 6-thioxanthine, inorganic sulfate, S-methyl-6 thioxanthine, and 6-thiouric acid in addition to other products. Small amounts of unchanged drug were also excreted. These studies suggest that -TGdR is merely a latent form of TG.Deceased, to whose memory this paper is dedicated     

Mechanism of captopril toxicity to a human mammary ductal carcinoma cell line in the presence of copper

Captopril (D3mercapto2methylpropanoylLproli ne) is an angiotensin converting enzyme (ACE) inhibitor, used widely in the treatment of hypertension and congestive heart failure. Captopril also inhibits proliferation of a variety of cell types, including several lacking ACE and renin acitvity. We have previously demonstrated that human mammary ductal carcinoma cells are among the cell types whose mitotic activity is inhibited by captopril. In those cells, captopril also reduces estrogen receptor (ER) and increases progesterone receptor (PR) concentrations. The present study evaluated the mechanism of captopril's antiproliferative action in an ER/PRnegative human mammary ductal carcinoma cell line, Hs578T. Cells grown in a 10% serum medium showed negligible changes in the presence of captopril alone. However, in the presence of subphysiologic concentrations of copper salts or copperloaded ceruloplasmin, captopril caused a dosedependent reduction in cell number, thymidine incorporation and mitochondrial dehydrogenase activity. In contrast, iron salts and ironsaturated transferrin had no effect on captopril activity. Catalase and horseradish peroxidase nullified the cytotoxic effects of captopril/Cu⁺⁺, whereas H₂O₂ mimicked those effects. These data are consistent with the notion of a coppercatalyzed oxidation of captopril, leading to the generation of H₂O₂ as the cytotoxin to this clinically important cell type.     

Involvement of breast epithelial-stromal interactions in the regulation of protein tyrosine phosphatase-γ (PTPγ) mRNA expression by estrogenically active agents

Background. Protein tyrosine phosphatase (PTP) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicate that estradiol-17 (E₂)-induced suppression of PTP may play a role in mammary tumorigenesis. Zeranol (Z), a nonsteroidal growth promoter with estrogenic activity that is used by the US meat industry, induces estrogenic responses in primary cultured breast cells and breast cancer cell lines. Methods. PTP mRNA expression in human breast tissues and cells isolated from surgical specimens of mammoplasty and breast cancer patients were detected and quantified by RT-PCR. Immunohistochemical staining was used to localize PTP in human breast tissues. Breast epithelial and stromal cells were isolated and co-cultured to determine the involvement of cell–cell interaction in the regulation of PTP mRNA expression by E₂ and Z. Results. PTP mRNA expression was lower in cancerous than in normal breast tissues. Both E₂ and Z suppressed PTP mRNA levels in cultured normal breast tissues by 80%, but had a lesser effect in cultured epithelial cells isolated from normal breast tissues. In the co-culture system, both E₂ and Z suppressed PTP mRNA to a greater degree in epithelial cells than in stromal cells. In whole breast tissues, PTP was immunolocalized to the epithelium. Treatment with E₂ or Z diminished PTP staining indicating reductions in PTP at the protein level. Conclusions. The results indicate that both E₂ and Z regulate PTP expression in human breast and that epithelial–stromal cells interaction is important in the regulation of PTP expression by estrogenically active agents.     

Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP‐470 on human breast cancer cells

The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDAMB231, T47D, MCF7, KPL1, and MKLF). In all five cell lines, EPA and TNP470 alone both showed tumor growth inhibition in a time and dosedependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bc1x_S, the apoptosisenhancing proteins, was more upregulated and that of Bcl2 and Bclx_L, the apoptosissuppressing proteins, was more downregulated compared to the use of EPA or TNP470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.     

Pharmacologic studies on the dibutyl and γ-monobutyl esters of methotrexate in the rhesus monkey

Summary The pharmacokinetics and metabolism of dibutyl methotrexate (DBMTX) and -monobutyl methotrexate (-MBMTX) were studied in Rhesus monkeys. When a bolus IV dose of either [³H]DBMTX or [³H]-MBMTX was given, the principal species in serum for up to 1 h was the monoester, with MTX accounting for < 10% of the total radioactivity. Products other than -MBMTX and MTX were formed in substantial amounts with DBMTX, but not with -MBMTX. Total radioactivity recovered in the bile 5 h after [³H]DBMTX injection accounted for 32% of the administered dose, indicating high hepatic extraction for this lipophilic compound. Serum and CSF levels of unchanged -MBMTX, as well as of MTX arising via esterase cleavage, were measured by HPLC after IV infusion of -MBMTX (10 g/m²). Efflux of monoester from CSF was slower than disappearance from serum. However, -MBMTX levels in CSF were no higher than could be attained by infusing MTX itself at the same dose rate. While CSF/serum ratios were ca. 10-fold higher for -MBMTX than for MTX, this difference could be explained on the basis of the very different affinities of the two compounds for serum proteins. HPLC analysis of serum processed by methanol precipitation as opposed to ultrafiltration of the proteins showed -MBMTX to be >99% bound, whereas for MTX this value was 50% or less. When -MBMTX and MTX levels measured after ultrafiltration were corrected for this difference in serum protein binding the total amount of the two drugs in serum became almost equivalent.The abbreviations used are MTX methotrexate (4-amino-4-deoxy-N ¹⁰-methylpteroyl-l-glutamic acid) - DBMTX dibutyl methotrexate (NSC-305985) - -MBMTX -monobutyl methotrexate (NSC-305986) - DDMP 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine (NSC-19494) - CSF cerebrospinal fluid - HPLC high-performance liquid chromatography - TLC thin-layer chromatography