Supplementary Figure 1 from Stepwise Neoplastic Transformation of a Telomerase Immortalized Fibroblast Cell Line

This study examined the dose-dependent efficacy of erythropoietin (EPO) for preventing and/or treating cisplatin (CDDP) induced peripheral neurotoxicity (CINP), and its influence on tumour treatment and growth. Rats received eight intraperitoneal (ip) injections of 2 mg/kg CDDP twice weekly. EPO co-administered (50 or 10 microg/kg ip, three times/week) had a dose-dependent effect, partially preventing CINP, but 0.5 microg/kg ip was not effective. The neuroprotective effect lasted at least 5 weeks after the last dose of EPO and CDDP. In addition, EPO (50 microg/kg ip three times/week) after the last injection of CDDP still induced a significant recovery of CINP. In a separate experiment in rats bearing mammary carcinoma EPO treatment (50 microg/kg ip) given concurrently with CDDP (1.0 and 1.5 mg/kg twice a week for four weeks) was neuroprotective without influencing the effectiveness of the treatment or tumour growth. EPO thus appears to be an effective neuroprotectant that does not interfere with tumour treatment.

PURPOSE:To investigate the cytotoxic and antitumor effects of the combination of the novel anticancer drug ET-743 and doxorubicin (Dx) and to determine whether any pharmacokinetic interaction occurs in sarcoma-bearing mice.METHODS:The cytotoxicity of each drug and of their combinations was assessed in the rhabdomyosarcoma cell line TE-671 by a clonogenic assay, and isobologram analysis was performed to detect any synergistic, additive or antagonistic effects. The antitumor activities of each drug and of the combinations were also evaluated in nude mice transplanted subcutaneously with human TE-671 rhabdomyosarcoma and in C3H female mice injected intravenously with UV2237 M fibrosarcoma or with the Dx-resistant subline UV2237 M-ADM which overexpresses Pgp. Antitumor activity was evaluated by monitoring the TE-671 tumor volume over time and, in the case of the murine fibrosarcomas, by evaluation of lung deposits at autopsy quantified by determining lung weight. Pharmacokinetic studies were performed in TE-671-bearing mice. ET-743 was determined in plasma by an HPLC-MS method and Dx in plasma and tissue by an HPLC method with fluorescence detection.RESULTS:The combination of ET-743 and Dx was found to be additive with the average combination index slightly lower than 1 at all survival levels, suggesting weak synergism. In TE-671 tumors in vivo the activity of ET-743 or Dx given alone was marginal, whereas the combination produced a significant antitumor effect. The log cell kill (LCK) values were 0.13 and 0.33 for ET-743 and Dx alone, whereas they ranged from 0.85 to 1.12 for the combination. Giving ET-743 1 h before Dx slightly enhanced the effect (LCK 1.12) compared with giving the drugs simultaneously (LCK 0.85) or in the opposite sequence (LCK 0.92). In UV2237 M fibrosarcoma, both Dx and ET-743 showed an effect in reducing the weight of lung metastases, although the combination of the two drugs was not superior to each drug alone. In UV2237 M-ADM tumors neither of the two drugs was active, whereas the combination, particularly when the two drugs were given simultaneously, produced a significant effect. Plasma levels of ET-743 and Dx were not significantly different when the drugs were given alone or in combination. The concentrations of Dx in tissues including tumor, liver, heart and kidney were found to be the same whether the drug was given alone or in combination with ET-743.CONCLUSIONS:These results indicate that ET-743 and Dx in combination produce an additive effect against human sarcoma cells, reinforcing the idea that they act by a different mechanism of action. In mice no pharmacokinetic interaction between the two drugs was found. The observed activity in UV2237 M-ADM and in human TE-671 sarcoma suggests that the combination of the two drugs could be effective for tumors displaying low sensitivity to each drug given alone. Based on these findings a phase I study on the combination of the two drugs was recently initiated.

The studies described herein were designed to determine whether doxorubicin (DOX) pharmacokinetics (PKs) could be described by a physiologically based PK model that incorporated macromolecule-specific binding and organ-specific metabolism and excretion. Model parameters were determined experimentally, or were gathered from the literature, in a species-specific manner, and were incorporated into a physiologically based description of DOX blood and tissue distribution for mice, dogs, and humans. The resulting model simulation data were compared with experimentally determined data using PK parameters calculated using compartmental or noncompartmental analysis to assess the predictability of the models. The resulting physiologically based PK model that was developed could accurately predict blood and tissue PKs of DOX in mice. When this model was interspecies extrapolated to predict DOX levels in dogs and humans undergoing treatment for cancer, predictions in dog plasma or human serum were also consistent with the actual clinical data. This model has potential utility for predicting the magnitude of PK interactions of DOX with other drugs, and for predicting changes in DOX PKs in any number of clinical situations.

Peliosis hepatis, characterized by the presence of blood- filled spaces within hepatic parenchyma, developed in C57B1 mice implanted subcutaneously with melanoma cells 23 days previously. The peliosis was associated with dilated hepatic sinusoids that were lined by prominent, proliferating endothelial cells. The development of peliosis hepatis was completely abrogated when melanoma growth was inhibited by administration of dexamethasone. These features support the concept that peliosis hepatis can be induced by a circulating tumor- derived endothelial growth factor such as vascular endothelial growth factor.

SDZ PSC 833 (PSC 833) is a cyclosporin A analogue that is under clinical investigation in combination with doxorubicin (Dx) or other anticancer agents as a type-1 multidrug resistance (MDR-1)-reversing agent. The present study was focused on the effects of PSC 833 on the distribution and toxicity of Dx in non-tumor-bearing CDF1 male mice. Mice were given PSC 833 i.p. at 30 min before i.v. Dx treatment. Dx levels were determined by a high-performance liquid chromatography (HPLC) assay at different times during a 72-h period following Dx treatment in the serum, heart, intestine, liver, kidney, and adrenals of mice. In all tissues, Dx area under the concentration-time curve (AUC) values were much greater in mice receiving 10 mg/kg Dx in combination with 12.5 or 25 mg/kg PSC 833 than in mice receiving Dx alone. The highest increase in Dx concentrations was found in the intestine, liver, kidney, and adrenals. Lower, albeit significant, differences were found in the heart. PSC 833 did not appear to influence either urinary or fecal Dx elimination or Dx metabolism to a great extent. Doses of PSC 833 devoid of any toxicity potentiated the acute and delayed toxicity of Dx dramatically. The mechanism responsible for this enhanced toxicity has not yet been elucidated but is likely to be related to an increased tissue retention of Dx due to inhibition of the P-glycoprotein (Pgp) pump by PSC 833, as has recently been proposed for cyclosporin A.

The pharmacokinetics of the anticancer agent p-(3,3-dimethyl-1-triazeno) benzoic acid (pCOOH-DMT), a drug now in phase I clinical trial in Europe, was investigated in C57Bl female mice with M5076 reticulum-cell sarcoma that were treated i.v. with 200 mg/kg pCOOH-DMT. The drug disappeared from plasma with a terminal half-life of about 2.5 h. Plasma clearance was approximately 6 ml/min per kg. Distribution studies showed some differences in drug levels in different tissues. The highest levels were found in the tumor, liver, kidney and lung; lower levels were found in the spleen and gut, and the lowest, in the brain. The N-desmethyl derivative of pCOOH-DMT was not detectable in plasma or tissues of mice treated with the drug. Therefore, the previous evidence of low N-demethylation of pCOOH-DMT was confirmed. pCOOH-DMT glucuronide was identified by mass spectrometry and quantified by high-performance liquid chromatography (HPLC) in plasma, tissues and urine samples. pCOOH-DMT glucuronide appears to be the major urinary metabolite of pCOOH-DMT in mice. Another metabolite identified by mass spectrometry and quantified by HPLC in some tissues and urine was pCOOH-DMT glycinate.

In this study we show that cytarabine given simultaneously with 5-aza-2'-deoxycytidine (Aza-dC) antagonized Aza-dC activity against L1210 mouse leukemia. This antagonism was seen after a single dose (on Day 3 or Day 5 after tumor implant) and after repeated doses. This observation suggests caution in combining cytarabine with Aza-dC in the treatment of human leukemias.

The covalent binding of hexamethylmelamine (HMM) and its metabolites was studied in liver, tumor, blood, kidney, spleen, lung, brain, heart, and small intestine after a single IP injection of 2,4,6-14C-hexamethylmelamine (50 mg/kg) to C57Bl/6J female mice bearing 20-day-old M5076/73A ovarian cancer. Covalent binding to tissue macromolecules was measured 2, 10, and 40 h after injection of the drug. At 2 h liver and small intestine showed the highest levels of irreversibly bound metabolites, the lowest being found in brain and heart. Except in the small intestine, where a decrease was observed between 2 and 10 h, the level of covalent binding was constant up to 40 h.

The pharmacokinetics of VP16 have been investigated in Lewis lung bearing mice after i.v. doses of 13 and 40 mg/kg. At both doses the plasma elimination half-life was around 30 min. The lowest VP16-213 levels were in brain and primary tumor. Drug concentrations were much higher in metastases than in primary tumor. The highest concentrations were in small intestine, liver and kidney. Drug levels in the liver were disproportionally higher after 40 mg/kg, the AUC value being approximately 12 times greater than after 13 mg/kg. Urinary excretion of VP16-213 as unchanged drug accounted for 20–30% of the administered dose in the 60 h after treatment. The concentration cytotoxicity curve was very steep and apparently similar for cells derived from primary tumor or metastases grown in vitro.

The differential distribution of doxorubicin (Adriamycin = AM) and daunorubicin (Daunomycin = DM) within the blood components, after an i.v. injection of 10 or 15 mg/kg of body weight, was investigated from its metabolites and quantified by means of the TLC scanning fluorescence technique. AM accumulated in the following order (of decreasing percentages): plasma and red cells (RBC), white cells (WBC), and platelets (PT), but the absolute amount of drug that reached each cell type was related to its relative volume. In the presence of higher blood concentrations (after injection of 15 mg/kg of body weight) the RBCs accumulated much more AM than the plasma, WBC, and PT; suggesting that the RBC fraction has a greater capacity to concentrate the drug. However, if the concentration of AM is expressed per unit volume of each component, a markedly higher value was observed for PT, and this was confirmed by in vitro results obtained by incubating blood in the presence of AM. DM seemed to be distributed on a percent basis to a greater extent than AM in the RBC fraction. Both compounds were taken up by blood cells, particularly platelets, to levels in excess of the extracellular concentration.

In spite of clinical activity in heavily-pretreated ovarian cancer, the antitumour s-triazine trimelamol [TM; tris(hydroxymethyl)-tris(methyl)melamine] had to be withdrawn from further clinical studies due to formulation difficulties related to instability. A synthetic programme has produced tris(hydroxymethyl) analogues containing electron-withdrawing groups in place of methyl-triscyanomethyl CB 7669, tristrifluoroethyl CB 7639, CB 7529 and trispropargyl CB 7547, all showing markedly superior stability to TM. Chemosensitivity testing of analogues (MTT assay, continuous exposure) using a panel of rodent and human cell lines showed activity close to that of TM, e.g. for the CHI human ovarian cancer cell line. IC50 values were TM 23.4 μM, CB 7639 30.5 μM, CB 7529 29.5 μM, CB 7547 28.5 μM and CB 7669 27.3 μM. CB 7669 and CB 7639 required prolonged exposure (> 12 h) in order to exhibit equivalent cytotoxicity to a 2-h exposure to TM. Thus, rather than administration as a single daily dose, the stable analogues may be more suited to prolonged infusion, which was suggested as being a more beneficial regimen in clinical trials with TM. In line with clinical observations indicating the efficacy of TM in platinum-refractory ovarian cancer, we saw no significant cross-resistance to TM or CB 7529 in a range of platinum-sensitive and :acquired-resistant cell line pairs or in an alkylating-agent resistant cell line, despite TM's ability to crosslink DNA. Data obtained using cell lines with acquired resistance to TM, CB 7669 and formaldehyde (released in the breakdown of TM) suggest a pivotal role for formaldehyde and a more minor role for alkylating activity in the mechanism of action of the N-(hydroxymethyl)melamines in vitro. Further clinical trials of these compounds are eagerly awaited, and their usefulness as second-line chemotherapy for heavily pretreated ovarian cancer deserves further investigation.

Concentrations of pentamethylmelamine (PMM) and some metabolites were determined in plasma of rats treated with 10 and 50 mg PMM/kg IV. The areas under the plasma levels curve after these doses were 241 and 1,827 μg/mlxmin; plasma clearances were 0.042 and 0.027 l·kg-1·min-1, respectively.

The relative distribution of adriamycin to plasma and blood cells after IV injection of 10 mg/kg was investigated in CD rats bearing intramuscular 256 Walker carcinosarcomas 15 days old. The drug was measured by a fluorimetric procedure and the amount of unchanged compound was separated from metabolites and quantitated by means of a TLC scanning fluorescence technique. In the presence of a tumor much lower hematocrit values are found, with marked anemia and thrombocytopenia associated with leukocytosis. These modified hematologic parameters account for an altered pattern of drug distribution. The low number of blood cells per milliliter results in a smaller amount of drug being present in the cellular fraction, so that more of the compound (even twice as much) is made available in plasma. Changes in adriamycin concentrations per unit volume or cell of each cell type are inversely related to changes in their relative number per milliliter. The only cell fraction where the drug increase per cell or cubic micrometer does not compensate the marked reduction in cell count observed in the presence of tumor is the platelet fraction, in which adriamycin amounts are 25% or less of those observed in the blood of normal rats, indicating that these blood cells become saturated in tumor-bearing animals.

Mice bearing the S-180 sarcoma displayed a depression of liver catalase and cytochrome P-450-dependent enzymes (ethoxycoumarin deethylase, ED) from day 6 following tumor implantation. Injection of serum obtained from tumor-bearing mice into normal mice caused depression of liver ED suggesting that a circulating factor was involved. Tumor-bearing mice did not show any significant change in serum triglycerides and food intake. By contrast, injection of endotoxin, interleukin-1 (IL-1) or tumor necrosis factor (TNF) caused not only a depression in liver ED but also a marked increase in serum triglycerides.To study the possible analogies between cancer-associated circulating factor and monokines, we studied the effect of dexamethasone (a known inhibitor of monokine synthesis) on liver ED activity in tumor-bearing mice. Dexamethasone (DEX) treatment increased (up to 60%) liver ED activity in tumor-bearing mice.We conclude that: (i) a circulating factor is involved in cancer-associated ED depression; (ii) that this mediator is not necessarily identical to TNF or IL-1 and (iii) that DEX reverses the depression of liver ED in cancer, possibly by inhibiting the synthesis, or the effects, of this factor.

Cyclophosphamide (CP) injected at different treatment schedules into CD male rats, prolongs narcosis after either parenteral or oral administration of pentobarbital. However, it takes 7 days before any change appears after CP pretreatment. The level of pentobarbital in blood and brain at different times after injection was higher in CP pretreated than in untreated animals. Since previous studies reported a decreased rate of inactivation of drugs after treatment with cytotoxic agents, experiments were performed to ascertain whether, beside the impairment of the metabolism, the animals respond differently to the same brain concentration of pentobarbital.