Pharmacokinetic study of doxifluridine given by 5-day stepped-dose infusion

Summary Doxifluridine (5-deoxy-5-fluorouridine, 5-dFUR) metabolism has been reported to be saturable and associated with a fall in clearance of the drug as the dose is increased. The aim of the present study was to determine the disposition of 5-dFUR and 5-fluorouracil (5-FU) when 5-dFUR was given as a 5-day infusion, with the infusion rate increased stepwise every 24 h. Measurement of plasma and urinary levels of 5-dFUR and 5-FU at steadystate for each infusion rate enabled the estimation of 5-dFUR renal (Cl_R) and nonrenal (Cl_NR) clearance and 5-FU renal clearance. A total of 28 patients with histologically proven malignancy received 5-day courses of 5-dFUR ranging in dose from 3.75 to 20 g/m² per 120 h. The lowest dose given over 24 h was 0.25 g/m², and the highest was 5 g/m². Steady-state plasma levels of 5-dFUR ranged from 167 to 6.519 ng/ml. At these plasma levels there was no evidence of significant saturation of 5-dFUR metabolism; steady-state plasma levels of 5-dFUR increased approximately linearly with dose, and nonrenal clearance did not change significantly with dose. There was also no evidence of nonlinearity in 5-dFUR renal clearance. The mean (±SD) Cl_R of 5-dFUR was 108.9±53.6 ml/min per m² (range, 45.7–210 ml/min per m²), and the Cl_NR was 728±181 ml/min per m² (range, 444–1,119 ml/min per m²). Renal clearance comprised 13% of the total 5-dFUR clearance. The mean renal clearance of 5-FU was 100.8±48.6 ml/min per m² (range, 23.5–198 ml/min per m²). There was considerable interpatient variability in 5-dFUR renal and nonrenal clearance, event at the same dose level. We concluded that the administration of 5-dFUR by the infusion method described avoided the saturation of nonrenal elimination processes reported to occur with shorter infusion schedules.This study was supported by a grant from F. Hoffmann-La Roche, Basel, Switzerland     

Activity of (-)-2'-deoxy-3'-oxacytidine (BCH-4556) against human tumor colony-forming units

Background: BCH-4556 ((-)-2-deoxy-3-oxacytidine) is an L-nucleoside analogue shown to have broad preclinical anticancer activity, particularly against solid neoplasms such as prostate, renal, and hepatoma in vitro and in vivo, in contrast to cytosine arabinoside (ara-C) which is preferentially active against leukemia.Materials and methods: The antitumor activity of BCH-4556 was evaluated using human tumor colony-forming unit (HTCFU) assay, in which fresh tumor specimens were taken directly from patients with and without prior chemotherapy.Results: Overall, in vitro responses (50% or less survival compared to untreated controls) were observed in 11% (two of 18), 29% (five of 17) and 50% (nine of 18) of specimens treated for one hour with BCH-4556 at 1, 10 and 100 µg/ml, respectively; and 16% (nine of 55), 32% (24 of 74), 48% (35 of 73) and 65% (11 of 17) of specimens treated continuously with BCH-4556 at 0.1, 1, 10 and 100 µg/ml, respectively. With the one-hour schedule, a significant difference in response rates was noted between 100 µg/ml and 1 µg/ml (P = 0.02). With the continuous schedule, significant differences in response rates were observed between 1 µg/ml and 0.1 µg/ml (P = 0.02), between 10 µg/ml and 0.1 µg/ml (P = 0.0001), as well as between 10 µg/ml and 1 µg/ml (P = 0.01). A trend suggesting the superiority of continuous exposure was observed in paired specimens (n = 18) at comparable drug concentrations. Activity was noted against ovarian (nine of 16 = 56%), renal (three of four = 75%), and melanoma (two of two = 100%) HTCFU at 10 µg/ml using the continuous schedule. Comparisons between BCH-4556 and paclitaxel were made in 32 specimens at 10 µg/ml using the continuous exposure. Twenty-three specimens showed similar responses with both drugs; seven showed better responses with BCH-4556; and two showed better responses with paclitaxel (P = 0.18).Conclusions: Promising activity was observed with BCH-4556 against ovarian, renal, and melanoma HTCFU. There appeared to be a positive relationship between BCH-4556 concentration and response using both one-hour and continuous exposures. Continuous exposure to BCH-4556 provided high response rates especially at concentrations above 10 µg/ml. For both one-hour and continuous exposures, BCH-4556 had similar, and at times, greater potency than paclitaxel against the same tumor specimens in the present study.     

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of 1 mM for six or more hours, significantly enhances the efficacy of 5-FU.Patients and methods: 5-FU/FA was given as follows: days 1 and 2 – FA 250 mg/m² (max. 350 mg) over two hours followed by 5-FU 400 mg/m² by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m² infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours.Results: Thirty patients were entered into the study. Median survival was nine months (range 1–51+ months). There were eight partial responses (28%, 95% CI: 13%–47%). The median duration of response was 6.5 (range 4–9 months). Grade 3–4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another.Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.     

Synergistic effect of 3′-deoxyadenosine N 1-oxide and adenosine deaminase inhibitors on growth of Ehrlich ascites tumor cells in vivo

Summary The simultaneous administration of 3-deoxyadenosine N ^l-oxide (3-dANO) and the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or 2-deoxycoformycin (2-dCF) to mice bearing Ehrlich ascites tumor cells resistant to 3-dANO resulted in 80%–90% inhibition of tumor growth in vivo. 3-dANO and 2-dCF increased the survival time of tumor-bearing mice by a factor of 2. In vitro studies showed that the 3-dANO resistant Ehrlich cells initiate the metabolism of 3-dANO by a reduction to 3-deoxyadenosine, which is converted primarily to 3-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3-dATP. By the addition of EHNA or 2-dCF it was possible to block the formation of 3-deoxyinosine, resulting in a profound stimulation in the accumulation of 3-dAtP. The development of resistance to 3-dANO was studied in cell cultures and found to be accompanied by changes in the enzyme activities of the reductase, the adenosine kinase, and the adenosine deaminase.Abbreviations 3-dANO 3-deoxyadenosine N ^l-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate - EHNA erythro-9-(2-hydroxy-3-nonyl) adenine - 2-dCF 2-deoxycoformycin This work was supported by the Danish Medical Research Council, Gerda and Åge Haensch Foundation, Dirktør Åge Henriksens Foundation, P. Carl Petersens Foundation and the Danish Cancer Society     

Studies on the mechanism of cytotoxicity of 3′-deoxyadenosine N1-oxide in different strains of Ehrlich ascites tumor cells

Summary The correlation between the metabolic processing of 3-deoxyadenosine N ¹-oxide (3-dANO) in vitro and its effect on tumor growth in vivo has been investigated in seven different strains of Ehrlich ascites tumor cells.The metabolism of 3-dANO is initiated by reduction to 3-deoxyadenosine (3-dA). This process is the rate-limiting process. The 3-dA does not accumulate, but is converted to 3-deoxyadenosine triphosphate (3-dATP) or 3-deoxyinosine (3-dI). The ratio between 3-dATP and 3-dI inosine corresponds to the ratio between the activities of adenosine kinase and adenosine deaminase in the cell.Two of the cell lines were markedly inhibited by 3-dANO in vivo. In these cells the accumulation of 3-dATP was 1.4–2.2 nmol/h per mg cells, which accounts for the major part of the metabolized 3-dANO. Five of the cell lines were not inhibited by 3-dANO and the formation of 3-dATP was 5–10 times less in these than in the sensitive strains. The low level of 3-dATP is caused primarily by a low ratio between the activities of adenosine kinase and adenosine deaminase, which is 15 time less than in the sensitive cell lines. The rate of reduction of 3-dANO seems to be of minor importance.These results indicate a correlation between the inhibition of tumor growth by 3-dATP and the ability of the cell to accumulate 3-dATP from 3-dANO and show that this conversion is determined solely by the rate of reduction of 3-dANO (3-dANO reductase activity) and the ratio between the activities of adenosine kinase and adenosine deaminase in the cell. Consequently, the estimation of these enzyme activities in cell lysate of a given tumor can be used to predict whether the tumor is susceptible to inhibition by 3-dANO.Abbreviation 3-dANO 3-deoxyadenosine N ¹-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate This work was supported by The Danish Medical Research Council, Gerda and Åge Haensch Fond, Direktor Åge Henriksens Fond, P. Carl Petersens Fond and the Danish Cancer Society     

Potentiation of the antitumor activity of 5-trifluoromethyl-2′-deoxyuridine by the use of depot forms of the parent compound

Summary 5-Trifluoromethyl-2-deoxyuridine (CF₃dUrd), an antitumor agent, is known to be short-lived in human plasma. Since its rapid elimination from the blood-stream seems to have descouraged the clinical evaluation of this drug, we explored the potential use of masked derivatives of CF₃dUrd as depot forms of the parent compound. First, we observed that the toxicity of CF₃dUrd against HeLA cells in culture was 10⁴ times greater for a 24-h treatment as compared with a 1-h treatment at identical concentrations of the drug, which suggests the importance of using a prolonged treatment period. In fact, the divided dosing of CF₃dUrd to L1210-bearing mice was markedly more effective than its single administration. 5-O-Hexanoyl-,N ³-p-butylbenzoyl-, 5-O-benzyloxymethyl-, and 3-O-benzyl-CF₃dUrd were found to be effective in maitaining the CF₃dUrd concentration in plasma. The oral doses of these agents required to achieve 50% growth inhibition (ED₅₀) in mice bearing sarcoma 180 tumors were 19, 34, 10, and 13 mg kg^–1 day^–1, respectively, whereas that of CF₃dUrd was 63 mg kg^–1 day^–1. The ED₅₀ values for these compounds were inversely correlated with the residence time of CF₃dUrd in plasma. The therapeutic indices of these compounds, calculated as the dose producing a 50% inhibition of body-weight gain (IB₅₀) divided by the ED₅₀ value (1.89, 1,21, 1.40, and 2.15, respectively), were significantly higher than that of CF₃dUrd (0.78). Consequently, these depot forms of CF₃dUrd, particularly 3-O-benzyl-CF₃dUrd, are expected to be more useful than the parent compound as antitumor agents.Abbreviations CF₃dUrd 5-trifluoromethyl-2-deoxyuridine - CF₃dUMP 5-trifluoromethyl-2-deoxyuridine-5-monophospate - S180 sarcoma 180 - L1210 L1210 leukemia - k_el elimination rate constant - T1/2 half-life time - AUC area under the curve - ILS increase in life span - TS thymidylade synthase - FdUMP 5-fluoro-2-deoxyuridine-5 monophosphate - FUra 5-fluorouracil     

Antitumor activity of 3′, 5′-diesters of 5-fluoro-2′-deoxyuridine against murine leukemia L1210 cells

Summary Antitumor activity of several 3,5-diesters of 5-fluoro-2-deoxyuridine (FUdR) against L1210 leukemia cells following intraperitoneal administration was examined. Esters of FUdR with aromatic acid or aliphatic acid of longer chain length were markedly active. Their activities, with respect to ILS₃₀, were as much as 100 times that of unesterified FUdR. 3,5-ditoluoyl FUdR also had an improved therapeutic effect: its therapeutic ratio was increased to 8.1, as against 2.0 for FUdR. On the other hand, 3,5-diesters of FUdR with aliphatic acid of shorter chain length do not appear to be as active as FUdR. The relationship between the antitumor activity and plasma levels has also been examined. After 3,5-diacetyl FUdR, which is one of the drug group showing low cytotoxicity, the plasma concentration rapidly decreased to unmeasurable level 3 h after dosing. This tendency is similar to that shown in FUdR. On the other hand, with 3,5-dipalmitoyl FUdR and 3,5-dibenzoyl FUdR, each of which has a marked antitumor effect, plasma concentrations decreased slowly and were maintained for as long as 48 h after dosing. The results show that the cytotoxicity of diesters of FUdR is correlated with the duration of a high plasma level of FUdR.     

Phase II study of 4′-iodo-4′-deoxydoxorubicin in non-resectable non-small-cell lung cancer

A total of 44 patients with previously untreated, non-resectable non-small-cell lung cancer (NSCLC) were treated with 4-iodo-4-deoxydoxorubicin (IDX), which is an analogue of doxorubicin with less cardiotoxicity. Patients received 80 mg/m² i. v. every 3 weeks. Dose reductions were carried out for haematological toxicity. Response was assessed prior to each treatment according to WHO criteria. Among the 43 evaluable patients, 1 (2%; 95% confidence limits, 0–8%) achieved a partial response. Leucocytopenia of WHO grade 3 or 4 occurred in 64% of patients and corresponding thrombocytopenia grade 3 or 4 occurred in 30%. Of the 26 patients who were evaluated by measurements of the left ventricular ejection fraction (LVEF), 4 had a decline in LVEF of more than 15%, and 2 patients developed congestive heart failure. Myocardial biopsies were not done. In conclusion, IDX is not active in NSCLC at the applied dose and on the schedule used. Moreover, it does not seem possible to increase the dose intensity further due to the observed toxicity.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

Pre-clinical and clinical study of QC12, a water-soluble, pro-drug of quercetin

Background:Quercetin is a naturally occurring flavonoid with manybiological activities including inhibition of a number of tyrosine kinases.A phase I, dose-escalation trial of quercetin defined the maximum tolerateddose (MTD) as 1700 mg/m² three weekly, but the vehicle, dimethylsulphoxide (DMSO) is unsuitable for further clinical development ofquercetin. Patients and methods:A water-soluble, pro-drug of quercetin(3(N-carboxymethyl)carbomyl-3,4,5,7-tetrahydroxyflavone), QC12has been synthesised. Six cancer patients received 400 mg of QC12 (equivalentto 298 mg of quercetin), orally on day 1 and intravenously (i.v.) in normalsaline on day 14. Results:Following oral administration of QC12 we were unable todetect QC12 or quercetin in plasma. After i.v. administration, we detectedpeak plasma concentrations of QC12 of 108.7 ± 41.67 µMolar(µM). A two-compartment model with mean t_1/2 of 0.31± 0.27 hours and mean t_1/2 of 0.86 ± 0.78 hoursbest described the concentration-time curves for QC12. The mean AUC was 44.54± 13.0 µM.hour and mean volume of distribution (Vd) of 10.0± 6.2 litres (l). Quercetin was found in all patients following i.v.infusion of QC12, with peak levels of quercetin 19.9 ± 11.8 µM.The relative bioavailability of quercetin was estimated to be20%–25% quercetin released from QC12. Conclusions:QC12 is not orally bioavailable. This water-solublepro-drug warrants further clinical investigation; starting with a formal phaseI, IV, dose-escalation study.     

Phase I/II tolerability/pharmacokinetic study with one-hour intravenous infusion of doxifluiridine (5′-dFUrd) 3 g/m2 VS 5 g/m2 QD x 5 per month

Summary Eighteen patients with advanced solid cancer were treated with daily 5-dFUrd infusions given over 1 h on days 1–5 of a 4-week cycle. Nine patients received 3 g/m² 5-dFUrd daily and another nine patients 5 g/m². One patient on 5 g/m² 5-dFUrd was not fully evaluable for tolerability due to early death (progressive disease) 4 weeks after the first cycle. A total of 48 cycles was given. The gastrointestinal and hematological toxicity was generally mild (grade 1–2). Central neurotoxicity (ataxia, unsteadiness, diplopia, dysarthria, sometimes confusion) was observed in 7 of 8 patients on 5 g/m² 5-dFUrd leading to premature discontinuation of treatment in 3 patients (after 2 cycles). Only 3 of the 9 patients in the 3 g/m² group had slight signs of cerebellopathy. Typically, the reversible neurological side effects started at the end of the 2nd week of a cycle. The serum elimination kinetics of 5-dEUrd and its metabolites 5-FU and 5-dFUH₂ have been investigated in the serum and showed very low intra- and interindividual variations. Peak concentrations of the 5-dFUrd at the end of the infusion approximated 500 mol/l and 1000 mol/l for the 3 g/m² and 5 g/m² group, respectively. The peak of the serum 5-FU was reached at the same time, the ratio 5-FU/5-dFUrd being around 10%. The elimination half-life time for 5-FU was protracted by a factor of 2–3 compared with the direct injection of 5-FU.Monthly infusion of 5-dFUrd 5 mg/m² per day on days 1–5 lead to an unacceptable frequency and degree of neurological toxicity. Similar infusions of 5-dFUrd 3 g/m² per day on days 1–5 were well tolerated.     

Drug distribution and pharmacokinetic/pharmacodynamic relationship of paclitaxel and gemcitabine in patients with non-small-cell lung cancer

Background:Gemcitabine and paclitaxel are two of the mostactive agents in non-small-cell lung cancer (NSCLC), and pharmacologicinvestigation of the combination regimens including these drugs mayoffer a valuable opportunity in treatment optimization. The presentstudy investigates the pharmacokinetics and pharmacodynamics ofpaclitaxel and gemcitabine in chemotherapy-naive patients with advancedNSCLC within a phase I study. Patients and methods:Patients were given i.v. paclitaxel 100 mg/m² byone-hour infusion followed by gemcitabine 1500, 1750 and 2000mg/m² by 30-min administration. Plasma levels of paclitaxel,gemcitabine and its metabolite 2,2-difluorodeoxyuridine(dFdU) were determined by high-performance liquid chromatography (HPLC).Concentration-time curves were modeled by compartmental andnon-compartmental methods and pharmacokinetic/pharmacodynamic (PK/PD)relationships were fitted according to a sigmoid maximum effect(E_max) model. Results:Paclitaxelpharmacokinetics did not change as a result of dosage escalation ofgemcitabine from 1500 to 2000 mg/m². A nonproportionalincrease in gemcitabine peak plasma levels (C_max, from 18.56± 4.94 to 40.85 ± 14.85 µg/ml) and area under theplasma concentration-time curve (AUC, from 9.99 ± 2.75 to 25.01± 9.87 h·µg/ml) at 1500 and 2000mg/m², respectively, was observed, suggesting the occurrenceof saturation kinetics at higher doses. A significant relationshipbetween neutropenia and time of paclitaxel plasma levels 0.05µmol/l was observed, with a predicted time of 10.4 h to decreasecell count by 50%. A correlation was also observed betweenpercentage reduction of platelet count and gemcitabine C_max,with a predicted effective concentration to induce a 50% decreaseof 14.3 µg/ml. Conclusion:This study demonstratesthe lack of interaction between drugs, the nonproportionalpharmacokinetics of gemcitabine at higher doses and the E_maxrelationship of paclitaxel and gemcitabine with neutrophil and plateletcounts, respectively. In addition, gemcitabine 1500 mg/m² isthe recommended dosage in combination with paclitaxel 100mg/m² for future phase II studies, due to its predictablekinetic behaviour and less severe thrombocytopenia than expected.     

Doxifluridine (5′-dFUrd) in patients with advanced colorectal carcinoma

Summary Infusion of 5-dFUrd (2.0–3.0 g/m² over 1 h on days 1–5 every 3rd week) resulted in one partial response in 21 patients with advanced and progressing colorectal cancer. No patient had received chemotherapy before the 5-dFUrd trial. Hematological and gastrointestinal toxicity were generally mild. In 4 patients peripheral neurotoxicity was diagnosed during treatment, whereas transient cerebellopathy was observed in one. Cardiac side effects (repeated angina pectoris following 5-dFUrd infusion) led to discontinuation of treatment after two courses in ont superior to conventional 5-FU treatment in colorectal cancer. Neurological and cardiac side effects are rare but may be a problem in individual patients.     

Saturation of 2′, 2′-difluorodeoxycytidine 5′-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine

Summary The plasma and cellular pharmacology of 2, 2-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m² per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange highpressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m² per 30 min, during which steady-state dFdC levels of 15–20 M were achieved in plasma. A comparison of patients infused with 800 mg/m² over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10–15 M dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.Abbreviations ara-C I--d-arabinosylcytosine - ara-C _ss steady-state concentration of ara-C - ara-CTP 5-triphosphate of ara-C - dFdC 2, 2-difluorodeoxycytidine, Gemcitabine - dFdC _ss steady-state concentration of dFdC - dFdCTP 5-triphosphate of dFdC Supported in part by grants CA28596 and CA32839 from the National Cancer Institute, Department of Health and Human Services, and by grant CH-130 from the American Cancer Society     

Reduced cardiotoxicity and increased cytotoxicity in a novel anthracycline analogue, 4′-amino-3′-hydroxy-doxorubicin

Summary The acute and chronic cardiotoxicity and cytotoxicity of the novel doxorubicin (DXR) derivative 4-amino-3-hydroxy-DXR were compared with those of 4-deoxy-DXR and DXR. In the acute cardiotoxicity study, the ECG and hemodynamic changes recorded in anesthetized rats that had been treated i.v. with 10 mg/kg 4-amino-3-hydroxy-DXR or 8.6 mg/kg 4-deoxy-DXR were significantly less severe than those caused by 13 mg/kg DXR. In the chronic cardiotoxicity study, rats received 3 weekly i.v. injections of 3 mg/kg DXR, 3 mg/kg 4-amino-3-hydroxy-DXR, or 2 mg/kg 4-deoxy-DXR during the first 14 days of the study and were observed for an additional 35-day period. DXR induced severe cardiomyopathy that was characterized by ECG changes in vivo (ST-segment widening and T-wave flattening) and by impairment of the contractile responses (F _max,±dF/dt _max) to adrenaline of hearts isolated from treated animals. 4-Deoxy-DXR caused a progressive enlargement of the ST segment in vivo and a significant impairment of the-dF/dt _max value in vitro, which were less severe than those produced by DXR. The least cardiotoxic drug was 4-amino-3-hydroxy-DXR, which induced minor ECG changes without causing significant alterations in the contractile responses of isolated hearts to adrenaline. On the basis of the drug concentration required to inhibit 50% of the colony formation (IC₅₀) of cell lines in vitro, 4-amino-3-hydroxy-DXR was less active than 4-deoxy-DXR but at least twice as active as DXR against human cancer and murine transformed cell lines. These data indicate that 4-amino-3-hydroxy-DXR is significantly less cardiotoxic and more cytotoxic than DXR.     

A feasibility study evaluating docetaxel-based sequential and combination regimens in the adjuvant therapy of node-positive breast cancer

Background and purpose:Docetaxel is an active agent in thetreatment of metastatic breast cancer. We evaluated the feasibility ofdocetaxel-based sequential and combination regimens as adjuvant therapies forpatients with node-positive breast cancer. Patients and methods:Three consecutive groups of patients withnode-positive breast cancer or locally-advanced disease, aged 70 years,received one of the following regimens: a) sequential A T CMF:doxorubicin 75 mg/m² q 3 weeks × 3, followed by docetaxel 100mg/m² q 3 weeks × 3, followed by i.v. CMF days 1 + 8 q 4weeks × 3; b) sequential accelerated A T CMF: A and T wereadministered at the same doses q 2 weeks; c) combination therapy: doxorubicin50 mg/m² + docetaxel 75 mg/m² q 3 weeks × 4,followed by CMF × 4. When indicated, radiotherapy was administeredduring or after CMF, and tamoxifen started after the end of CMF. Results:Seventy-nine patients have been treated. Median age was48 years. A 30% rate of early treatment discontinuation was observedin patients receiving the sequential accelerated therapy (23% duringA T), due principally to severe skin toxicity. Median relativedose-intensity was 100% in the three treatment arms. The incidence ofG3–G4 major toxicities by treated patients, was as follows: skintoxicity a: 5%; b: 27%; c: 0%; stomatitis a: 20%;b: 20%; c: 3%. The incidence of neutropenic fever was a:30%; b: 13%; c: 48%. After a median follow-up of 18months, no late toxicity has been reported. Conclusions:The accelerated sequential A T CMFtreatment is not feasible due to an excess of skin toxicity. The sequentialnon accelerated and the combination regimens are feasible and under evaluationin a phase III trial of adjuvant therapy.     

Similar changes in cardiac morphology and DNA synthesis induced by doxorubicin and 4′-epi-doxorubicin

Summary Doxorubicin is an antineoplastic agent whose clinical administration is limited by dose-dependent irreversible cardiomyopathy. Doxorubicin inhibits the rate of DNA synthesis in cultured rat myocardial cells after 1 h incubation with 16M, as is demonstrated by a decreased incorporation of [methyl-³ H]thymidine. An analogue of doxorubicin, 4-epi-doxorubicin, also inhibits the rate of DNA synthesis within 1 h after treatment with 16M, to the same extent as doxorubicin-treatment of myocardial cells. Furthermore, similarity between doxorubicin and 4-epi-doxorubicin in their effect on the myocardial thymidylate pool was also demonstrated by a significantly decreased incorporation of total [methyl-³ H]thymidine. The effect of doxorubicin on the rate of DNA synthesis in cultured rat skeletal muscle cells treated for 1 h with 16M was quantitatively the same as in myocardial cells. Light microscopy of doxorubicin-and 4-epidoxorubicin-treated myocardial cells and doxorubicin-treated skeletal muscle cells showed distinct nucleolar fragmentation and revealed no differences between the two drugs in their effect on either myocardial or skeletal muscle cells. Electron microscopy of myocardial cells following doxorubicin treatment showed increased nuclear pleomorphism and invaginations, along with a striking and distinctive clumping of nuclear chromatin. Furthermore, an apparent high density of the mitochondria due to an increased matrix volume and a concomitant decrease in the intermembrane compartment were observed. The results of this study indicate that doxorubicin-induced inhibition of cardiac DNA synthesis in cultured myocardial cells is nonpredictive of cardiotoxicity. The mechanism is at least bimodal, and the apparent minor toxicity of 4-epi-doxorubicin compared with that of doxorubicin in clinical trials cannot be distinguished by a difference in the inhibition of DNA synthesis in the rat heart.     

Antitumor effects of IST-622, a novel synthetic derivative of chartreusin,against murine and human tumor lines following oral administration

The antitumor effects of 6-O-(3-ethoxypropionyl)-3,4-O-exo-benzylidenechartreusin (IST-622), a new synthetic derivative of chartreusin (CT), were investigated. Following oral administration, IST-622 showed marked antitumor effects against various mouse tumors such as P388 and L 1210 leukemias, B 16 melanoma, Lewis lung carcinoma, Colon 26 and Colon 38 adenocarcinomas, and M5076 reticulum-cell sarcoma. The best antitumor effects were obtained by five intermittent treatments given every 4 days. In addition, IST-622 showed a significant growth-inhibitory effect against two human tumor xenografts, a large-cell lung cancer (Lu-116) and a gastric adenocarcinoma (St-4), among the seven lines tested. IST-622, which was rapidly metabolized into 3,4-O-exo-benzylidenechartreusin (A-132) and not into CT in vivo or in culture medium, exhibited remarkable growth-inhibitory activity against P388 leukemia in vitro, its 50% growth-inhibitory concentration (IC₅₀) being over 20-fold lower than that of CT. IST-622 showed an in vivo antitumor effect superior to that of authentic A-132, possibly resulting from a higher absorption ratio of IST-622 through the gastrointestinal tract. IST-622 is now under clinical phase I study in Japan.     

Toxicity and metabolism of 3′-deoxyadenosine N1-oxide in mice and Ehrlich ascites tumor cells

Summary The toxic effect of 3-deoxyadenosine N¹-oxide (3-dANO) on mice, on their different organs, and on Ehrlich ascites tumor cells was studied. In both healthy and tumour-bearing animals, the lethal dose for 10% of the mice receiving i. p. injections (LD₁₀) of 3-dANO was estimated to be about 300 mg/kg×4 days in one mouse strain (Theiller). In another mouse strain (NMRI), we obtained a markedly higher LD₁₀ value (675 mg/kg×5 days). At nonlethal doses (250 mg/kg×4 days), we observed reversible neurological symptoms on days 4–12 after treatment, but no macroscopical or microscopical changes was detected in the brain, heart, thymus, lung, lymph node, spleen, liver, kidney, bone marrow, or gastrointestinal tract. At doses of 450 mg/kg×4 days, severe neurological symptoms were observed, and atony of the gastrointestinal canal and damage to the kidney and liver were registered. Even at doses that were lethal to the mice, no histopathological change was observed in the bone marrow or in the gastrointestinal canal. Pharmacokinetics studies showed that after the i.p. injection of 3-dANO, the maximal plasma concentration was reached after 10 min, after which it declined showing a half-life of about 40 min. A transient accumulation of 3-deoxyadenosine triphosphate (3-dATP) was observed within 24 h in the liver and kidney, with the maximal concentration being reached after about 2–3 h. 3-dANO was excreted partly as the unchanged substance and partly as the metabolite 3-deoxyinosine within 24 h. Flow-cytometric DNA analysis of Ehrlich tumor cells treated either in vitro or in vivo with 3-dANO revealed no therapy-induced change in the cell-cycle perturbations, which indicates that cells were randomly killed during all phases of the cycle.Abbreviations 3-dANO 3-deoxyadenosine N¹-oxide - 3-dA 3-deoxyadenosine - 3-dI 3-deoxyinosine - 3-dATP 3-deoxyadenosine triphosphate This work was supported by grants from the Director Ib Hendriksens Foundation and the P. Carl Petersens Foundation     

In vivo antitumor activity by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone in a solid human carcinoma xenograft model

Previously we have shown that 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone (DMC), which is isolated from the buds of Cleistocalyx operculatus, significantly inhibits the growth of human liver cancer SMMC-7721 cells and is able to induce apoptosis of SMMC-7721 cells in vitro. Here we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft mouse model using human liver cancer SMMC-7721 cells. The average tumor weights in the control group and in mice injected with 150 mg/kg DMC were 1.42±0.11 g and 0.59±0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated an aneuploid peak (representing 33.60±0.80% of the total in mice injected with 150 mg/kg DMC). To our knowledge, this is the first time that chalcone compounds have been applied to a human tumor xenograft model.