Multivesicular liposomes containing bleomycin for subcutaneous administration

Summary Optimal cancer treatment with cell-cycle-specific agents requires maintenance of a cytotoxic drug level for a prolonged period. We explored the use of multivesicular liposomes as a slow-release depot of bleomycin for systemic administration via the s. c. route. The average volume-adjusted liposome size was 19.1 m, the half-life of leakage in human plasma was 32.1 h, and the half-life of s. c. liposomal bleomycin was 31.8 h. When tested against the s. c. B-16 melanoma model in BDF₁ mice, the therapeutic index of single-dose bleomycin given s.c. was significantly improved when the drug was encapsulated in multivesicular liposomes. The efficacy was improved as assessed by both inhibition of tumor growth and increased life span, and the toxicity appeared to be decreased.Supported by grants CH-484 (American Cancer Society) and CA-01082 (National Cancer Institute, PHS), and by the Clayton Foundation for Research, California Division, and the University of California Cancer Research Coordinating Committee     

Clinical pharmacology of cytarabine in patients with acute myeloid leukemia: a Cancer and Leukemia Group B study

The pharmacokinetics of cytarabine (ara-C) were determined in 265 patients with acute myeloid leukemia (AML) receiving ara-C (200 mg/m² per day for 7 days as a continuous infusion) and daunorubicin during induction therapy. The mean (standard deviation) ara-C concentration at steady-state (Css) and systemic clearance (Cl) were 0.30 (0.13) M and 134 (71) l/h per m² respectively. Males had a significantly faster ara-C Cl (139 vs 131 l/h per m²,P=0.025) than females. Significant correlations were noted between ara-C Cl and the pretreatment, peripheral white blood cell count (P=0.005) and pretreatment blast count (P=0.020). No significant differences in ara-C Css or Cl were noted in patients achieving complete remission compared with those failing therapy (P=0.315,P=0.344, respectively). No significant correlations were observed between ara-C pharmacokinetic parameters and several indices of patient toxicity. Our findings indicate that variability in ara-C disposition in plasma at this dosage level does not correlate with remission status or toxicity in patients with AML receiving initial induction therapy with ara-C and daunorubicin.This study was supported in part by grants from the National Cancer Institute (CA-03927, CA-37027, CA-59518, CA-47577, CA-32291, CA-07968, CA-16450, CA-16450, CA-26806, CA-47545, CA-12197, and CA-41287), the Upjohn Company, and the Coleman Leukemia Research Fund     

Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-β-d-arabinofuranosyluracil

Summary Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200–1000 m) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 m ara-C following the exposure of cells to ara-U (1mm) resulted in 4.5 pmol ara-C DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 m ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10–100 m) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.Abbreviations ara-C 1--d-arabinofuranosylcytosine - ara-U 1--d-arabinofuranosyluracil - ara-CTP 1--d-arabinofuranosylcytidine triphosphate - HiDAC high-dose ara-C - HiCAU high concentrations of ara-U - dCTP deoxycytidine triphosphate - HiDAU high-dose ara-U - FiTC Fluoroisothiocyanate - dUDP deoxyuridine diphosphate - dUTP deoxyuridine triphosphate - dTTP thymidine triphosphate - BrdUrd bromodeoxyuridine - dCyd kinase deoxycytidine kinase Supported in part by grant CH-35H from the American Cancer Society, by Public Health Service grant CA-12197 from the National Cancer Institute, National Institutes of Health, and by the Gaston Cancer Society     

Phenotypic analysis of 1-B-d-arabinofuranosylcytosine deamination in patients treated with high doses and correlation with response

Summary Two phenotypes for 1-B-d-arabinofuranosylcytosine (ara-C) deamination corresponding to a ratio of distribution for slow (ratio, 14) vs fast (ratio, >14) deaminators of 70%30%, have been determined on the basis of studies on plasma ratios of 1-B-d-arabinofuranosyluracil/ara-C (ara-U/ara-C) in 56 subjects treated with high-dose ara-C (3 g/m² infused i.v. over 3 h). A positive correlation of age with the concentration of ara-U was observed. In a subgroup of 36 patients with leukemia, the ara-U/ara-C pattern was similar to that observed for all 56 subjects. In these leukemic patients, who were treated with combinations of ara-C plus other conventional agents, a tendency toward a positive response (complete response +partial response) was found for those showing low ara-U/ara-C ratios (slow deaminators). The phenotypic effect of deamination in acute leukemia needs to be evaluated prospectively.This work was supported by the Don Monti Memorial Research Foundation     

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     

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     

Plasma and cerebrospinal fluid pharmacokinetics of cytosine arabinoside in dogs

Summary Cytosine arabinoside (ara-C) is a component of many protocols for the treatment of CNS (central nervous system) leukemia and lymphoma in humans and dogs. It is also used for the prophylaxis of CNS metastasis in acute lymphoblastic leukemia. Although ara-C enters the cerebrospinal fluid (CSF) of human cancer patients after i.v. administration, it is unclear whether a similar CNS distribution occurs in humans whose blood-brain barrier has not been compromised by invasive disease. No information on the penetration of ara-C into the CSF in dogs is available. We studied the plasma and CSF pharmacokinetics of 600 mg/m² ara-C in ten healthy male dogs after its administration as a rapid i.v. bolus (six dogs) or as a 12-h i.v. infusion (four dogs). Ara-C concentration in blood and CSF samples was determined by high-performance liquid chromatography (HPLC). After an i.v. bolus of ara-C, the mean plasma distribution half-life was 7.1±4.5 min and the mean elimination half-life was 69±28 min. The mean plasma clearance was 227±125 ml min^–1 m^–2. The peak concentration of ara-C in the CSF was 29±11 m, which occurred at 57±13 min after the ara-C bolus. The CSF elimination half-life was 113±26 min. During a 12-h infusion of ara-C (50 mg m^–2 h^–1), the plasma steady-state concentration was 14.1±4.2 m, the CSF steady-state concentration was 8.3±1.1 m, and the CSF: plasma ratio was 0.62±0.14. The plasma eleimination half-life was 64±19 min and the plasma clearance was 214±69 ml min^–1 m^–2. The CSF elimination half-life was 165±28 min. No clinically significant toxicity was observed over a 21-day period following drug administration in either of the treatment groups. Our data indicate that ara-C crosses the blood-brain barrier in normal dogs and that i.v. administration of this drug has potential as a treatment modality for neoplasia involving the CNS.Supported by the Canine Disease Research Fund and in part by the Elsa U. Pardee Foundation     

Relation between age and clearance rate of nine investigational anticancer drugs from phase I pharmacokinetic data

Aging influences the disposition and effects of several classes of drugs. Although drug clearance rate is correlated with toxicity for many anticancer drugs, few data have been published concerning the relationship of aging and clearance of chemotherapy. This study was performed to identify any relationship between age and clearance rate for anticancer drugs in phase I trials at the Johns Hopkins Oncology Center. In a retrospective study, we examined the clinical and pharmacokinetic data for 344 adults (aged 21–77 years) who received 9 phase I drugs with linear clearance in 13 clinical trials. We sought correlations between age and clearance for each drug and for the whole group. Data available for 9 of the 13 trials were used to compare age (<65 or >65 years) versus dose delivered [< the maximum tolerated dose (MTD) vs the MTD] or toxicity (< grade 3 vs grade 3). Of 344 patients, 81 (23.5%) were >65 years old, 34 (9.9%) were 70 years old, and 5 (1.5%) were 75 years old. There was no significant correlation between drug clearance and age for individual drugs or the group as a whole. There was no significant difference between patients of the older and younger age groups with regard to dose or toxicity. Although only a small number of patients aged 75 years were treated, our results suggest that the elderly do not experience greater toxicity even when treated at doses comparable with those given younger patients and should not be excluded from phase I trials on the basis of age. As the population of the United States ages, more elderly patients will be candidates for chemotherapy. A more thorough examination of the relationships between age, clearance rate, and toxicity can be accomplished as active drugs enter phase II/III studies.Supported in part by Public Health Service grants N01CM07302 and RR00035-33 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, and by a grant from the American Federation for Aging Research/Hartford Foundation     

Cellular pharmacology ofN 4-hexadecyl-1-β-D-arabinofuranosylcytosine in the human leukemic cell lines K-562 and U-937

The mechanisms of cytotoxicity, cellular drug uptake, intracellular drug distribution, cellular pharmacokinetics, formation of arabinofuranosylcytosine triphosphate (ara-CTP), and DNA incorporation ofN ⁴-hexadecyl-1--d-arabinofuranosylcytosine (NHAC), a new lipophilic derivative of arabinofuranosylcytosine (ara-C) formulated in small unilamellar liposomes, were determined in vitro in the human leukemic cell lines K-562 and U-937. Furthermore, the induction of erythroid differentiation by NHAC was tested in K-562 cells. The cytotoxicity of NHAC in both cell lines was not influenced by the deoxycytidine (dCyd) concentration or the presence of the nucleoside-transport-blocking agent dipyridamole as demonstrated in coincubations with dCyd and/or dipyridamole, where-as in contrast, the cytotoxicity of ara-C was decreased additively by both drugs. As compared with ara-C, the uptake of NHAC displayed up to 16- and 5-fold increases in K-562 and U-937 cells, respectively, depending on the drug concentration. Studies of the drug distribution and pharmacokinetics of NHAC revealed a depot effect for NHAC in the cell membranes, resulting in half-lives 2.6 and 1.4 times longer than those of ara-C in the two cell lines. The ara-CTP concentrations derived from NHAC were 150- and 75-fold lower at a drug concentration of 1 M in K-562 and U-937 cells, respectively. The DNA incorporation of the drugs observed after incubation with 2 M NHAC was 60- and 30-fold lower as compared with that seen at 2 M ara-C in the two cell lines. Furthermore, NHAC was capable of inducing irreversible erythroid differentiation to a maximum of only 22% of K-562 cells, where-as ara-C induced differentiation at a drug concentration 100-fold lower in 50% of the cells. These results indicate a mechanism of cytotoxicity for NHAC that is independent of the nucleoside transport mechanism and the phosphorylation pathway and suggest that the mechanisms of action of NHAC are significantly different from those of ara-C. Therefore, NHAC might be used for the treatment of ara-C-resistant malignancies.This work was supported by the Sassella Foundation, Stiftung für experimentelle Krebsforschung, Stiftung für Krebsbekämpfung, and the Swiss National Science Foundation (grant 32-29979.90)     

Short-termcis-diamminedichloroplatinum(II) accumulation in sensitive and resistant human ovarian carcinoma cells

Summary We examined the short-term accumulation of cisplatin (DDP) in sensitive 2008 human ovarian carcinoma cells and in a 2- to 3-fold DDP-resistant and accumulation-deficient variant. During the 1st min of exposure to 500 M DDP, sensitive cells accumulated platinum at a rate of 187±63 pmol/mg protein per min, whereas resistant cells accumulated platinum at 123±85 pmol/mg protein per min, a rate that was 66% that of sensitive cells. From 2–10 min of exposure, sensitive and resistant cells accumulated the drug at rates of 51.4±21.5 and 34.0±9.70 pmol/mg protein per min, respectively. In resistant cells, this rate again represented 66% that of sensitive cells. For each cell line, the DDP accumulation was 3.6 times faster during the 1st min that it was over 2–10 min. Initial DDP accumulation was linear with drug concentration in each cell line. Efflux measurements were made over a 50-min period after a 10-min load in 500 M DDP. The loss of platinum was biphasic in each cell line, with an initial, rapidly effluxing component being lost within 10 min in each cell line. The rate constant for loss of platinum from this rapidly effluxing pool, measured after a 10-min loading period in 500 M DDP, was 0.67±0.09 s^–1 in sensitive cells and 1.03±0.15 s^–1 (a 53% increase) in resistant cells. Between 5 and 50 min of an accumulation time course in 500 M DDP, the size of the rapidly effluxing platinum pool remained relatively constant in each cell line, with the major contribution to the increase in total platinum over time coming from growth of the slowly effluxing platinum pool. We conclude that diminished retention of platinum in the rapidly effluxing pool of resistant cells in a major determinant of decreased DDP accumulation in these cells.Abbreviations DDP cis-diamminedichloroplatinum(II) - DEP cis-dichloro(ethylenediamine)platinum(II) - RPMI Roswell Park Memorial Institute - PBS phosphate-buffered saline, consisting of (per liter) 8 g NaCl, 0.2 g KCl, 1.15 g Na₂HPO₄, and 0.2 g KH₂PO₄ Supported by a grant from Bristol-Myers Co., grants CA-35309 and CA-23100 from the National Institutes of Health, and grants CH-368 and CH-417 from the American Cancer Society. This work was conducted in part by the Clayton Foundation for Research, California Division. Drs. Mann, Andrews, and Howell are Clayton Foundation investigators     

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy

Summary The active 5-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose of ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.Abbreviations ara-C 9--d-arabinofuranosylcytosine - ara-CTP 9--d-arabinofuranosylcytosine 5-triphosphate - AUC area under the concentration-time curve - AUC_p inerease in the AUC caused by perturbation - CLL chronic lymphocytic leukemia - dCyd kinase deoxycytidine kinase - F-ara-A 9--d-arabinofuranosyl-2-fluoroadenine 5 - fludarabine F-ara-AMP, 9--d-arabinofuranosyl-2-fluoroadenine 5-monophosphate - F-ara-ATP 9--d-arabinofuranosyl-2-fluoroadenine 5-triphosphate - t _1/2 half-life of elimination This work was supported in part by grants CA32839, CA46452, CA53311, and CA57629 from the National Cancer Institute, Department of Health and Human Services, by the German Research Association (DFG), and by a contract from Berlex Laboratories, Inc.     

Mitoxantrone-induced DNA damage in leukemia cells is enhanced by treatment with high-dose arabinosylcytosine

Summary In a phase II study, patients with chronic myelogenous leukemia in blast crisis (CML-BC) were treated with intravenous (IV) mitoxantrone (5 mg/m²) per day given over 30 min x 5 days and high-dose arabinosylcytosine (ara-C) (3 g/m² IV q 12 h x 6).The effect of this treatment on DNA damage was studied in the leukemia cells of four patients using the alkaline elution technique modified to measure DNA in unlabeled human cells. A fluorescence assay using Hoechst 33258 dye was applied for the determination of eluted DNA.After a single infusion of mitoxantrone, neither frank nor protein-associated single-strand breaks (SSB) were observed.Even repeated treatment with mitoxantrone on 3 consecutive days did not induce significant SSB. However, after the combined sequential infusion of ara-C and mitoxantrone the DNA elution pattern changed, showing significant DNA damage.SSB remained apparent after 24 h and increased with subsequent doses of ara-C and mitoxantrone. Studies of other patients treated with ara-C alone did not reveal significant SSB (n=5).Following mitoxantrone infusion the median peak concentrations of intracellular ara-CTP (the triphosphate of ara-C) exceeded 900 M,a value greater than that observed in CML-BC patients receiving ara-C alone (230 M, n=15, P<0.02). The present study shows the applicability of the alkaline elution method for the assay of DNA damage in vivo. The enhanced DNA damage after combined treatment with mitoxantrone and high-dose ara-C suggests a synergistic drug effect.Abbreviations ara-C 1--D-arabinosylcytosine - ara-CTP the triphosphate of ara-C - SSB single-strand breaks - SSF strand scission factor - PK proteinase K - CML chronic myelogenous leukemia - BC blast crisis - AML acute myelogenous leukemia Supported by grants CA28596, CA32839, and CA23270 from the National Cancer Institute, Department of Health and Human Services     

A Phase I Trial of Continuously Infused Intratumoral Bleomycin for the Treatment of Recurrent Glioblastoma Multiforme

Intratumoral (IT) chemotherapy has theoretical advantages in the treatment of brain tumors. The blood–brain barrier is not a factor in drug delivery, and large concentrations of drug can be instilled in the tumor with little systemic toxicity. Bleomycin has activity against gliomas and is a cell cycle selective agent whose efficacy should be enhanced by continuous infusion. We performed a phase I trial to test the feasibility of IT chemotherapy using a refillable, sustained release device, and to determine the maximum tolerable dose of IT bleomycin. The study was an open-ended dose escalation study. A modified Ommaya reservoir (containing a semipermeable membrane) was implanted with the delivery tube in the center of the tumor. Groups of three patients with recurrent glioblastoma multiforme were entered at progressively higher dose levels of bleomycin. The study closed when all patients at a given starting dose level developed toxicity. Nine patients received doses ranging from 5 to 34U/wk; the median total cumulative dose was 195 U. No dose limiting systemic toxicity was detected. Neurologic toxicity occurred only at doses above 16U/wk. We conclude that continuously infused IT bleomycin is well tolerated; the MTD (and recommended dose for a phase II efficacy trial) of IT bleomycin is 16U/wk.     

Selectivity of the anthracyclines for negatively charged model membranes: role of the amino group

Summary The equilibrium-binding affinities of six adriamycin analogues and four daunomycin derivatives for negatively charged dimyristoyl phosphatidylcholine/dimyristoyl phosphatidic acid (DMPC/DMPA) small unilamellar vesicles are compared with values for electroneutral DMPC liposomes. Binding of the daunomycin series to negatively charged dimyristoyl phosphatidyl glycerol (DMPG) vesicles was also examined. Under physiological conditions of pH and ionic strength, substitution of the amino group of adriamycin or daunomycin resulted in a reduced affinity for negatively charged bilayers, even if the substituent enhanced the degree of ionization of the amine. Decreasing the ionic strength increases the binding affinity for acidic membranes but decreases the drug affinity for neutral membranes. We propose that the electrostatic bond of the phosphate-amino group that has been shown to exist between anthracyclines and phosphatidic acid is sterically destabilized by substitution of the amino group. The results are consistent with a mode of anthracycline binding to negatively charged membranes which is driven by hydrophobic and electrostatic considerations but is destabilized by steric bulk at the amino group. THe data also provide insight into the design of new anthracyclines with high membrane affinities and reduced uptake; such directed interaction with plasma membranes may enhance antineoplastic potential while reducing cardiac toxicity.Abbreviations DMPC l--dimyristoyl phosphatidylcholine - DMPG L--dimyristoyl phosphatidyl glycerol - DMPA l--dimyristoyl phosphatidic acid - TLC thin-layer chromatography - PBS phosphate-buffered saline containing 8 mM Na₂HPO₄, 1 mM KH₂PO₄ and the specified amount of KCl(pH 7.4) - T_m gel to liquid-crystalline phase transition temperature - DMPG l--dimyristoyl phosphatidyl glycerol - SUVs small unilamellar vesicles This research was supported in part by U. S. Public Health Service Grants CA-02817, CA-44729, and CA-01088 from the National Cancer Institute and by a grant from the American Cancer Society (CH-392).This research was supported in part by U. S. Public Health Service Grants CA-02817, CA-44729, and CA-01088 from the National Cancer Institute and by a grant from the American Cancer Society (CH-392).     

Clinical pharmacology of N4-palmitoyl-1-β-d-arabinofuranosylcytosine in patients with hematologic malignancies

Summary The pharmacokinetics of oral N⁴-palmitoyl-1--d-arabinofuranosylcytosine (PLAC), a lipophilic and deaminase-resistant derivative of 1--d-arabinofuranosylcytosine (ara-C), were determined in patients with hematologic malignancies. The concentration of ara-C and 1--d-arabinofuranosyluracil (ara-U), metabolites of PLAC, were measured by radioimmunoassay and gas chromatography-mass spectrometry-mass fragmentography, respectively. The concentration of PLAC was determined by measuring ara-C, which was derived from PLAC by hydrolyzation. In six patients given an oral bolus of PLAC (300 mg/m²), the plasma-disappearance curve of PLAC corresponded to a one-compartment open model, including first-order absorption. The peak plasma level was 22.9±6.4 ng/ml, and the predicted time to reach the peak level was 2.5±1.0 h. The elimination half-life was 3.8±2.7 h. The plasma ara-C level increased slowly to 6.9 ng/ml during the 1st 2–3 h after administration and remained over 1.0 ng/ml for 12 h. Plasma ara-U was detectable for at least 24 h, with a peak concentration of 376 ng/ml at 6 h. Urinary PLAC excretion was below the limit of detection (5 ng/ml) in all cases. Prolonged urinary ara-C and ara-U excretion was detected, but the total recovery rate was low (6.7% in 24 h) and varied between patients. In spite of the lipophilic nature of the drug, the PLAC concentration in the cerebrospinal fluid, measured at 3 or 6 h, was below the limit of detection in all four patients with no meningeal involvement. This study showed low but persistent levels of PLAC in plasma and tissues, with a continuous release of small amounts of ara-C, which demonstrated antitumor activity in patients with hematologic malignancies.This study was supported in part by Grants-in-Aid from the Ministry of Health and Welfare (62-18 and 63-3), Japan     

In vitro screening of crude extracts and pure metabolites obtained from marine invertebrates for the treatment of breast cancer

Summary A total of 15 samples (crude extracts and pure secondary metabolites) obtained from marine invertebrates collected from the offshore waters of British Columbia, Papua New Guinea, and Sri Lanka have previously been shown to exert cytotoxic activity in the in vitro L1210 leukemic bioassay. We screened these metabolites for in vitro cytotoxic activity against the drug-sensitive breast-tumor cell lines MCF-7, T-47D, ZR-75-1, and MDA-MB-231; the multidrug-resistant and P-glycoprotein (Pgp)-positive breast-tumor cell lines MCF-7 Ad^r and MDA-Al^r; and normal and malignant human breast epithelial cells (HBEC) in primary culture. Eight samples exhibited significant [drug concentration resulting in a 50% decrease in cell growth as compared with controls (ED₅₀),<25 g/ml] dose-dependent cytotoxicity against the drug-sensitive cell lines; the ED₅₀ values were as low as 0.004 g/ml. Five of the eight samples exhibited significant cytotoxicity against the multidrug-resistant cell lines; the ED₅₀ values were as low as 0.0006 g/ml. Incubation of MCF-7 Ad^r cells with varying concentrations of compounds in the presence of Adriamycin demonstrated that none of the compounds tested interfered with Pgp function. Results obtained using HBEC in primary culture showed a wide range of chemosensitivities for a given drug against tissue taken from different patients, demonstrating the uniqueness of the response of different individuals to chemotherapy.This work was supported by grants from the National Cancer Institute of Canada and the B. C. Health Research Foundation, and by funds donated by Ms. J. Harowitz     

A phase II study of bisantrene in malignant lymphomas

Summary Forty evaluable patients with malignant lymphoma (ML) were treated with bisantrene at a dose of 260 mg/m² every 3 weeks (18 patients) or 208 mg/m² every 3 weeks (22 patients). The initial dose rate was determined on the basis of expected myelosuppression. Patients were heavily pretreated and had advanced disease (92% had stage IV). The overall response rate was 10% and included 1 partial response (PR) in 17 patients with Hodgkin's disease (HD), 1 PR and 1 complete response (CR) in 5 patients with favorable histology in non-Hodgkin's lymphoma (NHL), and 1 PR in 18 patients with unfavorable histology in NHL. Neutropenia (WBC3000 cells/l) was the most common toxicity, occurring in 50% of patients. Phlebitis was a common side effect in patients treated with bisantrene administered by way of peripheral veins. Bisantrene has limited activity in heavily pretreated patients with HD or unfavorable histology in NHL. The role of bisantrene for treatment of NHL with favorable histology or for treatment at an earlier point in the natural history of ML is unknown.This investigation was supported in part by the following PHS Cooperative Agreement grant numbers awarded by the National Cancer Institute, DHHS: CA-13612, CA-22411, CA-13238, CA-04915, CA-20319, CA-28862, CA-12644, CA-27057, CA-21116, CA-03389, CA-22433, CA-12014 and CA-32102     

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.     

Intrathecal administration of topotecan in nonhuman primates

The cerebrospinal fluid (CSF) pharmacokinetics of topotecan were studied in a nonhuman primate model following intraventricular administration of 0.1 mg. Lactone and total drug concentrations were measured using a reverse-phase HPLC method with fluorescence detection. The mean peak concentrations of lactone and total drug in ventricular CSF were 83±18 M and 88±25 M, respectively. CSF drug elimination of the lactone was bi-exponential with a terminal half-life of 1.3 h. The mean clearance from ventricular CSF was 0.075 ml/min for the lactone and 0.043 ml/min for total drug. The ventricular CSF drug exposure (AUC) to lactone was 450-fold greater following intraventricular administration of 0.1 mg topotecan than after systemic intravenous administration of a 40-fold higher dose (10 mg/m²). Peak lumbar concentrations (n=1), which occurred 2 h after intraventricular drug administration, were 0.98 M and 2.95 M for the lactone and total drug, respectively. A transient CSF pleocytosis was observed in one animal following intraventricular topotecan administration and in one animal following intralumbar topotecan administration. No other acute or chronic neurologic or systemic toxicities were observed following a single intraventricular dose or weekly (×4) intralumbar topotecan. Compared with systemic topotecan, intrathecal administration provided a significant pharmacokinetic advantage in terms of CSF drug exposure and did not produce any significant neurotoxicity in a nonhuman primate model. Intrathecal topotecan should be evaluated clinically as a potential alternative therapy for refractory meningeal tumors.Pediatric Branch, National Cancer Institute, Building 10 Room 13N240, 9000 Rockville Pike, Bethesda, MD 20892, USA     

Etoposide in prostatic cancer: experimental studies and phase II trial in patients with bidimensionally measurable disease

Summary Etoposide, a semisynthetic derivative of podophyllotoxin, was evaluated concurrently in vitro against a human derived hormone-resistant cell line, PC-3, and in vivo in bidimensionally measurable hormone-resistant human prostatic cancer. In vitro, a dose-response relationship was observed, with 74% inhibition at 10 g/ml (1 h incubation) and >99% inhibition at 90 g/ml, both in the range of clinically achievable concentrations. In vivo, 1 PR (5%, 95% confidence limits 0–12%) of 18+ months was observed in 20 adequately treated patients. The results confirm the limited role of etoposide in hormone-refractory disease and the need for new model systems for evaluation of potential chemotherapeutic compounds in this disease.Supported in part by Public Health Service Grants CA-05826, CA-39203 and CA-90207 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, and the David H. Cogan Fund for Prostate Cancer ResearchRecipient of an American Cancer Society Clinical Oncology Career Development Award