An in silico screen links gene expression signatures to drug response in glioblastoma stem cells.

Cancer stem cells (CSCs) are thought to promote resistance to chemotherapeutic drugs in glioblastoma, the most common and aggressive primary brain tumor. However, the use of high-throughput drug screens to discover novel small-molecule inhibitors for CSC has been hampered by their instability in long-term cell culture. We asked whether predictive models of drug response could be developed from gene expression signatures of established cell lines and applied to predict drug response in glioblastoma stem cells. Predictions for active compounds were confirmed both for 185 compounds in seven established glioma cell lines and 21 compounds in three glioblastoma stem cells. The use of established cell lines as a surrogate for drug response in CSC lines may enable the large-scale virtual screening of drug candidates that would otherwise be difficult or impossible to test directly in CSCs.

Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Phase I and pharmacokinetic study of LY309887: a specific inhibitor of purine biosynthesis.

PURPOSE: In this phase I trial in humans the safety and pharmacology of LY309887 on a weekly schedule combined with daily oral 5-mg doses of folic acid were evaluated. BACKGROUND: LY309887 is an inhibitor of folate-dependent enzymes involved in de novo purine biosynthesis and has a broad preclinical antitumor activity. In murine systems, combining this agent with exogenous folic acid results in an enhanced therapeutic index. METHODS: This study was a single-institution, open-label, clinical trial of dose escalation with toxicity and pharmacokinetic parameters determined. The dose range studied was 0.5-4 mg/m2 per week x6 and then a modified schedule weekly x3 every 6 weeks. RESULTS: Dose-limiting toxicities were of delayed onset and associated with hematologic, neurologic, and mucosal effects. Pharmacokinetic parameters revealed dose linearity for AUC and Cmax. Low circulating levels of drug persisted for over 200 h. Urinary excretion accounted for approximately 50% of the parent drug but was highly variable. The urinary excretion was near maximal within 24 h of dosing. CONCLUSIONS: The modified dosing schedule allowed repetitive dosing in patients. Further evaluation of the 2 mg/m2 per week x3 every 6 weeks with daily oral folate supplement as a potential phase II dose may be warranted.

Clinical studies with MTA.

MTA (LY231514), a multi-targeted antifolate, is a classical antifolate undergoing intracellular polyglutamation. Polyglutamated MTA is a potent thymidylate synthase (TS) inhibitor and inhibits other folate-dependent enzymes, including dihydrofolate reductase and glycinamide ribonucleotide formyl transferase. Multifocal antifolates may overcome antifolate resistance, but it is not known whether the anti-tumour activity of MTA depends on its TS inhibition, its primary locus of action, or whether other loci contribute. MTA was examined in three phase I trials using different schedules: a 10-min i.v. infusion given once every 3 weeks, once weekly for 4 weeks every 6 weeks or daily for 5 days every 3 weeks. Dose-limiting toxicities were neutropenia and thrombocytopenia. Other consistently seen side-effects, which were manageable, included mucositis, skin rashes and transient elevations of transaminases. Toxicity was highly schedule dependent: the recommended dose for the 3-weekly schedule (600 mg m(-2)) was 30 times that for the daily x 5 schedule (4 mg m(-2)day(-1)). The 3-weekly dosing schedule was chosen for phase II evaluation. Phase II trials are underway to investigate the activity and toxicity of MTA in several tumour types, including colorectal, pancreas, breast, bladder and non-small-cell lung cancer (NSCLC) Further phase I trials will investigate MTA in combination with other agents, including gemcitabine, cisplatin, 5-fluorouracil and folate. Preliminary phase II trials results are encouraging; responses were seen in colorectal, pancreas, NSCLC and breast cancer.

A phase I and pharmacokinetic study of LY231514, the multitargeted antifolate.

LY231514 is a novel antifolate that principally inhibits thymidylate synthase, but with additional folate-dependent enzyme targets. A Phase I study of single-agent LY231514 administered as a daily i.v. infusion over 10 minutes for 5 days, repeated every 3 weeks, was conducted to evaluate the maximum tolerated dose, pharmacokinetic profile, and antitumor activity of the drug using this schedule. Thirty-eight patients with advanced malignancies that were refractory or not amenable to standard therapy were treated with a total of 116 courses of LY231514, escalating treatment doses through 10 dose levels, from 0.2-5.2 mg/m2/day. No objective clinical responses were observed, although minor antitumor activity not fulfilling the response criteria was seen in three patients. A maximum tolerated dose of 4.0 mg/m2/day was determined, with neutropenia as the predominant dose-limiting toxicity. Reversible disturbances of liver biochemistry, fulfilling the protocol definitions of dose-limiting toxicity, were also observed. Other toxicities included diarrhea, mucositis, skin rash, and fatigue. Pharmacokinetic studies were performed at all treatment levels. Analysis showed a linear relation between administered dose and both maximum plasma concentration (Cmax) and area under the plasma concentration/time curve. The drug was cleared with a day 1 total body clearance of 108.9 +/- 38.8 ml/min/m2, with plasma concentrations declining with a mean harmonic terminal half-life of 1.4 +/- 0.98 h. When given by this schedule, LY231514 is tolerable, and Phase II studies are in progress.

Advanced breast cancer: investigational role of gemcitabine.

There have been many recent advances in the treatment of advanced breast cancer including the introduction of novel drugs and the development of high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT). These innovations may offer significant hope for improvement in the treatment of breast cancer in the near future. Gemcitabine is a nucleoside analogue with significant antitumour activity in many human solid tumours. Conflicting results have been observed from studies evaluating gemcitabine in advanced breast cancer. Efficacy data for single-agent gemcitabine range from 25 to 46% depending on starting dose and whether patients have previously received chemotherapy for metastatic disease (as well as adjuvant use). Gemcitabine is extremely well tolerated, even in heavily pre-treated patients, and is easy to administer on an outpatient basis to both chemo-naive and previously treated patients. The most common toxicity is mild myelosuppression. Gemcitabine causes minimal nausea and vomiting, and significant hair loss is extremely uncommon. Combination chemotherapy studies with anthracyclines are underway and significant activity has been observed in combination with both doxorubicin and epirubicin. In view of its modest toxicity profile, and its novel mechanism of action, gemcitabine warrants further evaluation in breast cancer patients, both as a single agent and in combination chemotherapy schedules.

A phase I study of gemcitabine and carboplatin in non-small cell lung cancer.

Gemcitabine has shown phase II activity against a range of solid tumor malignancies. This study evaluated a combination of gemcitabine and carboplatin in a phase I trial in 13 chemotherapy-naive patients with non-small cell lung cancer to determine the maximum tolerated dose of carboplatin in combination with gemcitabine. Gemcitabine at a dose of 1,000 mg/m2 was administered on days 1, 8, and 15 of a 28-day cycle and carboplatin was given as a 30. minute infusion immediately before gemcitabine on day 1. Carboplatin dosing was escalated and determined using the Calvert formula, and three patients were treated at the initial predicted dose of area under the curve (AUC) 4 mg/mL/min. Subsequently the carboplatin dose was escalated to a predicted AUC 5.2 mg/mL/min. Pharmacokinetic studies were performed measuring gemcitabine and carboplatin (total platinum) concentrations. Responses were assessed following two cycles of treatment and patients with stable disease or objective responses proceeded to receive a maximum of six cycles. Dose-limiting myelosuppression was identified at a predicted carboplatin AUC 5.2 mg/mL/min, with two patients developing grade 4 neutropenia and two patients grade 4 thrombocytopenia. However, these grade 4 toxicities were not associated with any serious sequelae. In this preliminary study, four partial responses were observed and the median length of survival for all patients was 45 weeks. Pharmacokinetic parameters for gemcitabine were similar to those in patients receiving 1,000 mg/m2 as a single agent, and for carboplatin showed that the delivered dose was slightly below the calculated dose. There was one treatment-related death due to cerebral edema. The combination was well tolerated by the majority of patients, and symptomatic toxicity was similar to single-agent gemcitabine. The combination of gemcitabine and carboplatin is well tolerated in patients with non-small cell lung cancer, with myelosuppression being the dose-limiting toxicity. Symptomatic toxicities were rare and outpatient treatment was easy. In view of the antitumor activity observed, further examination of this combination is warranted. In the first instance, alternate sequencing of the combination with gemcitabine followed by carboplatin is planned in an extended phase I study prior to phase II evaluation.

Phase II activity of gemcitabine in advanced breast cancer.

Two phase II breast cancer studies have been completed with gemcitabine in patients with locally advanced or metastatic breast cancer. Gemcitabine was administered as a 30 minute intravenous infusion on days 1, 8, and 15 of a 28-day cycle. In a European study of 44 patients, 40 patients were evaluable for response, 26 having received chemotherapy (seven in the adjuvant setting). The mean number of completed cycles administered was 2.7 and 81% of doses were delivered as scheduled. There were three complete responses and seven partial responses, giving an overall response rate of 25.0% (95% confidence interval, 12.7% to 41.2%). Four patients were not evaluable for efficacy: one had insufficient therapy, two had no bidimensionally measurable disease, and one had insufficient therapy and no bidimensionally measurable disease. The median duration of survival was 11.5 months. Hematologic toxicity was generally mild, with World Health Organization grade 3 and 4 leukopenia occurring in 6.8% and 2.3% of patients and neutropenia in 23.0% and 7.0% of patients, respectively. Nonhematologic toxicity was minimal. Flu-like symptoms were mild and transient. Only one patient developed alopecia. In a US study, 18 of 21 heavily pretreated patients were evaluable, all of whom had stage IV disease. The median number of cycles administered was two, with 12% of injections omitted and 31% reduced by 50%. No responses were observed in this smaller study. The safety profile was similar to that in the European study, although myelosuppression was greater in the US study, in which patients were heavily pretreated. The differing results observed from single-agent studies of gemcitabine in advanced breast cancer may be explained in part by the amount of prior chemotherapy received and the lower mean dose of chemotherapy administered in the US study. Responses have been observed in both chemotherapy-naive and previously treated patients. The drug was extremely well tolerated in both studies, even in heavily pretreated patients. In view of its modest toxicity profile and its novel mechanism of action, gemcitabine deserves further evaluation in breast cancer patients and, in particular, because of its relative lack of myelotoxicity would be an ideal candidate for combination chemotherapy.

A phase I study of a 24 hour infusion of gemcitabine in previously untreated patients with inoperable non-small-cell lung cancer.

A phase I study to determine the maximum tolerated dose and toxicity of gemcitabine when given as a 24 h infusion to patients with inoperable non-small-cell lung cancer (NSCLC). A total of 24 patients with unresectable stage IIIa-IV NSCLC were entered into the study. Gemcitabine was administered as a 24 h infusion on days 0, 7 and 14. Courses of therapy were repeated every 28 days. There were 16 males and 8 females with a median age of 51 years (range 40-73 years). The WHO performance score was 1 (21 patients) or 2 (3 patients). The TNM stage was IIIa (6), IIIb (10) and IV (8). Three patients were entered at each dose level with six at the maximum tolerated dose (MTD). Dose levels were 10, 20, 40, 80, 120, 180 and 210 mg m-2. The MTD was 180 mg m-2 and dose-limiting toxicity was neutropenia and lethargy. Partial response was observed in five (21%) patients (95% CI 7-42%) lasting 10, 14, 18, 47 and 51 + weeks. The maximum tolerated dose of gemcitabine given as a 24 h infusion was 180 mg m-2.

Advanced breast cancer: a phase II trial with gemcitabine.

PURPOSE: In this phase II study, the efficacy and tolerability of gemcitabine were studied in 44 patients with locally advanced or metastatic breast cancer. PATIENTS AND METHODS: Of 40 patients assessable for response, 14 were chemotherapy-naive, seven had received adjuvant chemotherapy, and 19 had received one prior chemotherapy regimen for metastatic disease. Gemcitabine was administered as a 30-minute intravenous infusion once a week for 3 weeks followed by a 1-week rest every 4 weeks. The mean number of completed cycles administered was 2.7 and the mean dosage delivered was 725 mg/m2 per injection. Eighty-one percent of doses were delivered as scheduled. RESULTS: There were three complete responses and seven partial responses, for an overall response rate of 25.0% (95% confidence interval [CI], 12.7% to 41.2%). Four patients were not assessable for efficacy (one had insufficient therapy, two had no bidimensionally measurable disease, and one had neither). All responses were independently validated by an external oncology review board. Responses were observed early in treatment, with a median time to response of 1.9 months. The median survival duration for all 40 assessable patients was 11.5 months. Hematologic toxicity was generally mild, with World Health Organization (WHO) grade 3 and 4 leukopenia occurring in 6.8% and 2.3% of patients and neutropenia in 23.3% and 7.0%, of patients, respectively. The only other grade 4 toxicities were infection and nausea and vomiting in one patient each. One patient was withdrawn due to shortness of breath, possibly drug-related. Flu-like symptoms, which were mild, transient, and treatable with acetominophen, were reported in 6.8% of patients. Only one patient developed alopecia of severity greater than WHO grade 2. CONCLUSION: In view of the single-agent activity seen in advanced breast cancer, modest toxicity profile, and novel mechanism of action, gemcitabine deserves evaluation in breast cancer and is an ideal candidate for combination therapy.

Adoptive CD8+ T-cell immunotherapy of AIDS patients with Kaposi's sarcoma.

This article reviews published and original findings from two clinical trials of adoptive CD8+ T-cell immunotherapy of patients with acquired immunodeficiency syndrome (AIDS) and Kaposi's sarcoma (KS). In the first trial, AIDS patients with either KS or oral hairy leukoplakia (OHL) received five rounds of reinfusions of 10(8)-10(10) ex vivo expanded and activated autologous CD8+ T cells. Recombinant interleukin-2 (rIL-2) was coadministered only with the fifth and final infusion. Improvement, and in some cases, resolution of OHL, KS, and candidiasis was observed with no side effects. The observation that clinical improvement of KS was more pronounced when reinfusion of CD8+ T cells was followed by rIL-2 infusion led to a second clinical trial designed to examine the effect of repeated infusions of autologous CD8+ T cells with concomitant rIL-2 administration in the treatment of AIDS-related KS. Improvement of KS status was observed in four out of the eight patients studied (three partial and one complete response). The CD8+ T-cell immunotherapy protocol also provided the opportunity to comparatively study CD8+ T-cell-associated genetic programs. Baseline expression patterns of soluble and surface immune markers by CD8+ T cells from AIDS patients and uninfected controls were predominantly of the type 1 type and differed mainly at a quantitative or kinetic level. Deficiencies in immune mediator expression by CD8+ T cells from AIDS patients tended to dissipate with progression through the protocol. Findings are discussed in the context of current knowledge and therapeutic implications of CD8+ T-cell function in AIDS and neoplasia.

Assessment of immune mediator expression levels in biological fluids and cells: a critical appraisal.

The realization that dysregulation of the soluble immune mediator network can contribute to the progression of a wide variety of clinical disorders, including neoplasias, has opened up the possibility of designing new forms of therapy and has established the need for accurate methods of detection of immune mediator expression levels in biological fluids and cells. The purpose of this review is to provide a summary of current knowledge and experience in the area of immune mediator expression level assessment from the clinical, basic science, epidemiological, technical, and therapeutic perspectives.

Single-agent activity of weekly gemcitabine in advanced non-small-cell lung cancer: a phase II study.

PURPOSE: To evaluate the efficacy and safety of gemcitabine, a pyrimidine antimetabolite with activity against solid tumours. PATIENTS AND METHODS: Eighty-two patients with unresectable stage IIIa to IV non-small-cell lung cancer (NSCLC) were entered. The first 54 patients received gemcitabine 800 mg/m2, and subsequent patients 1,000 mg/m2, as a 30-minute intravenous infusion on days 0, 7, and 14. Courses of therapy were repeated every 28 days. Twenty percent dosage escalation was permitted after course no.1 if World Health Organization (WHO) toxicity was < or = 1. RESULTS: Sixteen (20%; 95% confidence interval [CI], 12% to 31%) of 79 patients assessable for response had independently validated partial responses, with a median duration of 7 months. The overall median survival duration was 7 months. Gemcitabine improved disease-related symptoms (70% patients) and increased WHO performance status (44%). Toxicity was generally mild and reversible. Patients experienced little WHO grade 3 and 4 toxicity, with anemia in four (5%), thrombocytopenia in one (1%), leukopenia in six (7%), and neutropenia in 18 (22%). Infection occurred in nine patients (12%) during the study (four were neutropenic), but there were no episodes of WHO grade 3 or 4 infection. WHO grade 3 and 4 biochemical toxicity occurred with transient elevations of transaminases in 10 patients (12%). Two patients had transient WHO grade 3 elevation of serum creatinine levels, and two developed acute renal failure 4 and 6 weeks after the last dose of gemcitabine. There was no WHO grade 4 symptomatic toxicity. WHO grade 3 vomiting occurred in 31 patients (38%) and grade 3 alopecia in one (1%). Flu-like symptoms were associated with gemcitabine administration in 36 patients (44%). Twenty-six patients (32%) experienced fever (1% WHO grade 3), 33 (40%) ankle edema not associated with cardiac failure, 31 (38%) lethargy, and 11 (13%) dyspnea. CONCLUSION: Gemcitabine is an active new agent in the treatment of NSCLC. This schedule was associated with little alopecia or myelosuppression. Gemcitabine warrants further investigation in other malignancies and in combination with other agents.

Clinical and immunological changes in AIDS patients following adoptive therapy with activated autologous CD8 T cells and interleukin-2 infusion.

OBJECTIVES: (1) To determine the safety and feasibility of repetitive reinfusions of activated autologous CD8 cells followed by low-dose continuous interleukin (IL)-2 infusion in patients with AIDS. (2) To study the relationships between clinical responses, surface marker phenotypic distributions and cytokine expression patterns of both cultured CD8 cells and lymphocytes in the peripheral blood compartment. DESIGN: Six adult patients with Centers for Disease Control and Prevention group IV HIV-1 disease ranging from mild to severe, were studied. All patients were receiving zidovudine prior to and during the study period, and had initial CD4 and CD8 cell counts > 50 and 200 x 10(6)/l, respectively. METHODS: Autologous CD8 T cells (10(8)-10(10)) were reinfused five times after ex vivo culture and stimulation with phytohemagglutinin and recombinant (r) IL-2. The fifth such infusion was followed by 5 days of rIL-2 infusion. Phenotypes and cytokine expression patterns of the expanded cells were determined as well as serum levels of immune mediators throughout the study. RESULTS: Patients showed stable CD4 and CD8 cell counts, p24 antigenemia, and minimal toxicity over the 24-week protocol study. Clinical improvement was observed in lymphadenopathy (six out of six), oral hairy leukoplakia (three out of four), and Kaposi's sarcoma (KS; two out of two) in the patients studied. In vivo induction of detectable levels of bioactive acid-stable interferon (IFN)-alpha, but not of other cytokines studied, upon activated CD8 cell reinfusion was associated consistently with improvement of oral hairy leukoplakia. However, partial regression of KS was observed after the CD8 cell infusion cycles and without IFN-alpha induction. In one of the two patients studied, KS regression was associated with decreased IL-1 alpha serum levels. In the other patient, who had failed previous IFN-alpha therapy, KS regression was observed after a decline in reinfused CD8 cell-associated gene expression of tumor necrosis factor (TNF)-beta. Both IL-1 alpha and TNF-beta are growth factors for KS cells. CONCLUSIONS: These observations demonstrate the feasibility and safety of ex vivo CD8 cell activation, expansion, and reinfusion, and rIL-2 infusion in AIDS patients. The findings in this Phase I trial suggest potential clinical efficacy and encourage Phase II trials. The correlations obtained between clinical and immunological states could contribute to an understanding of the relationship between CD8 T-cell function and HIV-1-associated disease progression.

Phase II trial of the novel sulphonylurea sulofenur in advanced breast cancer.

A total of 18 women with advanced breast cancer were treated with sulofenur [LY186641; N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)-urea], a diarylsulfonylurea that has broad-spectrum activity against a number of murine mammary tumour xenografts. The dosage chosen on the basis of pre-clinical and phase I studies was 700 mg/m2 given orally once daily for 14 days, with treatments being repeated every 3 weeks. There was no response. All patients experienced at least grade 1 anaemia, and two patients developed symptomatic methaemoglobinaemia. Two patients developed grade 4 rises in serum liver-function values along with histological changes consistent with drug-induced toxicity. The mean plasma concentrations of 176 micrograms/ml were lower than the levels required to exert anti-tumour effect in the mouse model.

A phase II study of sulofenur (LY186641) in gastric cancer.

Sulofenur is a novel diarylsulfonylurea with proven anti-tumor activity in murine tumor models. In this phase II study in patients with advanced gastric or gastroesophageal adenocarcinoma, 17 patients were treated with sulofenur orally at 700 mg/m2 for 14 days every 3 weeks. No tumor responses were seen. The main toxicities were anemia, methemoglobinemia and abnormalities in liver function tests. These toxicities precluded dose escalation. However, plasma levels of sulofenur, and its hydroxy and keto metabolites were probably insufficient to exert anti-tumor effect in comparison with data from murine studies. Further structure-activity studies are warranted.

A phase II study of sulofenur, a novel sulfonylurea, in recurrent epithelial ovarian cancer.

A total of 16 patients with recurrent epithelial ovarian cancer were treated with sulofenur (LY 186641), a novel oral sulfonylurea. All subjects had received previous chemotherapy. Anaemia occurred in all 16 patients, 14 of whom required a blood transfusion, and 2/16 patients received methylene blue for breathlessness due to methaemaglobinaemia. Treatment was discontinued in 2/16 cases due to rising liver enzyme values, which reverted to normal on cessation of the drug. There was no nausea or alopecia. Only two minor responses were seen. Plasma drug levels were insufficient to result in antitumour activity as extrapolated from animal data. Further studies that attempt to increase the bioavailability and improve the therapeutic index are warranted.

Role of the extracellular matrix on the growth and differentiated phenotype of murine colonic adenocarcinoma cells in vitro.

The growth and differentiation characteristics of MAC 15 murine adenocarcinoma cells, derived from routine passage in vivo for growth in vitro on a plastic substrate (MAC15j cells), were compared under conditions in which the cells were seeded onto a substrate of type-I collagen which was either attached to plastic or was released to float free in medium. Cells grown on a plastic substrate consisted of a heterogeneous, largely anaplastic population with a putative enterocytic morphology but with no evidence of junctional complexes or cell polarity typical of an epithelial phenotype. MAC 15j cells from cultures grown on a plastic substrate reestablished a moderate to well-defined degree of differentiation when transplanted back into NMRI mice. When MAC 15j cells were seeded from plastic onto type-I collagen, either attached to plastic or free-floating, tight junctional complexes were formed and the cells began to attain a more recognizable, columnar and polarised epithelial morphology. Cells grown on a type-I collagen gel which was free-floating showed a selective expression of alkaline phosphatase at the apical surfaces of approximately 10% of the cells. This expression was detectable by electron microscope histochemistry but could not be detected biochemically. Treatment of MAC 15j cells grown on a released collagen matrix with tetramethyl-urea (20mM) accelerated the expression of alkaline phosphatase activity at the apical surface as detected by microscopy.

High uptake of RSU 1069 and its analogues melanotic melanomas.

RSU 1069 and RSU 1164 are electron affinic agents that contain a nitro group together with a weakly basic alkylating aziridine moiety, and they represent lead compounds in the development of dual-function, bioreductive, hypoxic cell radiosensitizers. We studied the pharmacokinetics of these drugs in mice carrying KHT sarcoma. Lewis lung carcinoma, and B16 melanoma. Following an i.p. dose of 80 mg/kg, absorption was rapid and the elimination t1/2 was in the region of 30 min for both agents. Maximal tumour levels were 91, 16 and 19 microgram/ml for RSU 1069 and 109, 26 and 28 microgram/ml for RSU 1164 in. the B16, KHT and Lewis lung tumours, respectively. In B16 melanoma these levels corresponded to tumour:plasma ratios of 3.8 for RSU 1069 and 3.7 for RSU 1164. Cellular uptake of RSU 1069, RSU 1164 and a related compound, RB 7040, was measured in vitro as a function of extracellular pH. Melanotic cells from both B16 melanoma and HX118, a human tumour xenograft, showed substantially greater accumulation of these weakly basic sensitizers than any other cell type examined. Ratios of intra-:extracellular concentration (Ci/Ce) for RSU 1069 were around unity and independent of pH for Lewis lung cells and HX34 amelanotic melanoma cells, whereas ratios of up to 3 and 5 were obtained in B16 and HX118 cells, respectively. The highest measured value of Ci/Ce was 15 for RSU 1164 in HX118 cells at pH 8.4; this compares with a ratio of 1.5 for HX34 cells at the same pH. These studies indicate that the high levels of uptake of the weakly basic sensitizers into melanotic melanoma in vivo is a cell-mediated phenomenon and may be due to a lower average intracellular pH in the melanotic cells.