ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136.

ATP-binding cassette transporter P-glycoprotein (ABCB1) is responsible for the multidrug resistance (MDR1) phenotype observed in cancer cells. SJG-136, a new pyrrolobenzodiazepine dimer, is a sequence-dependent DNA crosslinking agent and substrate of ABCB1. We previously showed that colon cancer cell lines expressing high levels of ABCB1 showed a lower sensitivity to SJG-136. Here, we show that in 3T3 isogenic fibroblasts, ABCB1 genetic polymorphism differentially affects ABCB1 gene expression and transport function. However, this genotype-phenotype relationship was not observed in immortalized lymphocytes, which expressed 10- to 1000-fold less ABCB1 than colon cancer cell lines. Consistent with this, the cytotoxicity of SJG-136 in 3T3 fibroblasts was affected by ABCB1 genetic polymorphism but not in immortalized lymphocytes. ABCB1 genetic polymorphism is therefore likely to affect drug sensitivity in tissues expressing high levels of the transporter and in which significant variability is observed.

Short hairpin RNAs targeting Bcl-xL modulate senescence and apoptosis following SN-38 and irinotecan exposure in a colon cancer model.

Bcl-xL is an anti-apoptotic protein over-expressed in colorectal cancers acting on both the intrinsic and extrinsic pathways. We stably expressed four different short hairpin RNA (pSNG-xL1-4) targeting Bcl-xL in HCT 116 cells. HCT 116 pSNG-xL#1 produced a modest (30%) decrease in Bcl-xL expression whilst Bcl-2 levels were similar to the parental cell line, HCT 116 pSNG-xL#2 and 3 showed 50% decrease in Bcl-xL and stable Bcl-2. HCT 116 pSNG-xL#3 showed a concomitant decrease (50%) in Bcl-2. A decrease in Bcl-xL sensitised cells to the small molecule inhibitor of Bcl-xL, Antimycin A3 and the DNA topoisomerase I inhibitors, SN-38 and camptothecin, but not to doxorubicin. HCT 116 pSNG-xL#1 produced a moderate increase in both senescence and apoptosis and a limited increase in SN-38 induced cell death while HCT 116 pSNG-xL#2 produced an increase in apoptosis but reduced senescence. Finally, when both Bcl-xL and Bcl-2 were decreased to a similar degree (HCT 116 pSNG-xL#3), senescence was significantly increased but apoptosis was limited. This effect was confirmed in vivo after administration of irinotecan and was associated with greater anti-tumour effect. Optimal growth inhibitory effect was therefore observed when both Bcl-xL and Bcl-2 were decreased to a similar extent. Antimycin A3, in combination with SN-38 recapitulated this phenotype in HCT 116 cells, suggesting a potential role for small molecule inhibitors of Bcl-xL/Bcl-2 in the treatment of colorectal cancer, potentially in combination with irinotecan.

Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136.

SJG-136 is a novel pyrrolobenzodiazepine dimer analogue that acts as a minor-groove interstrand DNA cross-linking agent. The present study investigated the impact of ABCB1 (mdr-1) expression on the activity of SJG-136 using both in vitro and in vivo systems. SJG-136 was highly potent in the colon cancer cell lines HCT-116, HT-29 and SW620 (IC50 0.1-0.3 nM). However, HCT-8 and HCT-15 cells expressing significant levels of mdr-1 were less sensitive (IC50 2.3 and 3.7 nM, respectively) using a SRB assay. The cytotoxicity was increased in HCT-15 and A2780(AD) in presence of 5 microg/ml verapamil. Mdr-1 mRNA expression was determined by qRT-PCR and correlated to SJG-136 IC50s (r2=0.86, P=0.0001). Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were less sensitive to SJG-136 than the parental 3T3 cells (IC50 208 and 6.3 nM, respectively). Finally, SJG-136 (120 microg/kg/d dx5) was highly active against A2780 xenografts (SGD=275) but not A2780(AD) xenografts (SGD=67).

Investigation of the role of Bax, p21/Waf1 and p53 as determinants of cellular responses in HCT116 colorectal cancer cells exposed to the novel cytotoxic ruthenium(II) organometallic agent, RM175.

Ruthenium(II) organometallic complexes form monofunctional adducts with guanine in DNA in vitro and have a cytotoxic anticancer activity spectrum in preclinical models suggesting lack of cross-resistance with cisplatin. The primary cytotoxic lesion remains to be identified but the downstream mechanism of action is nevertheless of interest. Using isogenic derivatives of the HCT116 colorectal cancer cell line, we investigated the role of p53, p21/WAF1 and Bax in the cellular response to the novel ruthenium(II) organometallic complex RM175, [(eta(6)-C(6)H(5)C(6)H(5))RuCl (H(2)NCH(2)CH(2)NH(2)-N,N')](+) PF(6)(-). Western blotting demonstrated dose-dependent accumulation of p53, Bax and p21/WAF1 within 48 h of the start of RM175 treatment in wild-type HCT116 cells. HCT116 wild-type and Bax-null cells arrested in the G(1) and G(2) phases of the cell cycle. This pattern of cell cycle arrest was not observed in p53-null or in p21/WAF1-null cells. Following RM175 treatment, HCT116 wild-type and p21/WAF1 null cells underwent a dose-dependent induction of apoptosis (Annexin-V and sub-G(1) apoptosis assays). This apoptotic response was not observed in p53-null or Bax-null cells. In short-term sulphorhodamine B assays, the IC(50) for RM175 was 16 microM for p53-null HCT116, and 8 microM for wild-type cells (P<0.05). However, the sensitivity to RM175 in clonogenic assays at 16 days was independent of p53 status. These results identify determinants of the short-term in vitro response to RM175 demonstrating a role for p53 and p21/WAF1 in the growth arrest and for p53 and Bax in the apoptotic response. The mechanism of p53-independent suppression of long-term clonogenicity remains to be determined.

Cell cycle effects of the novel topoisomerase I inhibitor NU/ICRF 505 in a panel of Chinese hamster ovary cell lines.

The effect of the novel topoisomerase I inhibitor NU/ICRF 505 (20 microM, approximate IC50 concentration) on the cell cycle was studied by flow cytometry in four Chinese hamster ovary (CHO) cell lines. Postdrug treated cells were incubated with optimal concentrations of cytochalasin B to prevent re-entry of daughter cells into the cell cycle. NU/ICRF 505 had no significant effect on cell cycle distribution in the parent cell line (CHO-K1) and two mutants hypersensitive to topo II inhibitors (ADR-1, ADR-3), all of which express similar levels of topo I protein. In the drug-resistant variant ADR-r, which overexpresses topo I 2-fold, a significant accumulation of cells in G1 phase was recorded. These results are broadly consistent with the cell cycle effects expected in CHO cells by a classic topo I poison (camptothecin) and add weight to the view that NU/ICRF 505 induces cell death primarily through topo I inhibition.

Development of anthracenyl-amino acid conjugates as topoisomerase I and II inhibitors that circumvent drug resistance.

Anthracenyl-amino acid conjugates (AAC) represent a novel class of topoisomerase (topo) inhibitor. The relationship between mechanism of enzyme inhibition and in vitro cytotoxicity has been investigated in a panel of 5 Chinese hamster ovary (CHO) and 2 human ovarian cancer cell lines (A2780) shown to possess different drug resistance phenotypes associated with altered expression of topo I and topo II. From a total of 13 compounds, 4 displayed broad-spectrum activity (IC50 ranging from 3.5-29.7 microM). NU/ICRF 500 (topo II catalytic inhibitor) was 1.4-fold more active against CHO ADR-1, which overexpresses topo II and was essentially noncross-resistant in CHO ADR-r (13.9-fold resistant to doxorubicin (DOX)) and 2780AD (1,460-fold resistant to DOX). NU/ICRF 505, which stabilises topo I cleavable complexes, was noncross-resistant in CHO ADR-3 (3,4-fold resistant to camptothecin) and only 1.8-fold cross-resistant in 2780AD. Hypersensitivity was recorded in ADR-r that overexpresses topo I. The most active compound was NU/ICRF 506, a dual catalytic inhibitor of topo I and II. Hypersensitivity was observed in ADR-r (1.4-fold) but not ADR-1, indicating that topo I is the likely nuclear target, and a low level of resistance was seen in the CHO ADR-6 drug transport mutant and 2780AD. The topo II catalytic inhibitor NU/ICRF 513 only produced hypersensitivity in ADR-r. These data suggest that NU/ICRF 500, 505, and 506 induce cell death, at least partly, through topo inhibition. NU/ICRF 513 appears to be cytotoxic via a nontopo mechanism of action. In addition, NU/ICRF 505 significantly inhibited the growth of two human xenografts (HT-29 colon cancer and NX002 nonsmall-cell lung cancer) in nude mice after i.p. administration at a dose of 25 mg/kg. The important properties of noncross-resistance and in vivo antitumour activity merit further development of AAC as potential new anticancer drugs.

Determination of the novel topoisomerase I inhibitor NU/ICRF 505 and its major metabolite in plasma, tissue and tumour by high-performance liquid chromatography.

A high-performance liquid chromatographic technique is presented for the determination of the novel topoisomerase I inhibitor NU/ICRF 505 (a tyrosine conjugate of anthraquinone), its major metabolite (NU/ICRF 505/M) and an internal standard (NU/ICRF 513, dihydroxyphenylalanine conjugate). The method uses a reversed-phase (Apex ODS-2) stationary phase and a mobile phase consisting of 0.25 M ammonium acetate adjusted to pH 3 with 25% (v/v) trifluoroacetic acid and methanol with gradient elution. Between-day variation in retention times were less than 1% for NU/ICRF 505 and 513 and 2.4% for the metabolite. Selective detection was achieved at a wavelength of 545 nm giving a limit of detection of 2 ng on column and 50 ng/ml after sample preparation for all three components. Chromatograms were free from interfering peaks even at very high detector sensitivity. Sample preparation was based on incubation of biological specimens (0.5 ml plasma or homogenate) with dimethylsulphoxide and acetonitrile at 4 degrees C for 30 min followed by centrifugation. Liver and tumour were homogenised in phosphate buffered saline. Recoveries were consistently high (81.7-106.7% for NU/ICRF 505; 88.7-103.3% for NU/ICRF 513 and 83.7-98.7% for NU/ICRF 505/M) with between day coefficients of variation of normally less than 10%. The method will contribute significantly to the preclinical evaluation of NU/ICRF 505.

Induction of apoptosis in human cancer cell lines by the novel anthracenyl-amino acid topoisomerase I inhibitor NU/ICRF 505.

Anthracenyl-amino acid conjugates represent a novel chemical class of topoisomerase (topo) inhibitor. NU/ICRF 505 is a lead compound that stabilises topo I cleavable complexes and is actively cytotoxic at low microM concentrations. In this study, endonucleolytic DNA cleavage was used as a marker of apoptosis to investigate mechanisms of cell death produced by this compound. NU/ICRF 505 (5 microM) induced a substantial increase in the level of DNA fragmentation in HL60 cells (up to 30% of total extracted DNA) but only after a 48 and 72 h drug exposure (compared with 6 h after treatment with camptothecin), as determined qualitatively by conventional gel electrophoresis and quantitatively by spectrofluorimetry. This effect was substantially reversed by co-treatment with zinc (1 mM). Subsequent studies with the human lung (NX002), ovarian (A2780) and colon (HT29) cancer cell lines yielded evidence of formation of higher molecular weight DNA fragments in NX002 and A2780 cells in response to NU/ICRF 505 (5 microM). Co-treatment with zinc (1 mM) caused a small decrease in DNA fragmentation. These data suggest that the induction of apoptosis may play an important role in the mechanism of cytotoxicity of NU/ICRF 505 in HL60 cells and that other pathways of cell death may also be operative in NX002 and A2780 in conjunction with apoptosis.

Characterization of the major metabolite of the novel topoisomerase I inhibitor NU/ICRF 505.

NU/ICRF 505 is a tyrosine conjugate of anthraquinone modified at the C terminus of the amino acid as an ethyl ester and it stabilizes topoisomerase I (topo I)-cleavable complexes. It is active in vitro against a panel of human cell lines, including drug-resistant variants, and possesses in vivo antitumour activity. NU/ICRF 505 was rapidly metabolized in nude mice to a product which represented the sole detectable form of the drug present in plasma and a chemosensitive human xenograft (HT-29 colon cancer). The metabolite (codenamed NU/ICRF 505/M) was purified, characterized by mass spectrometry and UV-visible spectroscopy, and shown to be the free amino acid produced by hydrolysis of the ethyl ester bond. NU/ICRF 505/M stabilized topo I-cleavable complexes in assays with human enzyme and was equipotent to the parent drug. Nonetheless, the metabolite was inactive in vitro against a panel of human tumour cell lines (including HT-29) and was not significantly taken up into cells in drug-uptake studies. Levels of the metabolite measured in the HT-29 xenograft after administration of a therapeutic dose of NU/ICRF 505 (25 mg/kg i.p.) remained above 1 microM for 6 h, and exceeded 10 microM at 10 min and 2 h. These data suggest that NU/ICRF 505 is a prodrug in nude mice for its topo-active metabolite NU/ICRF 505/M which accumulates in the tumour.

Identification of anthracenyl-dipeptide conjugates as novel topoisomerase I and II inhibitors and their evaluation as potential anticancer drugs.

As part of an ongoing rational drug design programme aiming to develop monosubstituted anthracenyl-peptides as potential anticancer drugs, three novel dipeptide conjugates have been synthesized and evaluated as inhibitors of topoisomerase (topo) I and II. Each of the three conjugates (designated NU/ICRF 600-602) was shown to inhibit the catalytic activity of both topoisomerase I and II, of which NU/ICRF 602 was the most active [100% inhibition of both enzymes at 5 micrograms/ml (approximately 15 microM) or less]. In a topo I/DNA unwinding assay, none of the compounds bound to DNA, suggesting genuine inhibition of catalytic activity. NU/ICRF 600 stabilized topo I cleavable complexes, although none of the compounds induced topo II-mediated DNA cleavage. Using a panel of Chinese hamster ovary cell lines along with the human ovarian cancer cell line, A2780, none of the three compounds were actively cytotoxic at concentrations < 100 microM. Subsequent drug uptake studies with NU/ICRF 600 and 602, using a method developed to correlate the chemosensitivity of A2780 cells with the uptake of anthracenyl-amino acid conjugates, revealed a lack of cellular uptake for both dipeptide conjugates. The significance of this finding in relation to drug design and the future development of this series of compounds is discussed.

Cytogenetic evaluation of the mechanism of cell death induced by the novel anthracenyl-amino acid topoisomerase II catalytic inhibitor NU/ICRF 500.

Anthracenyl-amino acid/dipeptides are novel topoisomerase (topo) inhibitors which can be actively cytotoxic in the low microM range. The present studies have been performed to determine whether cells treated with the topo II catalytic inhibitor NU/ICRF 500 (serine derivative) would manifest cytogenetic lesions consistent with its proposed mechanism of enzyme inhibition. Three other compounds were included for comparison: NU/ICRF 505 (tyrosine) which stabilises topo I cleavable complexes, NU/ICRF 602 (gly-gly) a non-cytotoxic catalytic inhibitor of topo I and II and NU/ICRF 502 (alanine) a non-cytotoxic non-topo inhibitor. Chromosomal damage was measured using the micronucleus test. NU/ICRF 500 (7.5-30 microM) induced an increase in CREST negative micronuclei (11-15 per 500 cells) in human lymphocytes (HL) and blocked the traverse of HL through the cell cycle, with cells accumulating in G2/M at 15 microM drug and G1/S at 30 microM drug. NU/ICRF 502 was without effect in the micronucleus test. NU/ICRF 500 and 602 (90-150 microM) caused no block in passage of synchronised metaphase Chinese hamster ovary cells through mitosis whereas NU/ICRF 505 produced a significant delay. DNA measurements of post-mitotic cells revealed that after NU/ICRF 500 treatment nuclei had a 4C DNA content, indicative of a lack of chromosomal segregation. Normal (2C) DNA content was observed with NU/ICRF 505 and 602. Overall, the data for NU/ICRF 500 are consistent with the cytogenetic modifications expected after catalytic inhibition of topo II and suggest that cell death may be mediated, at least in part, through this mechanism.

Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates.

Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.

Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography.

Anthracenyl amino acid/dipeptide conjugates (AADC) represent novel structures rationally designed for their DNA-binding properties. A high-performance liquid chromatography method is described for simultaneous determination of five compounds that exhibit novel mechanisms of action as topoisomerase I and II inhibitors. The method uses an Apex ODS-2 column and a mobile phase of 0.25 M ammonium acetate/trifluoroacetic acid (pH 3) in methanol with gradient elution. Selective detection is achieved by monitoring at 545 nm, with limits of detection ranging between 2 and 4 ng on the column. AADC are recovered from cell sonicates by solid-phase extraction using C2 cartridges, with extraction efficiencies ranging from 84% to 95%. Drug uptake studies were performed with three active compounds in the human ovarian cancer cell line A2780 and its multi-drug-resistant counterpart 2780AD. Marked differences were observed in the pattern of cellular accumulation produced by each compound. NU/ICRF 505 (tyrosine derivative) was taken up most avidly, reaching plateau levels of 4000 pmol/10(6) cells after 2 h, with no difference being apparent between A2780 and 2780AD. NU/ICRF 510 (arginine derivative) accumulated slowly in A2780, failing to achieve an equilibrium after 4 h, and appeared to be completely excluded from 2780AD. NU/ICRF 500 (serine derivative) was most rapidly taken up by A2780, producing a plateau of 800 pmol/10(6) cells after only 30 min with approximately 3-fold less accumulation in 2780AD. These results are correlated to the chemosensitivity of the two cell lines to the three compounds.

The comparative disposition of the pyrolloquinone GR63178A and its 9-hydroxy metabolite GR54374X in sensitive and resistant mouse colon adenocarcinoma.

The novel anticancer compound GR63178A is being evaluated in the clinic, having demonstrated activity against a wide range of experimental tumour systems in animals without significant toxic side-effects being apparent. In this work, we have demonstrated significant antitumour action of this compound against one murine colon cancer model (colon 38 tumour in BDF-1 mice, specific growth delay = 1.2) when given at 10 mg/kg over 21 consecutive days and in contrast shown minimal sensitivity of another similar murine colon adenocarcinoma, MAC 26, in NMRI mice with the same dose regime. We investigated the disposition of both the parent drug and the 9-OH metabolite (GR54374X) in plasma, tissues and tumours, using solid phase extraction followed by reversed-phase high performance liquid chromatography. Although plasma clearance profiles of GR63178A were similar, significant differences were seen in the disposition of the drug to major organs in two mouse strains. Noteably, the liver and kidneys of the sensitive model had higher levels of parent drug and 9-OH metabolite at both 30 min and 4 h post-injection. However, this was not apparent in the tumours themselves, and the levels of 9-OH metabolite were lower in the plasma and higher in the urine of the sensitive mice, indicating possible rapid renal clearance of this compound. Neither GR63178A nor GR54374X proved cytotoxic in in vitro experiments. The data presented here have revealed considerable variation in drug handling by these two mouse strains, but this did not produce different levels of either parent drug or GR54374X in the tumours, which are the presumed targets, suggesting that differences in disposition are probably not responsible for the different sensitivities of the two tumours. Other possible explanations include the production of a hitherto undetected ultimate cytotoxic metabolite in the sensitive, but not in the resistant, mouse/tumour combination, or differences in inherent tumour sensitivity, or in host-mediated effects. These possibilities are discussed.

The disposition of TCNU (tauromustine) in human malignant glioma: pharmacokinetic studies and clinical implications.

Tauromustine (TCNU), 130 mg/sq m, was administered intraoperatively by nasogastric tube to 10 patients with malignant glioma (seven glioblastomas and three anaplastic astrocytomas). High-performance liquid chromatography analysis of 32 tumor specimens for TCNU revealed that tissue concentrations ranged from 0 to 554 ng/gm: TCNU was not detected in necrotic regions of the tumor. Levels of TCNU in brain adjacent to tumor were similar to those recorded within the gliomas (range 0 to 635 ng/gm). The variability in the tissue level of TCNU was partly attributable to variable absorption of the drug, since peak plasma TCNU levels ranged from 164 to 3333 ng/ml. There were close quantitative and temporal relationships between the times of peak plasma levels (median 456 ng/ml at 45 minutes after administration), peak tumor levels (median 250 ng/gm tissue at 55 minutes), and brain adjacent to tumor levels (median 256 ng/gm tissue at 50 minutes). Linear regression analysis of the ratio between tissue and plasma TCNU levels at particular times after drug administration suggest that plasma concentrations can be used to estimate tissue concentrations. This study demonstrates that TCNU enters malignant glioma. In view of the activity of TCNU against a range of tumors, a full clinical evaluation of this new nitrosourea in malignant glioma seems justified.

A pharmacokinetic study of high-dose metoclopramide suppositories.

The pharmacokinetics of high-dose rectal metoclopramide have been studied in nine patients after administration of 150-mg suppositories. The results have been compared to the pharmacokinetics of the drug in five patients who received the same dose of metoclopramide intravenously. Administration of a metoclopramide suppository achieved plasma drug concentrations that are associated with the effective treatment of cytotoxic drug-induced nausea and vomiting. In three patients who received the drug by both routes the systemic availability of the suppository appeared to be complete. High-dose metoclopramide suppositories are convenient and may be advantageous in the treatment of medical oncology out-patients.

Pharmacokinetics of tauromustine in cancer patients. Phase I studies.

The pharmacokinetic properties of tauromustine (TCNU) were studied in 31 cancer patients who participated in phase I trials. The patients received single oral doses of tauromustine in the range of 20-170 mg/m2. Plasma samples were taken over 24 h after administration and analysed for tauromustine by reversed-phase liquid chromatography. Parent TCNU could be demonstrated in the plasma of all patients. Its absorption was rapid (tmax = 38 +/- 22 min), the half-life was 57 +/- 22 min (mean +/- SD), and maximal concentration (Cmax) and AUC values were linearly related to the dose level. Thus, our study does not indicate dose-dependent pharmacokinetics for the drug in the range of 20-170 mg/m2. Thrombocytopenia was the dose-limiting toxicity of TCNU; the reduction of platelet counts appeared to be linearly related to the log dose and Cmax and AUC values. TCNU appears to exhibit pharmacokinetic properties that are different from those of other nitrosoureas, which might be important for the clinical effect of the drug.

Activity of a new nitrosourea (TCNU) in human lung cancer xenografts.

The activity of a new nitrosourea (TCNU) based on the endogenous amino acid taurine was assessed in three human lung cancer xenografts growing in immunodeficient mice. Moderate activity (specific growth delays of 0.63 and 1.13 compared with controls) was seen in two non-small cell tumours after a single oral administration of 20 mg-1kg. This dose was curative in a small cell xenograft. By using high performance liquid chromatography it was possible to detect parent drug in the tumours as well as the plasma and tissues after oral administration of TCNU. Drug sensitivity was correlated inversely with the amount of the DNA repair enzyme 0(6)-methylguanine-DNA methyltransferase assayed from extracts of the tumour cells but not with the levels of parent drug within the tumour. This compound appears to have unique pharmacokinetic properties compared with other chloroethylnitrosoureas.

Phase I study of TCNU, a novel nitrosourea.

TCNU is a chloroethyl nitrosourea based on the endogenous amino acid taurine. This paper reports its first evaluation in man. Eighty-four patients with refractory cancer received 12 dose escalations from 10-150 mg/m2 TCNU administered orally every 6 weeks. Clinical side-effects were predominantly gastro-intestinal but dose-limiting toxicity was thrombocytopenia. Pharmacokinetic monitoring with an HPLC assay sensitive to the nanogram range demonstrated unchanged TCNU in plasma for up to 8 h following administration. The mean half-life was 60 min. Clinical responses were seen in melanoma (four patients), lung cancer (two squamous, one small cell) and one patient each with renal and stomach cancer. These responses, together with the unusual pharmacokinetic profile of TCNU, warrant exploration in disease-orientated phase II studies at a recommended dose of 130 mg/m2 p.o. q 5 weeks.

Experimental cerebral and plasma pharmacokinetic studies of TCNU: implications for brain tumour chemotherapy.

This study of the pharmacokinetics of TCNU, a new nitrosourea, in the rodent has shown that TCNU concentrations in the plasma (ng/ml) and brain (ng/g) are equivalent from 15 min to 4 hours after drug administration. The absolute levels of TCNU obtained with a dose of 100 mg TCNU/kg bodyweight were at most time points, three to four times those obtained with dosage of 25 mg TCNU/kg. The profile of rodent plasma TCNU levels following drug administration is similar to that recorded in humans, with peak TCNU concentrations occurring around 45 min. Since TCNU crosses an intact blood brain barrier (BBB), and clinical Phase I trials have shown it to possess potent antitumour properties. It may be a useful agent in the management of primary and secondary cerebral neoplasia.