Breast cancer is the most prevalent type of cancer in pre- and postmenopausal women in most Western countries. In the treatment of metastatic breast cancer, doxorubicin has the broadest spectrum of antitumor activity of any drug currently available but produces a dose-dependent cardiomyopathy that limits its clinical usefulness. The aim of this research project was to target the affected tissues, which contain estrogen receptors (ERs). Initially, a series of estrogen derivatives with side chains linked at the 3- and 17-positions of estrone were synthesized, and then novel anticancer prodrugs were obtained from these by further linking these compounds to doxorubicin by means of various alkyl spacer groups. These estrogenic prodrugs were designed to target tumor cells containing ERs, found in human breast cancer cells, and to release the active anticancer moiety when internalized. The estrogenic prodrugs were then biologically evaluated using in vitro chemosensitivity assays against human ER-positive (MCF-7) and ER-negative (MCF-7ADR and MT-1) breast tumor cells and a leukemia (K562) cell line. The results showed that estrone derivatives with substituted amino-alcohol side chains of various lengths (2-6 carbons) linked to the 17-position of estrone were mostly inactive. Estronedoxorubicin prodrugs containing doxorubicin at the 3-position of estrone (CCRL 1042 and CCRL 1036) were relatively inactive and nonselective against all cell lines tested. However, when doxorubicin was linked to the 17-position of estrone, these prodrugs had at least an order greater activity than their 3-linked counterparts. Using a short aminoxy-spacer group (2 carbons) at this position produced CCRL 1035, which had a lower activity against all cell lines tested compared to doxorubicin. In contrast, the prodrug incorporating doxorubicin at the 17-position of estrone via a long spacer group (12 carbons, CCRL 1033) was both potent and selective against ER-positive cells MCF-7. These studies have shown that linking doxorubicin to the 17-position of estrone via a long alkyl spacer group conferred selectivity of cytotoxic action against ER-positive breast cancer tumor cells.

NAD(P)H:Quinone oxidoreductase-1 (NQO1) has been implicated in the bioreductive activation of the clinically active anticancer drug Mitomycin C (MMC) and a polymorphic variant of NQO1 which lacks functional enzyme activity (NQO1*2) has been linked with poor survival in patients treated with MMC. The relationship between NQO1 activity and cellular response to MMC is however controversial and the aim of this study was to determine whether the response of bladder cancer patients to MMC can be forecast on the basis of NQO1*2 genotype status. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue from 148 patients with low to intermediate grade (G1/G2) superficial (Ta/T1) bladder cancers and NQO1*2 genotype status determined by PCR-RFLP. NQO1*2 genotype status was retrospectively compared with clinical response to intravesical administered MMC with the primary end-point being time to first recurrence. NQO1 phenotype was determined by immunohistochemistry. Of the 148 patients genotyped, 85 (57.4%) were NQO1*1 (wildtype), 59 (39.8%) were NQO1*1/*2 (heterozygotes) and 4 (2.7%) were NQO1*2/*2. No NQO1 protein expression was detected in NQO1*2/*2 tumours. A broad spectrum of NQO1 protein expression existed in tumours genotyped as NQO1*1 and NQO1*1/*2 although tumours with NQO1*1 typically expressed higher NQO1 protein. A poor correlation existed between NQO1*2 genotype status and clinical response to MMC. The results of this retrospective study suggest that tailoring, MMC therapy to individual patients with superficial bladder cancer on the basis of NQO1 genotype status is unlikely to be of clinical benefit.

NQO1 is a cytosolic flavoprotein that plays a dual role in the detoxification of potentially carcinogenic compounds and the bioreductive activation of quinone based anticancer drugs. Two polymorphic variants of NQO1 exist (NQO1*2 and NQO1*3) which cause significant phenotypic reductions in NQO1 protein content and activity. Current methods for detecting NQO1 polymorphisms commonly use PCR-RFLP techniques and have exclusively used DNA isolated from fresh tissues. This study describes a method that is suitable for analysing NQO1 polymorphisms in genomic DNA isolated from formalin-fixed paraffin-embedded tissue. The method utilises two rounds of PCR amplification using a nested primer strategy that generates specific PCR products followed by RFLP analysis using either Hinf1 (for NQO1*2) or Msp1 (for NQO1*3). Whilst existing methods proved unsatisfactory (low product yield and poor specificity), the nested primer strategy produced good quality PCR products suitable for RFLP analysis and genotyping of NQO1*2 and NQO1*3 in archival tissue samples. The ability to utilise the vast archives of human tissue held by pathology laboratories would be of considerable benefit as retrospective studies comparing NQO1 genotype status, patient history and treatment outcomes could be conducted.

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Bioassay guided fractionation of the roots of Cyathostemma argenteum using the brine shrimp resulted in the isolation of two uncommon flavanones, 2,5-dihydroxy-7-methoxy flavanone 1 and 2,5-dihydroxy-6,7-dimethoxy flavanone 2 while the stem bark yielded the related compounds 5-hydroxy-7-methoxy flavone 3 and 5-hydroxy-6,7-dimethoxy flavone 4. The alkaloids liriodenine 5 and discretamine 6 as well as benzyl benzoate 7 were isolated from the roots and 6 was also isolated from the stembark. In cytotoxicity tests using four human breast cancer cell lines, 1 and 2 were weakly toxic to MCF-7 cells (IC(50) = 19.6 and 19.0 microM, respectively) but showed little activity against MCF-7 cells resistant to doxorubicin or against two oestrogen receptor-deficient cell lines. Compound 5, but not 6 and 7, was moderately cytotoxic against all four cell lines. These results are discussed in the context of the traditional use of C. argenteum in the treatment of breast cancer.

It is now generally accepted that activation of tamoxifen occurs as a result of metabolism to alpha-hydroxytamoxifen. In this study, alpha-hydroxytamoxifen was given to female Wistar/Han rats (0.103 or 0.0103 mmol/kg, intraperitoneally, daily for 5 days). This resulted in liver DNA damage, determined by (32)P-post-labelling, of 3333 +/- 795 or 343 +/- 68 adducts/10(8) nucleotides, respectively (mean +/- SD, n = 4). Following HPLC separation, the retention times of the major alpha-hydroxytamoxifen DNA adducts were similar to those seen following the administration of tamoxifen. However, after rats were treated with alpha-hydroxytamoxifen (0.103 mmol/kg) for 5 days and the animals kept for up to 13 months, no liver tumours developed (0/7 rats), even with phenobarbital promotion (0/5 rats). GST-P foci were detected in the liver, but only after 13 months was their number or area significantly increased over the corresponding controls. When alpha-hydroxytamoxifen was given to female lambda/lacI transgenic rats (0.103 mmol/kg orally for 10 days) and the animals killed 46 days later, there was an approximate 1.8-fold increase in mutation frequency but no significant increase in G:C to T:A transversions as described after tamoxifen treatment. It is concluded that DNA damage alone, resulting from the short-term administration of alpha-hydroxytamoxifen, is not sufficient to initiate liver tumours even with phenobarbital promotion. As with tamoxifen, long-term exposure may be required to allow promotion and progression of transformed cells.

The indolequinone EO9 demonstrated good preclinical activity but failed to show clinical efficacy against a range of tumours following intravenous drug administration. A significant factor in EO9's failure in the clinic has been attributed to its rapid pharmacokinetic elimination resulting in poor drug delivery to tumours. Intravesical administration of EO9 would circumvent the problem of drug delivery to tumours and the principal objective of this study is to determine whether or not bladder tumours have elevated levels of the enzyme NQO1 (NAD(P)H:quinone oxidoreductase) which plays a key role in activating EO9 under aerobic conditions. Elevated NQO1 levels in human bladder tumour tissue exist in a subset of patients as measured by both immunohistochemical and enzymatic assays. In a panel of human tumour cell lines, EO9 is selectively toxic towards NQO1 rich cell lines under aerobic conditions and potency can be enhanced by reducing extracellular pH. These studies suggest that a subset of bladder cancer patients exist whose tumours possess the appropriate biochemical machinery required to activate EO9. Administration of EO9 in an acidic vehicle could be employed to reduce possible systemic toxicity as any drug absorbed into the blood stream would become relatively inactive due to an increase in pH. © 2001 Cancer Research Campaign http://www.bjcancer.com

The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of beta-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg(-1) day(-1) ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (DeltaT(m) value) or toxicity in the mouse-malaria model.

C1311 has emerged as the lead compound from a novel group of anticancer agents, the imidazoacridinones, and will be entering clinical trials shortly. Previous murine pharmacokinetic studies have shown C1311 to be rapidly and extensively distributed into tissues including tumor. This study has identified two major metabolites of C1311 and describes their pharmacokinetics in mice. M1 is a glucuronide of the parent compound with high concentrations in both plasma and liver. Calculated area under the plasma concentration versus time curve values were 6-fold and 2-fold greater, respectively, than C1311, Based on these studies, we propose M2 to be a nonfluorescent oxidation product because electrospray ionization-mass spectroscopy/mass spectroscopy analysis gave a molecular ion at m/z 367, 16 U greater than the parent compound. It formed rapidly in liver preparations in vitro, both murine and human, by a cytosolic process in the presence of NADPH and in vivo was detected in liver tissues at concentrations equivalent to those of C1311 but was not detectable in plasma. Preliminary in vitro toxicity studies showed M2 to be as potent as C1311 against MAC15A tumor cells. Over the first 24 h, 39% of the administered dose is eliminated via the bile (28%) mostly as C1311 or the kidneys (11%) as the glucuronide (M1). This study has given valuable information as to the likely metabolic pathway to occur in humans, and the cytotoxic metabolite M2 may play a role in the antitumor activity or toxicity of C1311 in the clinic.

To investigate the activation mechanisms involved in tamoxifen carcinogenicity, analogues of tamoxifen isomers modified at the ethyl group were synthesized and assessed for their ability to induce hepatic DNA damage following their administration to female F344 rats. The cis isomer was prepared by acid-catalyzed isomerization of tamoxifen and isolated by preparative HPLC. The active metabolite alpha-hydroxytamoxifen and geometric isomers of bromotamoxifen and C-desmethylenetamoxifen, analogues in which the ethyl group has been replaced by a bromine atom and methyl group, respectively, were synthesized according to published procedures. The levels of hepatic DNA adducts induced were determined by 32P-postlabeling. Bromotamoxifen and tamoxifen 1,2-epoxide caused no detectable DNA damage relative to controls. Trans isomers of tamoxifen, C-desmethylenetamoxifen, and alpha-hydroxytamoxifen all produced DNA adducts at a 5-90-fold higher level than the corresponding cis isomers. In contrast, both the cis and trans isomers of alpha-hydroxytamoxifen showed similar reactivity toward calf thymus DNA in vitro. Molecular models of alpha-hydroxytamoxifen isomers suggest this difference in DNA adduct-forming ability is due to steric hindrance of the enzymes involved in the activation of this metabolite. There were high adduct levels in the liver, but no uterine DNA adducts were detected in rats treated with alpha-hydroxytamoxifen. This suggests that in contrast to the liver, alpha-hydroxytamoxifen is not further activated in rat uterus. This may help to explain the absence of uterine tumors in rats following long-term tamoxifen treatment.

The roots of Cyathostemm argenteum @I.) Sinclair are used in Malay tradnional medicine for the treatment of breast cancer. In order to evaluate the cytotoxicity of crude e m c t and compound isolated from these species, toxicrty against the brine shrimp (Artemia salina) was determined using a 96-well microplate method (Solis et al., 1993). This method has been previously shown to be a good model for the derectlon of cytotoxic compounds.

A reversed phase isocratic high-performance liquid chromatographic method is reported in which a formal structured procedure, the solvent selectivity triangle, was applied to predict the mobile phase composition giving baseline resolution of the clinically important triphenylethylene antioestrogenic agent (Z)-tamoxifen, its principal (Z)-metabolites, and also the clinically relevant (E)-geometric isomers of tamoxifen and 4-hydroxytamoxifen. The technique of solvent selectivity triangle was used to select the optimal organic modifier parameter for use with a Hichrom ODS 1 column, to achieve baseline separation of six triphenylethylene solutes. The detection system utilised post-column ultraviolet irradiation to convert solutes into their respective photocyclisation products, followed by fluorescence detection (lambda[ex] = 254 nm, lambda[em] = 360 nm), and the low detection limit for tamoxifen in serum of 0.1 microM. The optimal mobile phase composition was determined to be methanol-acetonitrile-water-trichloroacetic acid (50:31:18.9:0.1, v/v, pH 2.9). A single stage liquid-liquid extraction method for determination of triphenylethylene drugs in serum was developed. Reproducible recoveries for the (Z)-geometric isomers of tamoxifen (84 +/- 3%) and its principal metabolites including Metabolite Y (94 +/- 3%), N-desmethyltamoxifen (94 +/- 3%) and 4-hydroxytamoxifen (92 +/- 3%) were achieved, though more variable results were obtained for their corresponding (E)-geometric isomers (71 +/- 7% and 70 +/- 10%, respectively).