Idoxifene: report of a phase I study in patients with metastatic breast cancer.

Idoxifene, a novel antiestrogen with reduced estrogenic activity when compared to tamoxifen, has been given to 20 women with metastatic breast cancer, 19 of whom had received tamoxifen previously, in doses between 10-60 mg. Idoxifene had an initial half-life of 15 h and a terminal half-life of 23.3 days. At a maintenance dose of 20 mg, a mean steady-state level of 173.5 ng/ml was achieved. Significant falls in luteinizing hormone and follicle-stimulating hormone were seen, but the falls were not dose related. Idoxifene was well tolerated, with 11 patients complaining of mild symptoms similar to those seen with tamoxifen. Fourteen patients continued idoxifene therapy for 1-56 weeks; 4 patients showed stabilization of disease for 6-56 weeks and 2 patients showed a partial response.

Novel steroidal inhibitors of human cytochrome P45017 alpha (17 alpha-hydroxylase-C17,20-lyase): potential agents for the treatment of prostatic cancer.

Steroidal compounds having a 17-(3-pyridyl) substituent together with a 16,17-double bond have been synthesized, using a palladium-catalyzed cross-coupling reaction of a 17-enol triflate with diethyl(3-pyridyl)borane, which are potent inhibitors of human testicular 17 alpha-hydroxylase-C17,20-lyase. The requirement for these structural features is stringent: compounds having 2-pyridyl (9), 4-pyridyl (10), or 2-pyridylmethyl (11) substituents instead of the 3-pyridyl substituent were either poor inhibitors or noninhibitory. Reduction of the 16,17-double bond to give 17 beta-pyridyl derivatives diminished potency with 3-pyridyl substitution (3-->27; IC50 for lyase, 2.9-->23 nM) but increased it with a 4-pyridyl substituent present (10-->28; IC50 1 microM-->53 nM). In contrast, a variety of substitution patterns in rings A-C of the steroid skeleton afforded inhibitors having potencies similar to those most closely related structurally to the natural substrates pregnenolone and progesterone, respectively 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (3, Kiapp < 1 nM; IC50 for lyase, 2.9 nM) and 17-(3-pyridyl)androsta-4,16-dien-3-one (15; IC50 for lyase, 2.1 nM). Thus compounds having variously aromatic ring A (18), saturated rings A/B (21, 22), and oxygenated ring C (26) exhibited IC50 values for lyase (1.8-3.0 nM) falling within a 2-fold range. The most potent compounds are candidates for development as drugs for the treatment of hormone-dependent prostatic carcinoma.

Esters of 3-pyridylacetic acid that combine potent inhibition of 17 alpha-hydroxylase/C17,20-lyase (cytochrome P45017 alpha) with resistance to esterase hydrolysis.

Esters of 3- and 4-pyridylacetic acid have been prepared and tested for inhibitory activity toward the human testicular 17 alpha-hydroxylase/C17,20-lyase and human placental aromatase enzymes. The structural features required for optimal inhibition of the hydroxylase/lyase enzyme were a 3-pyridine ring, methyl substitution alpha to the carbonyl group, and a bulky alkoxycarbonyl substituent. The compounds with the greatest selectivity were isopinocampheyl 2-methyl-2-(3-pyridyl)propanoate, 9, 1-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 12, and 2-methyl-2-adamantyl 2-methyl-2-(3-pyridyl)propanoate, 14, which, while inhibiting the aromatase activity with IC50 values of 30, 35, and 40 microM, respectively, exhibited IC50 values toward hydroxylase/lyase of between 13 and 90 nM. For comparison, ketoconazole gave an IC50 value of 15 microM against aromatase and values of 65 and 26 nM for inhibition of the hydroxylase and lyase activities, respectively. Some of the structural features required for enzyme inhibition also conferred resistance to esterase hydrolysis, in vitro using rat liver microsomes as a source of the esterase activity. Therefore these esters are lead compounds in the development of inhibitors of androgen biosynthesis for the treatment of hormone-dependent prostatic cancer.

3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer.

Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer.

Rationally designed analogues of tamoxifen with improved calmodulin antagonism.

Computerized molecular modeling studies on the interactions of the antiestrogen tamoxifen (1) and its analogues bound to the calcium-binding protein calmodulin have guided the rational design of more potent antagonists. Compounds with either three or four methylene units in the basic side chain or slim lipophilic 4-substituents were expected to be more potent. All compounds were tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to the estrogen receptor from rat uteri. Some compounds were assayed for cytotoxicity against MCF-7 breast tumor cells in vitro. Introduction of lipophilic 4-substituents was accomplished by using palladium(0)-catalyzed coupling reactions with a 4-iodinated precursor. Both the 4-ethynyl (16 and 17) and 4-butyl (18 and 19) compounds were more potent calmodulin antagonists than tamoxifen. Extension of the basic aminoethoxy side chain of 4-iodotamoxifen (3) and idoxifene (2) ((E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phen yl-1- butene) by one or two methylene units resulted in modest gains in calmodulin antagonism (10-13). All the compounds assayed retained estrogen receptor binding characteristics. The compound possessing the optimal combination of calmodulin antagonism and estrogen receptor binding was 12 ((E)-1-[4-[3-(N-pyrrolidino)propoxy]phenyl]-1-(4-iodophenyl)-2-phe nyl-1 - butene) (IC50 = 1.1 microM, RBA = 23). Correlation between calmodulin antagonism and cytotoxicity was demonstrated for selected compounds.

Pharmacology of novel steroidal inhibitors of cytochrome P450(17) alpha (17 alpha-hydroxylase/C17-20 lyase).

Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P450(17) alpha, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3 beta-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3 beta-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoconazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to < or = 0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers.

Biochemistry and pharmacokinetics of potent non-steroidal cytochrome P450(17alpha) inhibitors.

Two potent non-steroidal inhibitors (CB7645 and CB7661) of human cytochrome P450(17alpha) were tested for in vivo activity in WHT mice. There were no signs of toxicity, but there was no effect on the androgen-dependent organs. The pharmacokinetics and biochemistry of the compounds in mice were investigated. Following i.p. administration of 0.5 mmol/kg of CB7645 and CB7661, peak plasma levels of 13.4 and 3.4 microM, respectively, occurred after 2-4 h, both compounds were cleared rapidly (terminal half-lives 2.7 and 3.3 h, respectively) and neither was detectable at 24 h. CB7645 produced some decrease in plasma testosterone at 4 h, but this was not sustained. When tested in vitro against the WHT testicular enzyme, the CB7645 and CB7661 were competitive inhibitors with K(i) values of 10 and 13 nM, respectively. However, the K(m) for the substrate progesterone was lower at 4.3 nM. These data indicate that, for effective and continuous inhibition of the murine cytochrome P450(17alpha) enzyme, higher peak levels of the compounds would be required, and these levels would need to be maintained throughout the treatment period.

Pharmacokinetics and biodistribution of radiolabelled idoxifene: prospects for the use of PET in the evaluation of a novel antioestrogen for cancer therapy.

With a view to evaluating the role of PET imaging in the development of new anticancer drugs, we are investigating the novel antioestrogen pyrrolidino-4-iodotamoxifen (idoxifene). [125I]idoxifene and [131I]idoxifene have been produced in no-carrier-added form using a tributyl stannylated precursor, and the bio-distribution and dynamic behaviour of the compound investigated using syngeneic transplantable mammary tumours in the rat. Our findings support the use of PET imaging with 124I to study the clinical pharmacology of idoxifene. Factors other than hormone receptor levels appear to influence tumour uptake and therefore, possibly the biological effects of this compound.

Non-haematological solid tumours as surrogate granulocytes: a possible mechanism for metastatic spread.

The concept that non-haematological solid tumours utilize normal leukocyte mechanisms, specifically those of granulocytes, to facilitate metastatic spread is presented here. In this article we will address the question of whether the process of metastasis is a phenomenon unique to tumour cells that utilize unique processes that are only found in neoplastic cell populations, or whether neoplastic populations 'turn on' or co-opt latent leukocyte genes normally not expressed by the parental, normal tissue of origin. We suggest that ectopic expression of leukocyte genes in epithelial tumours may be the simplest method for solid tumours to acquire the mechanisms for neoplastic spread through metastasis and may also account for the observed lack of host immunosurveillance of such tumours.

Effects of the potential chemopreventive agent DMU-135 on adenoma development in the ApcMin+ mouse.

DMU-135 (3,4-Methylenedioxy-3',4',5'-trimethoxy chalcone) is a novel anticancer prodrug designed to be activated into a potent tyrosine kinase inhibitor by the tumour selective enzyme activity of the cytochrome P450 enzyme CYP1B1. CYP1B1 is selectively expressed in a wide variety of tumours including colon. The hypothesis was tested that DMU-135 would inhibit Apc(Min/+) mouse gastrointestinal adenoma formation. From 4-18 weeks of age animals received DMU-135 (0.2% w:w) in AIN93G diet. DMU-135 was well tolerated, induced no systemic side-effects and reduced adenoma multiplicity by 46 +/- 18.3% compared to controls (p < 0.001). Further characterisation of this promising chemopreventive agent is required.

A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism.

A mechanism for the generation of electrophilic alkylating agents from tamoxifen by hepatic alpha-oxidation of its ethyl group is presented which accounts for the formation of covalent DNA adducts associated with its hepatic carcinogenicity. The key process leading to the generation of electrophilic alkylating agents from tamoxifen is postulated to be alpha-hydroxylation in conjunction with 4-hydroxylation or secondary metabolic conjugation. The mechanism explains the lack of DNA adducts observed for the tamoxifen analogues toremifene, droloxifene and idoxifene and accounts for the species variation in hepatic carcinogenicity of tamoxifen.

Reduced genotoxicity of [D5-ethyl]-tamoxifen implicates alpha-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen.

A proposed mechanism for the metabolic activation of tamoxifen to electrophilic species that form DNA adducts leading to liver cancer involves alpha-hydroxylation of the ethyl group in the critical first step. This mechanism predicts that tamoxifen deuterated at the alpha-position would be less genotoxic than the non-deuterated compound owing to an isotope effect that would reduce the rate of oxidative metabolism at this position. This hypothesis has now been tested with experiments conducted in rats in vivo and in human cells in vitro. It was found that the deuterated compound [D5-ethyl]-tamoxifen is significantly less genotoxic than tamoxifen. Administration of 0.06 and 0.12 mmol/kg [D5-ethyl]-tamoxifen to female Fischer rats by gavage resulted in a 2.5- and 1.7-fold reduction respectively in the levels of hepatic DNA adducts present 24 h after treatment, compared with the non-deuterated compound. Treatment of MCL-5 cells with [D5-ethyl]-tamoxifen resulted in a 2- to 3-fold decrease, compared with tamoxifen, in the number of micronuclei induced in cells arrested in cytokinesis by cytochalasin-B. Further evidence is provided by UV irradiation of the major hepatic DNA adducts isolated from tamoxifen-treated rats, which caused a large shift in the chromatographic mobility of the adducts, consistent with the presence of a 1,2-olefinic linkage in the tamoxifen residue of the adduct leading to cyclization to a phenanthrenylic compound, and consistent with this adduct having arisen from reaction with DNA at the alpha-position of tamoxifen. Taken together, these data strongly suggest that tamoxifen is metabolized to a liver carcinogen by alpha-hydroxylation of its ethyl group.

Development of a high performance zinc-62/copper-62 radionuclide generator for positron emission tomography.

Clinical utilisation of positron emission tomography could be enhanced by the availability of short-lived radionuclides derived from generator systems. The zinc-62/copper-62 combination is one such system which could be used as a source for a number of copper-62 radiopharmaceuticals. We have developed and optimised a high activity (5.6 GBq, 150 mCi) zinc-62/copper-62 generator to provide 62Cu in a form that is suitable for direct labelling of pyruvaldehyde-bis-(N4-methylthiosemicarbazone)-copper(II), Cu(PTSM). The distribution coefficients of Zn(II) and Cu(II) between anion-exchange resin and various hydrochloric acid/organic solvent mixtures were measured. Based on these measurements a generator eluent of 0.3 M HCl/40% ethanol provided 62Cu in greater than 90% yield in a 3-ml volume. A very low 62Zn breakthrough of less than 3 x 10(-7)% was achieved. Copper-PTSM was successfully labelled with the no-carrier-added 62Cu eluent directly from the generator with 94% radiochemical yield.

The deuterium isotope effect for the alpha-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen.

This study describes the application of on line HPLC-electrospray ionization MS in the structural determination of the metabolites formed following incubation with rat liver microsomes of an equimolar mixture of the anticancer drug tamoxifen and its [D5-ethyl]-analogue. The ratio of ca 3:1 between unlabelled and D4-labelled alpha-hydroxytamoxifen, indicating a large isotope effect for this metabolic process, accounted for the previously observed lower yield of DNA adducts formed in the livers of rats treated with D5-tamoxifen compared with unlabelled drug. The loss of one deuterium atom on metabolism discriminated hydroxyethylated metabolites from others and enabled two further such metabolites to be detected, namely alpha-hydroxytamoxifen N-oxide and alpha-hydroxy-N-desmethyltamoxifen of which the latter is novel. Furthermore, the use of [alpha-D2-ethyl]- and [beta-D3-ethyl] tamoxifens discriminated alpha- from beta-hydroxylated metabolites and proved that the metabolites described here were alpha-hydroxylated. In contrast to the alpha-hydroxylated metabolites, the other metabolites identified, namely tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen and their deuterated counterparts were not depleted in the deuterated components. The use of on line HPLC-electrospray ionization MS combined with isotopic labelling is a powerful technique for probing the structures of metabolites, and, applied to tamoxifen, has provided further evidence that alpha-hydroxylation is an important pathway for the conversion of the drug into a DNA-reactive metabolite.

The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1.

Resveratrol is a cancer preventative agent that is found in red wine. Piceatannol is a closely related stilbene that has antileukaemic activity and is also a tyrosine kinase inhibitor. Piceatannol differs from resveratrol by having an additional aromatic hydroxy group. The enzyme CYP1B1 is overexpressed in a wide variety of human tumours and catalyses aromatic hydroxylation reactions. We report here that the cancer preventative agent resveratrol undergoes metabolism by the cytochrome P450 enzyme CYP1B1 to give a metabolite which has been identified as the known antileukaemic agent piceatannol. The metabolite was identified by high performance liquid chromatography analysis using fluorescence detection and the identity of the metabolite was further confirmed by derivatisation followed by gas chromatography-mass spectrometry studies using authentic piceatannol for comparison. This observation provides a novel explanation for the cancer preventative properties of resveratrol. It demonstrates that a natural dietary cancer preventative agent can be converted to a compound with known anticancer activity by an enzyme that is found in human tumours. Importantly this result gives insight into the functional role of CYP1B1 and provides evidence for the concept that CYP1B1 in tumours may be functioning as a growth suppressor enzyme.

Pharmacokinetics in mice and growth-inhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4'-tetramethoxystilbene.

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a naturally occurring polyphenol with cancer chemopreventive properties in preclinical models of carcinogenesis, including those of colorectal cancer. Recently, a variety of analogues of resveratrol have been synthesised and investigated in in vitro assays. One analogue, 3,4,5,4'-tetramethoxystilbene (DMU 212), showed preferential growth-inhibitory and proapoptotic properties in transformed cells, when compared with their untransformed counterparts. As part of a chemoprevention drug development programme, the pharmacokinetic properties of DMU 212 were compared with those of resveratrol in the plasma, liver, kidney, lung, heart, brain and small intestinal and colonic mucosa of mice. DMU 212 or resveratrol (240 mg kg(-1)) were administered intragastrically, and drug concentrations were measured by HPLC. Metabolites were characterised by cochromatography with authentic reference compounds and were identified by mass spectrometry. The ratios of area of plasma or tissue concentration vs time curves of resveratrol over DMU 212 (AUC(res)/AUC(DMU212)) for the plasma, liver, small intestinal and colonic mucosa were 3.5, 5, 0.1 and 0.15, respectively. Thus, resveratrol afforded significantly higher levels than DMU 212 in the plasma and liver, while DMU 212 exhibited superior availability compared to resveratrol in the small intestine and colon. Resveratrol was metabolised to its sulphate or glucuronate conjugates, while DMU 212 underwent metabolic hydroxylation or single and double O-demethylation. DMU 212 and resveratrol inhibited the growth of human-derived colon cancer cells HCA-7 and HT-29 in vitro with IC(50) values of between 6 and 26 microM. In the light of the superior levels achieved in the gastrointestinal tract after the administration of DMU 212, when compared to resveratrol, the results provide a good rationale to evaluate DMU 212 as a colorectal cancer chemopreventive agent.