Michael Jarman

Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide

Abstract Evidence is presented in support of the following metabolic pathways, in the liver, of the antitumour agent cyclophosphamide 2-[bis(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide. The drug is first converted, presumably by the mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down by elimination of acrolein from its tautomeric form, aldophosphamide, to yield phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid], a known cytotoxic agent. In competition with this process is the enzymic conversion of 4-hydroxycyclophosphamide (by dehydrogenation) and aldophosphamide (by oxidation) into the known in vivo metabolites of cyclophosphamide, 4-ketocyclophosphamide and carboxyphosphamide, respectively, each of which has low cytotoxicity. 4-Hydroxycyclophosphamide, which was too unstable to allow identification directly by conventional procedures, was trapped by reaction with ethanol. The resulting two, apparently isomeric, ethyl derivatives, (1) were amenable to mass spectrometry, (2) yielded acrolein 2,4-dinitrophenylhydrazone on treatment with acidic 2,4-dinitrophenylhydrazine, (3) were hydrolysed in water (pH 4.3), each isomer apparently regenerating 4-hydroxycyclophosphamide, (4) were highly toxic to Walker tumour cells in culture. Phosphoramide mustard was also isolated after in vitro metabolism of cyclophosphamide. On the basis of a bioassay involving Walker tumour cells in whole animals it appeared that, of the known metabolites of cyclophosphamide, only phosphoramide mustard possesses the cytoxicity and biological half-life appropriate to the active antitumour metabolite. Four other metabolites of low cytotoxicity were isolated and identified, namely, 4-ketocyclophosphamide, carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, and 3-hydroxypropyl-N,N-bis(2-chloroethyl)phosphorodiamidate. The significance of metabolic detoxification processes in relation to the selective cytotoxicity of cyclophosphamide towards tumour cells in vivo is discussed. The metabolic activation of isophosphamide appears to follow a pathway similar to that of cyclophosphamide.

Hormonal impact of the 17 alpha-hydroxylase/C-17,C-20-lyase inhibitor abiraterone acetate (CB7630) in patients with prostate cancer

A series of three dose escalating studies were conducted to investigate the ability of the 17α-hydroxylase/C17,20-lyase inhibitor abiraterone acetate, to cause maximum suppression of testosterone synthesis when delivered to castrate and noncastrate males with prostate cancer. Study A was a single dose study in castrate males. Study B was a single dose study in noncastrate males and study C was a multiple dose study in noncastrate males. The drug was given orally in a once-daily dose and blood samples taken to assess pharmacokinetic (PK) parameters and hormone levels in all patients. The study drug was well tolerated with some variability in PKs. Suppression of testosterone levels to <0.14 nmol l−1 was seen in four out of six castrate males treated with a single dose of 500 mg. At 800 mg given days 1–12 in noncastrate males, target suppression was achieved in three out of three patients, but a two- to three-fold increase of Luteinising Hormone (LH) levels in two out of three patients overcame suppression within 3 days. All patients in the multiple dose study developed an abnormal response to a short Synacthen test by day 11, although baseline cortisol levels remained normal. This is the first report of the use of a specific 17α-hydroxylase/17,20-lyase inhibitor in humans. Repeated treatment of men with intact gonadal function with abiraterone acetate at a dose of 800 mg can successfully suppress testosterone levels to the castrate range. However, this level of suppression may not be sustained in all patients due to compensatory hypersecretion of LH. The enhanced testosterone suppression achieved in castrate men merits further clinical study as a second-line hormonal treatment for prostate cancer. Adrenocortical suppression may necessitate concomitant administration of replacement glucocorticoid.

Pharmacology of novel steroidal inhibitors of cytochrome P45017α (17α-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.

Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP–responsive element binding protein–binding protein–associated factor (PCAF) and p300 (1 to >50 μmol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 μmol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2–48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of α-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

Phase I and pharmacokinetic study of D-limonene in patients with advanced cancer

Purpose: d-Limonene is a natural monoterpene with pronounced chemotherapeutic activity and minimal toxicity in preclinical studies. A phase I clinical trial to assess toxicity, the maximum tolerated dose (MTD) and pharmacokinetics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. Methods: A group of 32 patients with refractory solid tumors completed 99 courses of d-limonene 0.5 to 12 g/m2 per day administered orally in 21-day cycles. Pharmacokinetics were analyzed by liquid chromatography-mass spectrometry. Ten additional breast cancer patients received 15 cycles of d-limonene at 8 g/m2 per day. Intratumoral monoterpene levels were measured in two patients. Results: The MTD was 8 g/m2 per day; nausea, vomiting and diarrhea were dose limiting. One partial response in a breast cancer patient on 8 g/m2 per day was maintained for 11 months; three patients with colorectal carcinoma had prolonged stable disease. There were no responses in the phase II study. Peak plasma concentration (Cmax) for d-limonene ranged from 10.8 ± 6.7 to 20.5 ± 11.2 μM. Predominant circulating metabolites were perillic acid (Cmax 20.7 ± 13.2 to 71 ± 29.3 μM ), dihydroperillic acid (Cmax 16.6 ± 7.9 to 28.1 ± 3.1 μM ), limonene-1,2-diol (Cmax 10.1 ± 8 to 20.7 ± 8.6 μM ), uroterpenol (Cmax 14.3 ± 1.5 to 45.1 ± 1.8 μM ), and an isomer of perillic acid. Both isomers of perillic acid, and cis and trans isomers of dihydroperillic acid were in urine hydrolysates. Intratumoral levels of d-limonene and uroterpenol exceeded the corresponding plasma levels. Other metabolites were trace constituents in tissue. Conclusions:d-Limonene is well tolerated in cancer patients at doses which may have clinical activity. The favorable toxicity profile supports further clinical evaluation.

A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells.

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.

The importance of tamoxifen metabolism in tamoxifen-stimulated breast tumor growth

The acquired ability of tamoxifen to stimulate tumor growth has been suggested as one mechanism for the development of treatment failure in breast cancer. We have reported that tamoxifen-stimulated MCF-7 breast tumors in nude mice display reduced tamoxifen levels as compared with tamoxifen-inhibited tumors and an altered metabolite profile with isomerization oftrans-4-hydroxytamoxifen to a weak antiestrogen and the production of metabolite E, an estrogenic metabolite. To investigate further the importance of tamoxifen metabolism in this model, we quantified levels of tamoxifen and major metabolites in tamoxifen-stimulated as compared with tamoxifen-inhibited MCF-7 tumors growing in nude mice and employed tamoxifen analogs resistant to metabolism. Tamoxifen-stimulated tumors have a relative abundance ofcis-4-hydroxytamoxifen and metabolite E. However, in vivo treatment of mice carrying tamoxifen-stimulated tumors with fixed-ring nonisomerizable tamoxifen analogs or with nafoxidine, a nonsteroidal antiestrogen with a different structure, nonetheless resulted in tumor growth stimulation. Tumors were also stimulated by a deoxytamoxifen analog resistant to conversion to metabolite E. Growth of tamoxifen-stimulated tumors was inhibited by a pure steroidal antiestrogen, ICI 182, 780, suggesting the need for clinical trials of this drug in patients with tamoxifen resistance. Growth of tamoxifen-stimulated tumors was further stimulated by estrogen replenishment, and this estrogen stimulation could be blocked by tamoxifen indicating that tamoxifen has both agonist and antagonist properties in these tumors. This study suggests that tamoxifen-stimulated tumor growth in this model is not due to isomerization or metabolism of tamoxifen to less antiestrogenic or more estrogenic metabolities. The mechanisms by which tamoxifen acquires more potent in vivo agonist properties, resulting in tumor growth stimulation over time, remain to be defined.

4-Hydroxyandrostenedione treatment for postmenopausal patients with advanced breast cancer.

4-hydroxyandrostenedione, a potent inhibitor of the aromatase (oestrogen synthetase) system, was given to 11 patients with metastatic breast cancer. After a single 500 mg intramuscular injection a sustained reduction of serum oestradiol was observed for at least 1 week in all patients in whom the steroid was measured. 4 patients responded to treatment for periods of up to 4 months, and healing of bone metastases and reduction in size of soft-tissue metastases was evident. The only side-effects were pain at the injection site and hot flushes. 4-hydroxyandrostenedione is a new and specific aromatase inhibitor which shows promise in the treatment of patients with metastatic breast cancer.

Variation of the inhibition of calmodulin dependent cyclic AMP phosphodiesterase amongst analogues of tamoxifen; Correlations with cytotoxicity

The ability of a variety of analogues of tamoxifen to inhibit calmodulin dependent cyclic AMP phosphodiesterase has been determined. Effective inhibition requires that the aminoethoxy side chain bears a positive charge at physiological pH and is not too bulky. Amongst 4-substituents, inhibitory potency increases with lipophilicity. The stereochemistry about the olefinic linkage is not important. The most potent agent found (IC50 1.4 microM, compare tamoxifen = 6.75 microM) has a 4-iodine substituent and pyrrolidino in place of dimethylamino. This analogue is also more cytotoxic than tamoxifen against MCF-7 human breast cancer cells as determined in a 24-hr assay, but there was no correlation found between calmodulin inhibition and cytotoxicity against the L1210 murine leukaemia or Walker rat carcinosarcoma cells in culture. The results are consistent with the possibility that calmodulin is important to the functioning of oestrogen receptor mediated growth in MCF-7 cells.

Hydroxy derivatives of tamoxifen

In the exploration of the structural features that affect the RBA (binding affinity for the estrogen receptor of rat uterus relative to that of estradiol) in the tamoxifen [trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl ]-1,2-diphenyl-1-butene] series, several derivatives variously substituted in the 1-phenyl group have been synthesized. [In the tamoxifen series, the descriptors E and Z, which define the configuration of the geometrical isomers and depend on the location and nature of substituents in the aromatic moieties and the ethyl group, may vary, although the relative configuration (cis or trans) does not. In order to avoid confusion the terms cis and trans will be used in this paper to refer to the relative positions of the 4-[2-(dimethylamino)ethoxy]phenyl and ethyl (or hydroxyethyl, hydroxypropyl, or bromo) substituents attached to the ethene moiety.] The final stage of each synthesis involved acid-catalyzed dehydration of a tertiary alcohol, and, in contrast to the known 3- and 4-hydroxy derivatives which were obtained as near-equimolar cis,trans mixtures, only the trans forms of the 2-hydroxy, 2-methyl, 2,4-dihydroxy, and 4-hydroxy-2-methyl derivatives were obtained. Also, in contrast to the trans forms of the 3- and 4-hydroxy derivatives, which are readily equilibrated to cis,trans mixtures, the trans 2-hydroxy derivative could not be isomerized. Tamoxifen and 2-methyltamoxifen had similar RBAs (approximately 1% of that of E2), but that of 2-hydroxytamoxifen was much lower (0.1%). Introduction of a second hydroxyl group (2,4-dihydroxy derivative) enhanced the RBA, and for the 4-hydroxy-2-methyl derivative, the RBA and growth inhibitory activity against the MCF-7 mammary tumor cell line in vitro were high and comparable to those of 4-hydroxytamoxifen, a metabolite of the parent drug. Tamoxifen derivatives hydroxylated at positions 3 or 4 of the 1-butene moiety and the 5-hydroxy-1-pentene analogue were also synthesized, but they had very low RBA values.