P. J. Nicholls

Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer

Fresh organically grown pomegranates (Punica granatum L.) of the Wonderful cultivar were processed into three components: fermented juice, aqueous pericarp extract and cold-pressed or supercritical CO2-extracted seed oil. Exposure to additional solvents yielded polyphenol-rich fractions (‘polyphenols’) from each of the three components. Their actions, and of the crude whole oil and crude fermented and unfermented juice concentrate, were assessed in vitro for possible chemopreventive or adjuvant therapeutic potential in human breast cancer. The ability to effect a blockade of endogenous active estrogen biosynthesis was shown by polyphenols from fermented juice, pericarp, and oil, which inhibited aromatase activity by 60–80%. Fermented juice and pericarp polyphenols, and whole seed oil, inhibited 17-β-hydroxysteroid dehydrogenase Type 1 from 34 to 79%, at concentrations ranging from 100 to 1,000 μg/ml according to seed oil ≫ fermented juice polyphenols > pericarp polyphenols. In a yeast estrogen screen (YES) lyophilized fresh pomegranate juice effected a 55% inhibition of the estrogenic activity of 17-β-estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. Inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (MCF-7) ≫ estrogen- independent (MB-MDA-231) > normal human breast epithelial cells (MCF-10A). In both MCF-7 and MB-MDA-231 cells, fermented pomegranate juice polyphenols consistently showed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. Pomegranate seed oil effected 90% inhibition of proliferation of MCF-7 at 100 μg/ml medium, 75% inhibition of invasion of MCF-7 across a Matrigel membrane at 10 μg/ml, and 54% apoptosis in MDA-MB-435 estrogen receptor negative metastatic human breast cancer cells at 50 μg/ml. In a %% murine mammary gland organ culture, fermented juice polyphenols effected 47% inhibition of cancerous lesion formation induced by the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The findings suggest that clinical trials to further assess chemopreventive and adjuvant therapeutic applications of pomegranate in human breast cancer may be warranted.

Inhibitors of steroidogenesis as agents for the treatment of hormone-dependent cancers

Oestrogens and androgens stimulate growth in hormone-dependent breast and prostate cancers respectively. Modern strategies seek to remove the influence of hormones on tumour growth using anti-oestrogens (breast) or anti-androgens (prostate) which block the action of the hormone on its receptor. For prostate cancer patients LHRH (luteinising hormone releasing hormone) agonists, which decrease testes synthesis of testosterone are also used. Newer alternative (or sequential) strategies aim to deplete circulating and tissue levels of the respective mitogenic hormone by inhibition of a specific target enzyme involved in its steroidogenic pathway: for breast cancer - aromatase (P450AROM) or 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 isoenzyme or the metabolic enzyme oestrogen sulfatase; for prostate cancer - 17α-hydroxylase: 17, 20-lyase (P450 17) or 5α-steroid reductase (5α-SR) or 17β-HSD Type 3 isoenzyme. The use of P450AROM inhibitors as sequential agents in breast cancer patients is well established; inhibitors of the other target enzymes are under development and could be sequential or combinational agents. Certain P450 17 inhibitors for prostatic cancer treatment protect the metabolism of retinoic acid (RAMBAs) and 1α,25-dihydroxy vitamin D-3, thereby being cellular differentiation and antiproliferative mimics. These new cytochrome P450 targets and the built-in action of the P450 17 inhibitors may be shown at a later date to alter or delay the progression of the disease from hormone-dependent to hormone-independent. This review discusses the progress made in developing of clinically useful steroidogenesis inhibitors for the relevant disease and some of the difficulties encountered in maintaining/achieving remission due to the changing nature of the disease.

Recent developments in aromatase inhibition as a potential treatment for oestrogen-dependent breast cancer

Publisher Summary This chapter discusses recent developments in aromatase inhibition as a potential treatment for oestrogen-dependent breast cancer. Breast cancer is one of the most common malignancies in women. Statistics in the U.S.A. alone show that over 100,000 new cases are diagnosed, and 40,000 deaths occur each year. The incidence increases with age, 75% of cases occurring between the ages 40 and 75. Eight out of ten women with the disease will die from it. Although 90% of patients present with a cancer that is clinically localized to the breast and the auxillary lymph nodes, the majority who eventually die of the disease suffer from systemic metastases in spite of local control. The influence of ovarian hormones, especially oesterogens, on the development of breast cancer has long been recognized. In laboratory animals, oestrogen administration is an effective means of inducing mammary tumours while in human patients the introduction of artificial menopause for reasons other than breast cancer can reduce the subsequent incidence of breast cancer by up to 75%. Oestrogens interact with the specific receptors to form an oestrogen-receptor complex, which migrates to the nucleus and interacts with the chromatin.

Liquid chromatographic determination of six antiepileptic drugs and two metabolites in microsamples of human plasma.

A simple, rapid, sensitive, and reproducible high-performance liquid chromatographic (HPLC) method for simultaneous determination of the antiepileptic drugs (ethosuximide, primidone, lamotrigine, phenobarbital, phenytoin, and carbamazepine) and two metabolites (carbamazepine-diol and carbamazepine-epoxide) in human plasma is described. The procedure involves extraction of the drugs from human plasma (100 microL) with ether using 9-hydroxymethyl-10-carbamyl acridan as an internal standard. The extract was evaporated and reconstituted with mobile phase and then injected onto the chromatograph. The drugs and the internal standard were eluted from a Supelcosil LC-18 stainless steel column at ambient temperature with a mobile phase consisting of a 0.01M phosphate buffer/methanol/acetonitrile (65/18/17, v/v/v) adjusted to a pH of 7.5 with phosphoric acid and a flow rate of 1 mL/min. The effluent was monitored at 220 nm. Quantitation was achieved by using peak area ratio of each drug to the internal standard. The intraassay and interassay coefficients of variation (CV) ranged from 2.43% to 6.25% and from 3.02% to 5.85%, respectively. The absolute (extraction) and relative (analytical) recoveries for the drugs ranged from 70.7% to 104.4% and from 88.3% to 106.1%, respectively. Stability tests showed that the drugs were stable in plasma for at least 4 weeks when stored at -20 degrees C. The method was applied clinically for monitoring the AEDs in epileptic patients.

A rapid liquid chromatographic method for the determination of lamotrigine in plasma

A rapid sensitive and simple high-performance liquid chromatographic (HPLC) method for the determination of lamotrigine in plasma is described. The drug was extracted from 100 microliters of plasma with chloroform:isopropanol (95:5% v/v) in the presence of 100 microliters of phosphate buffer (10 mM). The extract was evaporated and the residue was reconstituted with mobile phase and injected onto the HPLC system. The drug and the internal standard (chloramphenicol) were eluted from a Symmetry C18 stainless steel column at ambient temperature with a mobile phase consisting of 0.01 M potassium-acetonitrile-methanol (70.20:10% v/v/v), adjusted to pH 6.7, at a flow rate of 1.3 ml min-1 and the detector was monitored at 214 nm. Quantitation was achieved by measurement of the peak-area ratio of the drug to the internal standard and the lower limit of detection for lamotrigine in plasma was 20 ng ml-1. The intraday precision ranged from 3.34 to 6.12% coefficient of variation (CV) and the interday precision ranged from 2.15 to 8.34% CV. The absolute and relative recoveries of lamotrigine ranged from 86.93 to 90.71% and from 95.18 to 107.13%, respectively. The method was applied in studying the pharmacokinetics of lamotrigine administered orally to rabbits. This reliable micro-method would have application in pharmacokinetic studies of lamotrigine where only small sample sizes are available, e.g. paediatric patients.

Aminoglutethimide and ketoconazole: Historical perspectives and future prospects

Aminoglutethimide and ketoconazole, although originally developed as an anticonvulsant and antifungal agent respectively, have both been used to suppress steroid biosynthesis in patients with hormone-sensitive cancer. Aminoglutethimide inhibits several enzymes involved in the synthesis of corticosteroids as well as the aromatase enzyme which converts androgens to oestrogens. About one third of patients with breast cancer show objective improvement with aminoglutethimide, and it may also be of use in the treatment of adrenal carcinoma. However, its toxicity, and the need for concomitant cortisol replacement, severely limit its usefulness. Ketoconazole also inhibits several steroidogenic enzymes, notably C17,20-lyase, and has been used to treat carcinoma of the prostate. Again however, its toxicity and limited efficacy limit its value, although it may be useful in the treatment of certain endocrine conditions such as precocious puberty. Several aromatase inhibitors similar in structure to aminoglutethimide have been developed in an attempt to create more selective and efficient inhibitors. Some of these compounds have been tested in animals but none have as yet been subjected to clinical trials. Attempts to produce imidazole inhibitors of steroidogenesis are less advanced, although one compound (CGS 16949A) has been reported to be a more selective and potent aromatase inhibitor than aminoglutethimide. Selective and effective compounds could be of great value in the treatment of hormone-sensitive carcinoma.

Biological half-lives of [4–14C]testosterone and some of its esters after injection into the rat

Biological half‐lives for 14C from labelled testosterone in muscle and whole body of rats have been measured after intramuscular injection of [4‐14C]testosterone and its lower esters (formate to valerate). A relation has been observed between ethyl oleate‐water distribution coefficients, biological half‐lives in the rat and “times of maximum effect” in the rat and fowl.

The Measurement and Health Impact of Endotoxin Contamination in Organic Dusts from Multiple Sources: Focus on the Cotton Industry

Endotoxin is derived from Gram-negative bacterial membranes, and its inflammatory effects following inhalation are well characterized. The significance of this fact becomes apparent when the wide-ranging environments containing high levels of this microbial product are considered. Endotoxin is present in numerous industrial environments, especially where organic fibers are processed. Microbial contamination of these fibers mainly occurs at the agricultural stage. Materials such as flax and hemp are affected in this way, but the most important product in this context is cotton, from which chronic dust inhalation causes the disease byssinosis. Despite the fact that endotoxin constitutes a significant threat to public health, there are currently no occupational exposure limits for this toxicant. This communication describes the toxicology of endotoxin, and its role in inhalation-induced disease, focusing on measurement of airborne endotoxin in the occupational and domestic environments using the Limulus amebocyte lysate (LAL) enzyme assay. Following the success of the LAL assay for measuring endotoxin in dusts, our laboratory has examined its application to aqueous washes from cotton fibers. Reproducibility of the results was high, and data are presented displaying levels of endotoxin contamination in fibers from different cotton producing countries. Hence, worldwide comparison of industrial endotoxin concentrations can be readily made using this test. It would be highly desirable if the performance of the LAL assay facilitated introduction of industrial endotoxin safety limits, and in spite of minor surmountable shortcomings, the test is accurate, reliable, and well field-tested, so its continued widespread use may achieve this goal.

Substituted 1-[(benzofuran-2-YL)-phenylmethyl]-imidazoles as potent inhibitors of aromatase in vitro and in female rats in vivo

Substituted 1-[(benzofuran-2-yl)-phenylmethyl]-imidazoles are a new class of potent aromatase inhibitor with in vitro IC50 values < 10 nM for certain members using human placental enzyme. At a dose of 2 mg/kg in PMSG-stimulated rats, selected compounds effectively reduce the oestradiol levels by 82-98%.

Measurement of specific airway conductance in guinea pigs. A noninvasive method.

A constant volume plethysmographic technique has been developed to measure specific airway conductance (sGaw) in unanesthetized spontaneously breathing guinea pigs. The technique utilizes a specially designed animal restraining device and mask piece. sGaw is measured at end-expiration and does not require knowledge of thoracic gas volume. Control values are within the range reported previously for this species. The method is noninvasive with minimum stress to the animals. Exposure of guinea pigs to an aerosol of cotton dust extract produces similar qualitative changes (a fall) in sGaw to those observed in human volunteers exposed to the same aerosol. The method is proposed as a suitable model for the study of byssinosis (the occupational lung disease associated with chronic exposure to cotton dust). The technique may also be applied to the acute and chronic study of the airway response to other airborne pharmacological and toxicological agents.