Chromatographic techniques for the determination of putative dietary anticancer compounds in biological fluids.

Although a great number of papers demonstrate an association between high intake of fruits and vegetables and reduced risk of certain types of cancer, the epidemiological evidence is not conclusive. The identification and quantification of specific dietary anticancer compounds in plasma, urine and tissues is an important aspect of this research. We surveyed the recent literature for original papers which involved the use of separation techniques for the detection and quantification in biological fluids and tissues of putative anticancer compounds which are present in the diet. The compounds included in this review are flavonoids, phytoestrogens, carotenoids, retinoids, vitamin E and ascorbic acid. The review covers papers published in the last 3 years. For each class of compounds we discuss the sample preparation, chromatographic conditions, and validation of the methods used, in order to identify current trends in the bioanalysis of each class of these substances.

Studies on the metabolism and disposition of the new retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl] benzoic acid. 4th communication: absorption, metabolism, excretion and plasma protein binding in various animals and man.

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid (CAS 94497-51-5, Am-80) is a new synthetic retinoid which has been shown to have a potent topical antipsoriatic activity. Pharmaco-kinetic profiles of Am-80 were studied in dogs, mice and rabbits after percutaneous or subcutaneous administration of 14C-Am-80. Plasma protein binding of 14C-Am-80 was also studied in rats, dogs and humans. After topical application of 14C-labeled Am-80 by occlusive dressing technique at a dose of 1 mg 14C-Am-80/1,000 mg ointment/kg, the blood and plasma levels of radioactivity were below the detection limit in normal-skin dogs. In normal skin mice and rabbits, the plasma radioactivity peaked at 8 h (40.8 ng eq./ml) and at 12 h (34.0 ng eq./ml) after application, respectively. Percutaneous absorption of 14C-Am-80 was less than 2% of the dose for dogs, 34% for mice and 23% for rabbits. After subcutaneous administration at a dose of 1 mg/kg to mice, dogs and rabbits, plasma levels of radioactivity peaked at 1, 4 and 4 h after dosing with a concentration of 614.0, 902.9 and 757.7 ng eq./ml and then it declined with half-lives of 2.4, 7.2 and 4.1 h, respectively. Urinary and fecal excretion of radioactivity after subcutaneous administration at a dose of 1 mg/kg was 3.5 and 94.7% of the dose in dogs, 27.0 and 73.2% in mice and 43.5 and 45.6% in rabbits. A possible gastrointestinal secretion, which might lead to excretion into feces, was suggested from the results with bile-duct-cannulated dogs. Unchanged Am-80 was present in high amounts in the plasma and bile or feces of all animal species tested except in rat bile, in which Am-80 was predominantly detected in the form of its taurine conjugate (M-6). Hydroxylation of Am-80 to yield 7-hydroxy-Am-80 (M-4) and 6-hydroxy-Am-80 (M-3), which lead to the formation of 6-oxo-Am-80 (M-5), were commonly observed in all animal species. Taurine conjugation reaction of unchanged Am-80 and hydroxy-Am-80 (to form M-6 and both M-1 and M-2, respectively) was distinct in rats and dogs, but, hardly detected in mice and rabbits. The presence of tetrahydro-tetramethyl-naphtylamine (TTNA) was most marked in mice, followed by rabbits and rats, but it was almost absent in dogs. HPLC-RIA analysis of human samples obtained from the phase II and phase III clinical trials of Am-80 ointment suggested that fecal excretion was the major elimination route, and that hydroxylation and taurine conjugation reaction of unchanged and hydroxy-Am-80 also occurred. Unchanged Am-80 was predominant in human plasma as compared with metabolites M-1 to M-6. In vitro binding of 14C-Am-80 to the plasma protein was found to be more than 99% in rats, dogs and humans. In vivo plasma protein binding of 14C-Am-80 and/or its radioactive metabolites was also found to be more than 98% in rats and dogs after subcutaneous administration of 14C-Am-80. In both dogs and humans, in vitro. 14C-Am-80 appeared to be bound predominantly to serum albumin. The binding of 14C-Am-80 to human serum albumin was scarcely affected in the presence of diazepam, digitoxin or warfarin, indicating that there are no specific binding sites for Am-80 on serum albumin.

Studies on the metabolism and disposition of the new retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl] benzoic acid. 2nd communication: absorption, distribution and excretion after single and consecutive subcutaneous administration in rats.

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid (CAS 94497-51-5, Am-80) is a new synthetic retinoid which has been shown to have a potent topical antisporiatic activity. The accumulation characteristics of Am-80 were examined in rats after a single and consecutive subcutaneous administration of 14C-labeled Am-80 once a day for 24 days, at a daily dose of 0.2 mg/kg. As compared with the single administration, Tmax (1-2 h) and Cmax (about 50 ng eq./ml) of the blood radioactivity were not altered markedly after the consecutive administration. During the daily subcutaneous dosing, the blood level of radioactivity at 24 h after each dosing was also very low. These findings suggested that accumulation in the blood was low after long term consecutive administration of Am-80. The plasma levels of total radioactivity and the proportion of unchanged Am-80 to the total plasma radioactivity, being about 80% at 2 h after administration, and plasma elimination half-life of Am-80, being approximately 3 h, appeared to be hardly affected by the consecutive administration. The cumulative excretion of radioactivity at 168 h after the final dosing was 6.7% and 89.1% in the urine and feces, respectively. The radioactivity remaining in the carcass at this time was about 3% of the total dose. The excretion profile was not altered by the consecutive administration. In most tissues, the concentration of radioactivity at 24 h after each dose reached a steady-state within 24 doses. At 2 h after the consecutive administration for 24 days, the highest concentration of radioactivity was found in the liver followed by the adrenal gland. Accumulation and delayed elimination of radioactivity in most tissues, especially in the adrenal gland, fat, skin and epididymis, were evidently observed as predicted from the previous study where 14C-Am-80 was administered at a dose of 1 mg/kg. The profile of accumulation and retention of radioactivity after the consecutive administration may be considered as a common characteristic of retinoids, such as etretinate.

Systemic pharmacokinetics of acetylenic retinoids in rats.

In order to widen the therapeutic index of retinoids, one approach is to synthesize retinoids with reduced systemic distribution. Sixteen acetylenic retinoids were evaluated for their systemic disposition kinetics in rats after iv doses. Four pharmacokinetic parameters (i.e., total body clearance, volume of distribution at steady state, mean residence time, and the elimination half-life) were calculated for all retinoids tested. These compounds were categorized into four groups according to their functional head group. Retinoic acids having the trimethylcyclohexenyl head group as isotretinoin most mimicked isotretinoin in disposition profiles among all retinoic acids examined. They had volumes of distribution similar to and mean residence times shorter than those of isotretinoin. Retinoic acids containing the tetramethyltetralinyl head group as arotinoid had extensive tissue distribution and small body clearance. They had extended elimination half-lives similar to those observed for etretinate. Dimethylchromanyl and dimethylthiochromanyl retinoic acids were more polar; their terminal half-lives were reasonably short and no extensive tissue distribution was noted. The ethyl retinoates rapidly converted to their corresponding retinoic acids after iv doses. All ethyl esters had limited systemic residence times. The ethyl nicotinates tended to have much larger body clearance (10- to 25-fold) than the ethyl benzoates. After iv administration of ethyl retinoates, the ethyl esters disappeared rapidly, while their corresponding retinoic acids became the major drug-derived species in blood. The study results demonstrated different pharmacokinetic behaviors of acetylenic retinoids with different functional head groups.

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma.

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.