Biological Activity and Metabolism of the Retinoid Axerophthene (Vitamin a Hydrocarbon)

[CANCER RESEARCH 38, 1734-1738, June 1978] Biological Activity and Metabolism of the Retinoid Axerophthene (Vitamin A Hydrocarbon) Dianne L. Newton, Charles A. Frolik, Anita B. Roberts, Joseph M. Smith, Michael B. Sporn, Axel NUrrenbach, and Joachim Paust National Cancer Institute, Bethesda, Maryland 20014 [D. L. N., C. A. F., A. B. R., J. M. S., M. B. S.], and BASF Aktiengesellschaft, 6700 Ludwigshafen am Rhein, Germany Â¡A.N., J. P] ABSTRACT ity and properties of this molecule. In this study we report a detailed investigation of the biological activity and metabo Biological properties of axerophthene, the hydrocarbon lism of axerophthene. Subsequent studies will deal with the analog of retino!, have been studied both in vitro and in possible effectiveness of this compound for prevention of vivo. In trachea! organ culture axerophthene reversed experimental breast cancer. keratinization caused by deficiency of retinoid in the culture medium; its potency was of the same order of magnitude as that of retinyl acetate. Axerophthene sup MATERIALS AND METHODS ported growth in hamsters fed vitamin A-deficient diets although less effectively than did retinyl acetate. Axer Axerophthene was synthesized as follows: 1100 g (1.75 ophthene was considerably less toxic than was retinyl mol) of crystalline all-E-retinyltriphenylphosphonium bisul acetate when administered repeatedly in high doses to fate (16) were dissolved in 1500 ml of dimethylformamide at rats. Administration of an equivalent p.o. dose of axer about 25Â°.A solution of 140 g (3.5 mol) of sodium hydroxide ophthene caused much less deposition of retinyl palmi- in 1100 ml of water was added while the temperature of the tate in the liver than did the same dose of retinyl acetate, mixture was kept at about 25Â°.After being stirred for 3 hr at while a greater level of total retinoid was found in the about 25Â°,the mixture was extracted with n-hexane (3 x mammary gland after administration of axerophthene. 800 ml). The n-hexane solution was washed twice with 500 ml of 60% aqueous methanol and concentrated in a vacuum INTRODUCTION to give 660 g of light yellow oil that partially crystallized upon standing overnight at room temperature. The semi- Both natural and synthetic retinoids have recently been solid material was stirred with 500 ml of methanol at 50Â°for shown to inhibit mammary carcinogenesis in the experi several min and filtered to obtain 143.5 g (30%) of axero mental animal (9, 14, 15). Although these studies indicate a phthene with m.p. 75-77Â°. An analytically pure sample was new approach to the prevention of breast cancer in human obtained by recrystallization from poly(oxyethylene):castor subjects, the potential chronic toxicity of the retinoids that oil (4). This pure sample had the following physical con thus far have been used in the experimental studies (retinyl stants: m.p. 81-82Â° (4); UV (cyclohexane): \max 323 nm, acetate and retinyl methyl ether) precludes their clinical use E'cm = 1850; H-nuclear magnetic resonance (CDCI,t, 220 in women at high risk for development of this disease. The MHz): 1.03 (1-CH:i, 6H), 1.71 (5-CH,), 1.76 (14-CH,, J = 6.6 identification of new retinoids that will have enhanced Hz), 1.95 (9-CH.,), 1.81 (13-CH,), and 6.11 ppm (7- and 8-H), activity, better pharmacokinetic properties, or lesser toxic in accordance with the literature (17). ity is therefore essential. In a previous study we showed Methods for assay of reversal of keratinization in hamster that after p.o. dosing of equivalent amounts the less polar trachÃ©al organ cultures have been described in detail (6, analog, retinyl methyl ether, yielded a greater concentration 19). The procedure and diet used for measurement of of retinoid in the breast of the rat when compared to retinyl growth promotion in vitamin A-deficient hamsters have acetate (20). Retinyl methyl ether is also more effective than been described at length (5). In studies on metabolism and is retinyl acetate for inhibition of mammary carcinogenesis tissue distribution, retinoids were extracted from tissue induced in Sprague-Dawley rats by 7,12-dimethylbenz- with peroxide-free ethyl ether after grinding of the tissue (a)anthracene (9). Further studies on relatively nonpolar with anhydrous sodium sulfate (1). In some experiments, retinoids therefore appear warranted. One such compound, total retinoid in tissue extracts was measured with trifluo- which has had little biological evaluation, is the retinoid roacetic acid (7). For the measurement of total retinoid, 10 hydrocarbon axerophthene (Chart 1). Early reports on the parts of reagent (trifluoroacetic acid:chloroform, 1:2, v/v) putative synthesis of the hydrocarbon with the structure were added to 1 part of tissue extract (redissolved in shown in Chart 1 were subsequently shown to be incorrect, chloroform after removal of the ether with nitrogen). Ab- since these early preparations were found to have the retro sorbance was then measured at 30-sec intervals at either structure (10, 11). Although the synthesis of the hydrocar 574 nm (\max for axerophthene, e = 125,000) or 616 nm (Amax bon compound shown in Chart 1 was subsequently re for retinol or retinyl esters, e = 160,000). Values used for ported in the patent literature (2, 3), there has been no molar extinction coefficients have been extrapolated to extensive published report dealing with the biological activ- zero time. Molar extinction values for retinol, retinyl ace tate, and retinyl palmitate in the trifluoroacetic acid reaction Received December 2, 1977; accepted March 8, 1978. have all been reported to be identical (7). Separation of 1734 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. Biological Activity and Metabolism of Axerophthene of growth than did those treated with axerophthene. At the higher dose level, the rats treated with retinyl acetate showed a markedly greater suppression of growth as early R=CH3=AXEROPHTHENE as 7 days after treatment; by 14 days there were no survivors R=CH,OH=RETINOL in the group treated with retinyl acetate, while 5 of 6 of the R=CH2OCOCH3=RETINYL ACETATE group treated with axerophthene survived. The differences Chart 1. Structures of axerophthene, retlnol, and retinyl acetate. between axerophthene and retinyl acetate are highly signif icant statistically; at the higher dose levels, the confidence retinoids by HPLC1 on Spherisorb ODS columns has re levels are greater than 99.9%. cently been described (8). In the present studies Levels of Metabolites in Liver and Breast after Multiple Dosing with Axerophthene and Retinyl Acetate. The avail methanoliwater solutions have been used for elution of columns rather than acetonitrileiwater solutions, as re ability of new methods for the accurate determination of ported previously. Use of methanol allows more effective retinoids and their metabolites in tissue samples has ena analysis of lipid-rich extracts on Spherisorb columns. bled us to determine significant differences between axer ophthene and retinyl acetate, both in terms of their relative distribution to the liver and breast and in terms of their RESULTS Reversal of Keratinization in Trachea! Organ Culture by Axerophthene. This assay is a highly sensitive measure of the ability of retinoids to control normal epithelial cell differentiation and has been used routinely to measure the relative potency of different retinoids (20). Dose-response curves for both axerophthene and all-frans-retinoic acid are shown in Chart 2. All-frans-retinoic acid has been used as a reference substance in this assay since it is the most potent retinoid found to date for/n vitro reversal of keratinization. Although axerophthene completely reversed keratinization at 10~6 and 10~7 M, it was ineffective in 3 of 17 trachÃ©al cultures at 10~8 M and in 15 of 22 cultures at 10 9 M, and it was totally ineffective at 10~10 M. In contrast, all-frans- io-9 retinoic acid was completely effective in reversing keratini CONCENTRATION (Ml zation at concentrations as low as 10~9 M. The activity of Chart 2. Dose-response curves for reversal of keratinization in organ axerophthene found in these studies is approximately equal cultures of retinoid-deficient trachÃ©alepithelium by application of axer ophthene or all-trans-retinoic acid. Tracheas (1/dish) were treated with either to that previously reported for retinyl acetate (20), although careful titration in the 10~e- to 10~9-M range would be axerophthene or retinoic acid for 7 days before scoring for the presence of both keratin and keratohyaline granules. A total of 20 cultures was treated with no retinoid; of these, 17 (85%) were keratinized. Numbers of cultures required for a more exact comparison of these 2 com for each point shown were as follows: axerophthene, 10 6 M, 8; 10"' M. 22; pounds. 1Q-" M, 17; 10"' M, 22; 10"'Â°M, 17; retinoic acid. 10~' M, 7; 10 â€¢M,15; 10~'Â° Effect of Axerophthene on Survival and Weight Gain of M, 14. Hamsters Fed Vitamin A-deficient Diets. A comparison of the relative ability of axerophthene and retinyl acetate to Table 1 maintain survival and weight gain in animals deprived of Comparison of effects of small doses of axerophthene and retinyl acetate on weight gain and survival of hamsters maintained on dietary retinoids is shown in Table 1. When hamsters were vitamin A-deficient diets given only 0.01 Â¿imol of either retinoid per week, retinyl Syrian golden hamsters were maintained on a vitamin A-defi acetate was clearly superior to axerophthene, since ham cient diet from birth and were weaned at 21 days of age. At 36 days sters did not survive on this low dose of the hydrocarbon.

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