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

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. of age, they were randomized into 5 groups, each containing 9 However, at the higher dosage level of 0.10 ^mol/week, hamsters, with each group having a mean weight of 54 to 55 g. They were treated once weekly p.o., as shown below. The vehicle there was no significant difference in survival or in weight was ethanol:trioctanoin (1:3), 0.1 ml. gain between the 2 groups. Wt at 5 wk Comparative Toxicity of Axerophthene and Retinyl Ace Survivors Wt at 3 wk (g)VehicleTreatment at 3 wk at 5 wk(g)0/90/98/9 tate. In 2 sets of experiments, axerophthene was clearly less toxic than was retinyl acetate when given p.o. to rats. aloneAxerophthene,0.01 The 2 retinoids were given in high doses to young rats, and Â¿imol/wkRetinyl inhibition of growth or lethality was measured over a 2- acetate,0.01 6"9/9 81 Â± 79/9 95 Â± week period (Table 2). At the lower dose level, a significant /Â¿mol/wkAxerophthene,0.10 2*9/9 98 Â± Â±2b9/9 114 difference between the 2 retinoids was evident only in terms /Â¿mol/wkRetinyl of growth suppression; by the end of 14 days, the rats acetate,0.10 101 Â±2Survivors 118 Â±2 treated with retinyl acetate had a much greater inhibition /imol/wk1/90/98/9 " Mean Â±S.E. ' The abbreviations used are: HPLC, high-pressure liquid chromatogra- 6 Not significantly different from respective group treated with phy; ODS, octadecyl silane. retinyl acetate; p > 0.2.

JUNE 1978 1735

Table 2 (12, 13). After repeated dosing of rats with either axero Comparison of toxic effects of large doses of axerophthene and phthene or retinyl acetate, the parent compound was not retinyl acetate given to rats found in the liver in any appreciable amount (Chart 4, E and Four-week-old female Sprague-Dawley rats fed Wayne laboratory f). Treatment with retinyl acetate caused marked elevation chow were randomized into 6 groups, each containing 6 rats, with of liver stores of retinyl palmitate with only a small increase each group having a mean weight of 58 to 60 g. They were treated 5 times/week (Monday through Friday) P.O., as shown below. The in liver retinol (Chart 4E). The increase in peak sizes at 18 vehicle was cottonseed oil containing 4% CHCI3. The experiment and 23 min (Chart 4E) probably represents the formation of was terminated at the end of 14 days. fatty acid esters of retinol other than the palmitate ester, such as the linoleate, oleate, and stÃ©arateesters (A. B. Survivors at 14 at 14 Roberts, unpublished observations). Comparison of Chart daysVehicleTreatment at 7 days(g)88 days6/66/64/66/65/6*0/6Wtdays(g)120 4, E and F, makes it clear that an equivalent p.o. dose of 0.2ml,alone, Â±72 Â±495 axerophthene caused much less deposition of retinyl 5times/wkAxerophthene,30.4 palmitate in the liver than did the same dose of retinyl Â±63 Â±6"65 5times/wkRetinyl/Â¿mol, acetate. A different pattern of metabolites was found in breast acetate,30.4 Â±86 Â±9117 extracts. Chart 4A shows that, compared to the liver, there 5times/wkVehicleÂ¿Â¿mol, is little endogenous retinoid in the breast. Treatment with retinyl acetate (Chart 46) caused the appearance of a major 0.3ml,alone, Â±67 Â±389 5times/wkAxerophthene,45.6 peak with retention time identical with retinol, with no peak Â±50 Â±5 seen in the retinyl palmitate region of the chromatogram. 5times/wkRetinyl/j.mol, Axerophthene treatment (Chart 4C) resulted in major depo sition of this substance in the breast with 2 much smaller acetate,45.6 Â±3Â°3523C2Survivors peaks seen in and immediately in front of the retinol region. 5times/wk6/66.66/66/65/65/6Wtat7Â¿imol, The identity of this last, most polar peak is unknown. Most " Mean Â±S.E. of the retinoid found in the breast after axerophthene 6 Significantly different from respective group treated with reti treatment is axerophthene itself, which is in marked con nyl acetate; p <0.05. trast to the situation found in the liver. c Significantly different from respective group treated with reti The total levels of retinoids found in both liver and breast nyl acetate; p < 0.001. after treatment with axerophthene were also quantitated with trifluoroacetic acid, and the results are shown in Table metabolism in these tissues. Reverse-phase HPLC with 3. Since retinol, retinyl acetate, and retinyl palmitate all Spherisorb ODS columns (8) eluted with methanohwater yield the same molar absorbance with trifluoroacetic acid gradients provides excellent resolution of retinol, retinyl at 616 nm (7), the determination of the molar concentration acetate, axerophthene, and retinyl palmitate, as shown in of total retinoid, expressed as retinol and/or retinyl ester, Chart 3. Retention times varied slightly depending on the age of the columns and on the particular Spherisorb ODS column used. Recovery of these retinoids is essentially quantitative with the methods used. Chart 4 shows the 0.7 profiles of parent substances and metabolites found in either the liver or breast after repeated p.o. dosing of rats 0.8 with either axerophthene or retinyl acetate. Tissue samples were obtained 24 hr after the last of 9 doses, and extracts 0.5 were applied to Spherisorb ODS columns. Since liver ex tracts from both treated and untreated animals contain 10 0.4 to 100 times more retinoid than do comparable breast extracts, a smaller aliquot can be applied to columns to 0.3 give chromatograms that have excellent base lines and sharp, symmetrical peaks, with no interference from tissue 0.2 lipids or other endogenous tissue constituents. Thus, anal ysis of liver extracts (Chart 4 D to F) provides chromato 0.1 grams with resolution essentially identical with that ob tained by the direct application of the pure marker retinoids shown in Chart 3. In contrast, breast extracts contain much 4 8 12 16 20 24 28 less retinoid, and larger volumes of sample must be applied TIME, MINUTES to the columns for adequate detection. Lipids in these Chart 3. Separation of retinoids by HPLC. A solution containing 0.36 /ig extracts, as well as the larger sample volumes, caused of retinol, 0.90 ng of retinyl acetate, 0.52 /Â¿9ofaxerophthene, and 4.74 Â¿ig of retinyl palmitate in methanol was applied to a 5-ftm Spherisorb ODS loss of resolution during HPLC analysis, as shown in Chart column (3 x 250 mm). A linear gradient, beginning with 83% methanol in 4, B and C. water and ending with 97% methanol in water, was run over a period of 12 Liver extracts from rats treated with solvent alone (Chart min, with a flow rate of 1.2 ml/min. Elution was continued with 97% methanol in water to remove retinyl palmitate from the column. Recorder 4D) showed a small peak of retinol and a major peak of artifact during the first 3 min, resulting from solvent injection, has not been retinyl palmitate, which is in accord with classical studies plotted.

1736 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 Table 3 â€žOB1S Comparison of tissue levels of retinoids 24 hr after the last of 9

0.4ftS p.o. doses Female Sprague-Dawley rats were dosed p.o. with retinoids (30 030.20.1MOÃ•0.7Ã•Â°-6KWITHSOLVENT WITHRETINYL ALONE'0UVER,TREATMENT ACETATE/ly\â€” /Â¿mol/dose)dissolved in 0.4 ml of ethanol:trioctanoin (1:3). The PHTHENE^yvv^|tiVFUVER.TREATMENT rats were treated 3 times weekly and killed 24 hr after the final â€”â€”-^~EUVER.TREATMENTdose. Tissue levels were determined with the trifluoroacetic acid method after extraction of tissue with ethyl ether. Numbers are expressed as total Â¿imolof retinoid found per g, wet weight, of tissue.

RetinoidSolvent rats5 control Â±0.17"- * Â±0.0004" 9.73 Â±0.51" 0.062 Â±0.006" WTTH W[TH WITH Retinyl acetate 56Liver0.843.22 Â±0.29"- <"Breast0.0043 0.5g ALONE1'_jSOLVENT RETINYLACETATEi AXEROPHTHENE*\1 AxerophtheneNo.of 0.295 Â±0.019e-rf 0.4Â§03OL2ai0ABREAST.THEATMENT " Expressed as /umol of retinol and/or retinyl ester. "Mean Â±S.E. r Significantly different from corresponding value for retinyl acetate; p < 0.001. LBBREAST,TREATMENTAJiCBREAST,TREATMENTWITHAXEROrf Expressed as /^mol of axerophthene. 4 8 12 16 20 terminal groups (20), the relatively low toxicity of axero TIME, MINUTES phthene that we have found in this study is not surprising. Chart 4. Analysis of retinoids in breast and liver after dosing with retinyl However, axerophthene is not devoid of ability to increase acetate or axerophthene. Female Sprague-Dawley rats were dosed p.o. with retinoids (30 /imol/dose) dissolved in 0.4 ml of ethanol:trioctanoin (1:3). liver storage of retinyl palmitate and therefore has some They were treated 3 times weekly and killed 24 hr after the ninth dose. potential for causing liver damage. The relatively high SamÃ³lesof breast and liver tissue were taken and ground with 5 parts of concentration of axerophthene found in breast tissue after anhydrous sodium sulfate. The dried tissue powders were then extracted with ether (10 ml for breast samples, 20 ml for liver samples), and 4-ml p.o. dosing is likewise not surprising; it was expected that aliquots of the ethereal extracts were taken. The ether was removed with this very nonpolar retinoid would concentrate in a tissue nitrogen, and the extract was redissolved in 400 /il of chloroform to which with a high lipid content. 500 /il of acetonitrile were subsequently added. Aliquots of these redissolved extracts were then applied to Spherisorb ODS columns; for breast extracts More striking is the relatively high biological activity of 100-Â¿ilsampleswere applied to the columns; for liver extracts 10-/il samples axerophthene in trachÃ©al organ culture and in growth were used. Gradient elution of columns was performed as shown in Chart 3. promotion in the hamsters fed vitamin A-deficient diets. Chromatograms are shown from representative single animals. The original amounts of tissue samples taken were as follows: A, 0.23 g; B, 0.18 g; C, Although the assumption that axerophthene must be me 0.37 g; D. 1.26 g; E. 0.64 g; F, 1.22g. Recorder artifact during the first 3 min. tabolized to a biologically active form remains to be proven, resulting from solvent injection, has not been plotted. Detection for breast samples (A, B, and C) was with a Spectra-Physics Model 8200 detector with it is probable that this is the case from the demonstrated a312-nm filter; for liver samples (D,E, and F) a Schoeffel Model 770 variable increase in retinyl esters in the liver following axerophthene wavelength detector set at 325 nm was used. The larger cell volume in the administration. In particular, the biological activity of axer 8200 detector resulted in a more stable base line for analysis of the breast extracts. Absorbance values for Breast Samples A, B, and C have been ophthene in trachÃ©alorgan culture suggests that enzymatic multiplied by 25 (0.04 AUFS). mechanisms for activation of retinoids are not confined to the liver and that nonhepatic tissues may play an important may be validly performed in a tissue in which these com role in the metabolism of retinoids. pounds are the only retinoids present in significant amount. Treatment of rats with retinyl acetate resulted in a 3-fold greater deposition of retinoid in the liver than did equivalent ACKNOWLEDGMENTS treatment with axerophthene. In contrast, axerophthene We thank Doris Little for assistance in preparation of the paper. William was more effective than was retinyl acetate in elevating Henderson has provided valuable assistance with the animal experiments. retinoid levels in the breast, although in this case breast retinoid after axerophthene treatment was mostly axero REFERENCES phthene itself; after retinyl acetate treatment an increased 1. Ames, S. R., Risely, H. A., and Harris, P. L. Simplified Procedure for amount of retinol was found. Extraction and Determination of Vitamin A in Liver. Anal. Chem., 8: 1378-1381, 1954. 2. Badische Anilin- und Soda-Fabrik A. G. Vitamin A Series Compounds. DISCUSSION Chem. Abstr.. 52: 14719, 1958. 3. Badische Anilin- und Soda-Fabrik A. G. Axerophthene. Chem. 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Dianne L. Newton, Charles A. Frolik, Anita B. Roberts, et al.

Cancer Res 1978;38:1734-1738.

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