The synthesis of a new retinoid, N-(4-hydmoxyphenyl)-alltrans-retinamide, which has useful biological properties, isdescribed. This metinoid was more potent than retinyl acetate in reversing kematinization caused by retinoid deficiency in tracheal organ culture. It was markedly less toxicthan retinyl acetate when fed p.o. to rats over 2-week on 6-month periods. It was an effective agent for inhibition of thedevelopment of breast cancer induced in matsby N-nitrosoN-methylurea, although it was not as potent as retinylacetate in this regard. However, the lesser toxicity of 4-hydroxyphenylmetmnamide makes it a superior agent forprevention of breast cancer. High-pressure liquid chromatographic analyses of liver and breast extracts from ratstreated for 6 months with retinoids show the pharmacokinetic basis for the superiority of 4-hydmoxyphenylmetinamide; this retinoid and its metabolites were found in highconcentrations in breast tissue, without any measurableaccumulation in the liver or evident liver toxicity. In contrast, chronic feeding of metinyl acetate caused markeddeposition of retinyl esters in the liven and severe hepatotoxicity. Whole mounts of nat mammary glands, made afterchronic feeding of 4-hydroxyphenylretmnamide, showed thatit had a marked antiproliferative effect on mammary epithehum.
INTRODUCTION
Breast cancer continues to be the leading cause of deathfrom cancer among American women (18). Although manywomen at extremely high genetic risk for development ofpremenopausal breast cancer can be identified, there is noeffective preventive measure, short of prophylactic mastectomy. While vitamin A acetate (retinyl acetate) has beenshown to be highly effective in preventing breast cancerinduced in the rat by carcinogens such as 7,12-dimethylbenz(a)anthracene (14) or N-nitroso-N-methylurea (15), thetoxicity of retinyl acetate, particularly its ability to causesevere liver damage when given chronically in high dosage(16, 19), precludes its clinical use as an effective means ofbreastcancerprevention.We reportherethesynthesisandbiological activity of a new synthetic retinoid, N-(4-hydroxyphenyl)retinamide (Chart 1), which is shown to be markedly less toxic than retmnyl acetate and to have usefulproperties for prevention of breast cancer in the experimentalanimal.
I To whom requests for reprints should be addressed.
Received November 1, 1978; accepted January 12, 1979.
Retinoids.N-(4-hydmoxyphenyl)-aII-trans-retinamidewassynthesized (using foil-wrapped flasks throughout) as follows: a 1-liter 3-necked flask was fitted with a water condenser bearing a Drienite tube, a dropping funnel, a nitrogen inlet tube, and a motor-driven stirrer. Retinoic acid (45g, 0.150 mol) and 600 ml of benzene were added and stirredvigorously while the flask was swept with nitrogen. Asolution of 13.8 g (0.101 mol) of PCI3in 150 ml benzene wasadded slowly (5 to 8 mm). Stirring under nitrogen wascontinued until the retinoic acid had dissolved, forming adeep orange to red solution of retinoyl chloride (2.0 to 2.5hr), which was then decanted through a plug of glass woolinto a dropping funnel. A 2-liter 3-necked flask containing81 .9 g (0.750 mol) of p-aminophenol dissolved in 240 ml ofN,N-dimethylformamide was connected as above. The solution was cooled in an ice water bath to 15°, stirred , sweptwith nitrogen, and the retinoyl chloride solution added over6 to9 mm; theratewas regulatedtomaintainthetemperature at 15°.After addition was complete, the contents wereheatedto 25°,stirredatthistemperaturefor2 hr undernitrogen, and then heated at 60°for 45 mm. The reactionmixture, in which some solid p-aminophenol hydrochloridewas suspended,was cooled to room temperature,transferred to a 5-liter flask, and diluted with 2000 ml of ethylether. The ether suspension was filtered rapidly on a coarsesintered-glass Buchner funnel with suction to remove solidp-aminophenol hydrochloride. The ether filtrate was transferred to a 4-liter separatory funnel where it was washedwith two 375-mI portions of cold 5% HCI, and then with four200-mI portions of cold water. The ether solution was driedovernight at room temperature with anhydrous sodiumsulfate and then filtered and evaporated on a rotary evaporator,maintainingthe solutionat room temperatureorslightly lower. Drying at 0.5 to 1.0 mm (oil pump) yielded ayellowsolidweighing55 to58 g.
This crude product was dissolved in methanol (5 ml/g) byrefluxingfora few minuteson thesteam pot.The solutionwas filtered hot, and crystallizationinducedby cooling inice and scratching. After standing overnight in the refrigator, the crystals were filtered and dried for 3 hr at roomtemperature in a vacuum desiccator (0.1 mm). The drycrystals weighed 44 to 46 g and were recrystallized a secondtime from a blend of chloroform and hexanes. A batch of 909 was heated under reflux on the steam pot with chloroform(4.1 mI/g) until it dissolved (10 to 15 mm), filtered, andreheated; hexanes (5 mh/g) were then added graduallythrough the condenser, keeping the temperature near the
APRIL1979 1339
N-(4-Hydroxyphenyl)retinamide, A New Retinoid for Preventionof Breast Cancer in the Rat
Richard C. Moon,' Henry J. Thompson, Peter J. Becci, Clinton J. Grubbs, Robert J. Gander, Dianne L.Newton, Joseph M. Smith, Sandra L. Phillips,William R. Henderson,LarryT. Mullen, Charles C. Brown,and Michael B. SpornlIT Research Institute, Chicago, Illinois 60516(R. C. M., H. J. T., P. J. B., C. J. G.); Johnson & Johnson, New Brunswick, New Jersey 08903 (R. J. G.J; andNational Cancer Institute, Bethesda, Maryland 20014(0. L. N., J. M. S., S. L. P., W. R. H., L. T. M., C. C. B., M. B. 5.1
tnioctanoin (3 parts), to which 0.05 ml each of the antioxidants, Tenox 20 (Eastman Chemicals, Kingsport, Tenn.)and DL-a-tocopherol, were added. The retinoid solutionwas thenblendedfor30 mm ina foodmixerwith950 g ofWayne laboratory chow. Batches of diet were made fresheach week and stored at 2-4°; food cups were changedtwice weekly. Analysis of samples of diet showed completestability of the netinoid under these conditions, even whenfood cups were kept at room temperature for 7 days. Retinylacetate was fed in a similar manner using the ethanol:tnioctanoin vehicle on a vehicle of corn oil (Mazola) containing2% CHCI3,0.1% Tenox 20,and 0.1% DL-a-tocopherol;equivalent results have been obtained with either of these 2vehicles.
Inductionof BreastCancerwithNitrosomethylurea.Vimgin female Sprague-Dawley matswere obtained from ARS/Sprague-Dawley, Madison, Wis., at 42 days of age. Asshown in Table 3, 15 groups of mats(a total of 350 animals)received an i.v. injection of either 0.85% NaGI solution (pH5.0) or 50 or 15 mg N-nitroso-N-methylumea (Ash Stevens,Detroit, Mich.) dissolved in 0.85% NaCI solution (pH 5.0)per kg body weight at both 50 and 57 days of age, accordingto a previously published modification (15) ofthe method ofGulhino et a!. (9). Three days after the second injection of0.85% NaCI solution or nitrosomethylurea, animals wererandomizedintogroupsand fedeithera controldietor adiet supplemented with either 328 or 656 mg netinyl acetateor 391 or 782 mg N-(4-hydroxyphenyl)retmnamide per kg ofdiet.Animalswere palpatedfortumors twiceeach weekand weighed weekly; they were checked twice daily forexternalsignsofmetinoidtoxicity.
At necropsy, all tumors as well as areas of uninvolvedmammary tissue were fixed in 10% buffered fommahin,stained with hematoxylin and eosin, and subjected to histopathological evaluation. Whole mounts of the right abdominal mammary glands were prepared from 3 animals ineach of the 5 groups of animals that received 0.85% NaCIsolution injections and then fed either control diet or diet
supplemented with metinoid (Table 3). The entire gland wasdissected from the animal, attached to a microscope slide,and fixed using 4% fommaldehyde:1% glutamaldehyde in a176 mOsM phosphate buffer (13). After overnight fixation,the glands were stained with alum carmine, dehydrated in agraded ethanol series, and cleared in toluene (2).
RESULTS
Reversalof KeratinizationinTrachealOrganCulturebyN-(4-Hydroxyphenyl)retinamide.This assaymeasurestheintrinsic ability of a retinoid to control epithelial cell differentiation and is a valuable screening procedure for initialevaluation of the biological activity of new metinoids. Table1 shows that N-(4-hydroxyphenyl)metinamide is highly active
inthisassay.At10@ and 10' M,kematinizationwas reversedcompletely; even at 10@ M, this netinoid was active. N-(4-Hydmoxyphenyl)retinamide was more active than metinylacetate at 10@ M, while both compounds were definitely lessactive than all-trans-retinoic acid. Thus, neither N-(4-hydmoxyphenyl)retinamide nor retinyl acetate were found tohave any significant activity at 10'° M; in contrast, in alarge series of cultures performed in our laboratory during
boiling point. Precipitation of the retinoid began whenaboutone-thirdofthehexane had been added.The crystalslurry was cooled in the refrigerator overnight, filtered anddried at room temperature in a vacuum desiccator for 1 hron the water pump, then for 2 hr longer with an oil pump(0.1 mm). The final product weighed 80 to 82 g, m.p. 162-163°,(uncorrected); UV (CHCI3): Amax370 nm, €44,500; UV(CH3OH):Xmax362 nm, €47,900.
C2H33NO2
Calculated:C 79.8,H 8.49,N 3.58Found: C 79.5, H 8.67,N3.56
Nuclear magnetic resonance (CDCl@:&):1.03 (@,26H, 1,1-gem-dimethyl), 1.45 to 1.64 (m, 4H, C-2 and C-3 protons),1.71 (5, 3H, C-5 methyl), 1.98 (5, 5H, C-9 methyl plus C-4protons), 2.38 (5, 3H, C-13 methyl), 5.76 to 6.41 (m, 5H, C-7,C-8,C-10,C-12,C-14protons),6.64to7.50(m,6H,amideproton, 4 benzenoid protons, C-il proton).
Retinyl acetate, retinoic acid, and retinol were generouslyprovided by BASF Aktiengesellschaft, Ludwigshafen , Germany, and Hoffmann-La Roche Inc., Nutley, N. J.
Analytical Methods. Methods for assayof reversalofkeratinization in hamster tracheal organ cultures have beendescribed in detail (4, 20). The procedure and diet used formeasurement of growth promotion in vitamin A-deficienthamsters have been described at length (3). In studies onmetabolism and tissue distribution, retinoids were extracted from tissue with peroxide-free ethyl ether aftergrinding the tissue with anhydrous sodium sulfate (1). Insome experiments, total retinoid in tissue extracts wasmeasured with trifluoroacetic acid (6). For the measurementof total retinoid, 10 parts of reagent (trifluoroacetic acid:chloroform, 1:2, v/v) were added to 1 part of tissue extract(redissolved in chloroform after removal of the ether withnitrogen). Absorbance was then measured at 30-sec intervals at either 564 nm (Ama,, for N-(4-hydroxyphenyl)-retinamide, €88,000) or 616 nm (Amaxfor retinol or retinylesters, €130,000). Values used for molar extinction coefficientshavebeenextrapolatedtozerotime.Molarextinctionvalues for retinol, retinyl acetate, and retinyl palmitate inthe trifluomoacetic acid reaction have all been reported tobe identical (6). Separation of retinoids by high-pressureliquid chromatography on Sphenisorb octadecylsilane columns has recently been described (7).
Feeding of Retinoids in Experimental Breast CancerStudies. N-(4-Hydroxyphenyl)metmnamide (1 or 2 mmol) wasdissolved in 50 g of a solution of ethanol (1 part) and
2 The abbreviations used are: s, singlet; m, multiplet.
Comparison of activity of retinyl acetate andN-(4-hydroxyphenyl)retinamidein hamster trachealorgancultureAssays
were performed under standard test conditions(20),usinga total culture period of 10 days. At the end of thistime,tracheas
were fixed in 10% buffered formahin, sectioned,andscoredwith a microscope for the presenceof keratin and kerato
hyahinegranules. A test result was scored as ‘‘inactive'‘ifbothkeratinand keratohyaline granules were seen; it was scoredas“active―if neither keratin nor keratohyalinegranuleswere seen,orif
amideConcentration (M) Active Inactive ActiveInactive10-i
36/375/510-.31/3123/2310-i12/3224/2810-10
4/4 19/21
N-(4-Hydroxypheny!)retinamide and Breast Cancer
over a 2-week period without evident toxic effects; theirbody weights more than doubled over this time. As will bedescribed below, N-(4-hydnoxyphenyl)metinamide was alsofound to be markedly less toxic than metinyl acetate when
fed chronically over a 6-month period to rats.Prevention of Mammary Carcinogenesis by N-(4-Hy
droxyphenyl)retinamide. N-(4-Hydnoxyphenyl)retinamidewas found to be an effective agent for prevention of mammary cancer induced in the rat by nitrosomethylunea, asshown in Chart 2 and Tables 3, 4, and 5, which comparethis synthetic retinoid with metinylacetate, both in terms ofefficacy of prevention of cancer, as well as freedom fromtoxic side effects when fed chronically. Two doses ofcarcinogen and 2 doses of each retinoid were used in thesechronic experiments. The proportions of animals with atleast one mammary cancer and the average numbers ofcancers per animal for each experimental group are shownin Tables 3 and 4. The statistical procedures used tocompare the differences between treatment groups are theFisher exact test (10) for comparing incidence rates and anonparametric test proposed by Mantel (11) for comparisons of the average numbers of cancers per animal. Tables3 and 4 show that the experimental system used is anexcellent one for studying dose-response relationships; inall sets of experiments, the animals given the high dose ofnitrosomethylurea had a greater incidence of mammarycancers than the respective animals given the low dose ofthis carcinogen. The system also shows striking dose-response with respect to the level of retinoid treatment.
For those animals given the high dose of carcinogen, theanimals not treated with retinoid had a mammary cancerincidence of 100%, with an average of 5.2 cancers/animalafter sacrifice at 129 days. These figures were significantlyreduced in each group fed retinoids: 67% incidence (p <0.01) with an average of 1.6 cancers/animal (p < 0.001 ) forthe low-doseretinylacetategroup;13% incidence(p <0.001) with 0.12 cancers/animal (p < 0.001) for the highdose retinyl acetate group; 80% incidence (not significant)with 2.9 cancers/animal (p < 0.05) for the low-dose N-(4-hydmoxyphenyl)retinamide group; and 65% incidence (p <0.01) with 2.3 cancers/animal (p < 0.01) for the high-doseN-(4-hydroxyphenyl)retinamide group.
For those animals given the low dose of carcinogen, theanimals not treated with retinoid had a mammary cancerincidence of 30% along with an average of 0.35 cancers!animal aftersacrificeat 182 days.These figureswerereduced in each of the groups with diets supplemented withretinoid, except for the low dose of N-(4-hydroxyphenyl)retmnamide. No cancers were found in 40 animalstreated with the high dose of retinyl acetate (p < 0.001).
The effect of treatment with the retinoids upon cancerlatency (time until appearance of the first tumor) was alsoexamined. Chart 2 shows the cumulative incidence ofmammary cancer as a function of time since cessation ofcarcinogen treatment. These figures clearly demonstratethat the retinoids increased the latency period after treatment with both high and low doses of carcinogen. Astatistical procedure proposed by Mantel (12) was used tocompare these latency curves. For the groups treated withthe low dose of carcinogen, compared to the untreatedanimals, significant differences were found for the low dose
Table 1
the past several years, metinoic acid has shown activity in134 of 256 cultures at 10b0.3
EffectofN-(4-Hydroxyphenyl)retinamideonSurvivalandWeightGainof HamstersFedVitaminA-deficientDiets.N-(4-Hydroxyphenyl)metmnamide was found to have some ability to maintain survival and weight gain in animals fedvitamin A-deficient diets. The activity of N-(4-hydmoxyphenyl)metinamide in promotion of growth was definitelyless than that of metinylacetate and metinoic acid, althoughthe activity measured depended on the dosage scheduleused. In a standard test system (3), hamsters were dosedtwice weekly p.o. with 0.05 @moIof N-(4-hydroxyphenyl)metinamide, which allowed weight gain equivalent tothat found in animals receiving one-tenth the same amountof metinyl acetate or metinoic acid. Hamsters dosed twiceweekly with either 0.005 j.@molof N-(4-hydroxyphenyl)-metinamide or the metinoid solvent (ethanol:tnioctanoin, 1:3)lost weight.
Comparative Toxicity of N-(4-Hydroxyphenyl)retlnamide, RetinoicAcid, and RetinylAcetate. In 2 setsof experiments, N-(4-hydroxyphenyl)metinamide was significantlyless toxic than metinoic acid or retinyl acetate, when givenP.O. to rats. The 3 retinoids were administered in massivedoses to young rats and inhibition of growth or lethality wasmeasured over a 2-week period (Table 2). Experiments wererun at 2 dose levels, and it is clear that N-(4-hydroxyphenyl)retinamide in massive doses does not suppressgrowth to the same extent as either retinoic acid or retinylacetate. All rats fed N-(4-hydroxyphenyl)retinamide sumvived, while 4 of 12 and 5 of 12 of the matsfed retinoic acidor retinyl acetate, respectively, died from retinoid toxicity(p < 0.05). The differences between N-(4-hydroxyphenyl)metinamide and the other 2 retinoids in terms ofweight gain as measured on the 14th day are highly significant statistically; confidence levels are greater than 99%.Moreover, no difference was found at 14 days between theweights of the matstreated with the lower dose of N-(4-hydroxyphenyl)retinamide and the rats treated with thesolvent alone (p > 0.4). Thus, young rats could be dosedwith more than 100 mg of N-(4-hydroxyphenyl)retinamide
3 D. L. Newton and M. B. Sporn, unpublished observations.
FemaleSprague-Dawleyrats, 4 weeks old, fed Wayne laboratory chow, were randomized into 6 groups, eachcontaining 6 rats, with each group having a mean weight of 57 to 58 g. They were treated 5 times/wk (Monday toFriday)p.o. , as shown below. Vehiclewas ethanol:trioctanoin (1:3).The experimentwas terminated at the end of 14days.Survivors
at Wt (g) at 7 Survivorsat Wt (g) at 14Treatment(5times/wk) 7 days days 14daysdaysVehicle
Table 2Comparisonof toxic effectsof large dosesof N-(4-hydroxyphenyl)retinamide,retinoic acid, and retinyl acetategiven
to rats
aMean±SE.b Significantly different from respective group treated with retinoic acid or retinyl acetate, p < 0.05.(. Significantly different from respective group treated with retinoic acid or retinyl acetate, p < 0.01.
alsobeenobserved.4Although N-(4-hydroxyphenyl)retinamide was found to be
somewhat less effective than retinyl acetate for preventionof breast cancer induced by nitrosomethylurea, it wasclearly less toxic when fed chronically over a 6-monthperiod. Table 5 shows the weight gains of rats on thevarious diets. Feeding of N-(4-hydroxyphenyl)retinamide,even at 2 mmol/kg of diet, did not cause any significantfailure to grow; matsfed this high dose of retinoid after0.85% NaCI solution injection i.v. had final weights whichwere 98% of those of rats fed no retinoid. In contrast, thehigh dose of retinyl acetate caused marked failure to grow(p < 0.001) in all groups treated for 6 months. At autopsy,the livers of rats treated with N-(4-hydroxyphenyl)-retinamide were essentially normal when evaluated by conventional histological methods (5-sm sections stained withhematoxyhin and eosin). The livers of animals receiving thehigh dose of retinyl acetate had moderate to severe generahized perilobular degenerative changes, with necrosis ofrandom hepatocytes in the degenerative regions. Thesechanges were characterized by cloudy swelling, hydropicdegeneration, and vacuolization that is consistent in appearance with fatty change. There were traces of fibrosisand mild hyperplasia of Kupffer cells in the degenerativeregions.
Levelsof Metabolitesin Liverand Breastafter ChronicFeeding of N-(4-Hydroxyphenyl)retinamideand RetinylAcetate. An ideal retinoid for prevention of breast cancerwould be one that selectively targets to the mammaryepithelium and does not accumulate or cause toxic effectsin any other tissue of the body. In particular, it is desired toobtain an effective level of retinoid in the mammary glandwithout a concomitant deposition of retinoid in the liver,which would eventually cause serious hepatic damage. Forexample, although feeding of retinyl acetate has beenshown to markedly elevate retinoid levels in the rat breast(17, 21), and to be markedly effective in prevention ofmammary cancer in the rat (8, 14, 15), the excessive
4 R. C. Moon, P.J. Becci, and H.J.Thompson, unpublished observations.
Uz4
4
0
Uz0U2
zU
0 20 40 60 ai 100120
DAYS AFTERFIRSTDOSEOF NMU
Chart 2. Effect of retinoids on latency for development of breast cancerafter iv. nitrosomethylurea (NMU). Conditions are described under Materials and Methods―and in Table 3. 0, no retinoid; t@,4-hydroxyphenylretinamide, 1 mmol/kg of diet; A, 4-hydroxyphenylretinamide, 2 mmol/kg of diet;0, retinyl acetate, 1 mmol/kg of diet; @,retinyl acetate, 2 mmol/kg of diet.All cancers were confirmed by histological diagnosis.
of retinyl acetate (p < 0.05), the high dose of retinyl acetate(p < 0.001), and the high dose of N-(4-hydmoxyphenyl)retinamide (p < 0.05). For the groups treated withthe high dose of carcinogen, compared to the untreatedanimals, significant differences were found for the low doseof retinyl acetate (p < 0.001), the high dose of retinylacetate (p < 0.001), and the high dose of N-(4-hydroxyphenyl)retmnamide (p < 0.001).
Whole mounts of the rat mammary gland clearly indicatethat the retinoids have a potent antiprohiferative effect onmammary epithehium, as seen in Figs. 1 to 3. Evaluation ofmammary whole mounts from rats treated with retinoids for182 days showed that both retinyl acetate (Fig. 2) and N-(4-hydroxyphenyl)retinamide (Fig. 3) decreased ductalbranching and end bud proliferation when compared tothose rats receiving no retinoid (Fig. 1). For both retinoids,a dose-response was observed; the glands from animals fedthe higher retinoid doses were less proliferative than thosefed the lower doses. Similar effects of N-(4-hydmoxyphenyl)retinamide on mouse mammary epithehium have
Incidence of mammary cancer after treatment withretinoidsRats
were dosedat 50and 57dayswith either 0.85%NaCIsolution or nitrosomethylurea,and then placedonspecialdiets,as described under “Materialsand Methods.―They were sacrificed after either 129 or 182 days, andtheincidence
of mammary cancer was established by histopathological examination.Tumor
incidenceRetinyl
acetate 4-HydroxYl@enyI
None (retiCarcinogen noid vehicle) High dose° Low dose@@ High dose― Lowdose@@None―
b@ mmoh/kg of diet.C All animals sacrificed at 182 days.
d Numbers in parentheses, percentage.E, All animals sacrificed at 129 days.
I Given twice, each dose 50 mg/kg body weight.0 Significantly different from respective groups that received no retinoid, p < 0.001 (one-sided).
h Significantly different from respective group that received no retinoid, p < 0.01 (one-sided).I Given twice, each dose 15 mg/kg body weight.
Table 4Averagenumber of mammarycancersper animal after treatmentwith retinoids
Conditions are described in Table3. All cancerswere confirmed by histological diagnosis.
a Significantly different from respective group that received no retinoid, p < 0.001 (one-sided).
b Significantly different from respective group that received no retinoid, p < 0.01 (one-sided).C Significantly different from respective group that received no retinoid, p < 0.05 (one-sided).
Conditions are described in Table 3. Starting weight of rats at 50 days of age was 160 ±3 g.
a All animals sacrificed at 182 days.
b Mean ±S.E.C Numbers in parentheses, percentage comparison of a particular group with the respective control group that received no retinoid.
d All animals sacrificed at 129 days.
e Significantly different from respective control group that received no retinoid, p < 0.001.
accumulation of retinyl esters in the liver that occurs withretinyl acetate feeding (17, 19) is of serious concern. However, N-(4-hydroxyphenyl)retinamide has a markedly different pharmacokinetic profile, as shown in Table 6 and Charts3 and 4.When ratswere fedthisretinoidforas longas 6months, there was marked elevation of total retinoid in the
breast (Table 6; Chart 4C), without any detectable increasein liver retinoid levels (Table 6; Chart 4F). High-pressureliquid chromatography analyses of breast extracts afterchronic feeding of N-(4-hydroxyphenyl)retinamide (Chart40) indicatethat the predominantmaterialspresentaremetabohites of N-(4-hydroxyphenyl)retmnamide, rather than
Comparisonof tissue levelsof retinoids after 6 months offeedingConditionsare described in Table 3. Samples of liver and breast tissue were obtained when rats were sacrificed (182 days after startofexperiment),and tissue levelsof retinoids weredeterminedwith tnifluoroaceticacid, asdescribed under “AnalyticalMethods.―Absorbance
units/g, wet wt, oftissueLiver
retinoid, Breastretinoid, measuredatmeasured at 616
Chart 3. Separation of retinoids by high-pressure liquid chromatography.Reference samples of retinoids (approximately 10' to 10'@ mol of each)were applied to a 10-sm Spherisorb octadecylsilane column (3 x 250 mm). Alinear gradient, beginning with 31% acetonltrile in water and ending with97%acetonitrllein water,wasrun overa periodof 60mm,with a flow rateofI .2 mI/mm. Elution was continued with 97% acetonitrile in water to removeretinyl palmitate from the column. Absorbance was measured at 325 nm (A)or 350 nm (B); values in both charts have been multiplied by 25 (0.04absorbance unit full scale).
the parent compound itself. The chemical identity of thesemetabohites remains to be determined, although it is ofinterest that one of them cochromatographs with the alkylated derivative, N-(4-methoxyphenyl)retinamide. Table 6shows that the accumulation of N-(4-hydroxyphenyl)-retinamide and its metabohites in the breast is dose dependent, whether or not rats have been pretreated with nitrosomethylurea. Similarly, accumulation of retinyl acetate andits metabohites in the breast is also dose dependent (Table6). However, feeding the higher (and toxic) dose of retinylacetate (2 mmol/kg of diet) did not result in a greaterconcentration of liver retinoid after 6 months than feeding1 mmol/kg of diet; this may be a manifestation of liverdamage as well as severe anorexia resulting from chronicoverdosage with retinyl acetate. Table 6 indicates that N-(4-hydroxyphenyl)retinamide did not increase liver retinoidlevels after 6 months of feeding, whether at the level of 1 or2 mmol/kg of diet and whether or not rats had beenpretreated with nitrosomethylumea.
1344 CANCER RESEARCH VOL. 39
DISCUSSION
The data presented here clearly indicate that N-(4-hydroxyphenyl)retmnamide has useful new properties for prevention of breast cancer in the experimental animal. While it isa biologically active retinoid, it is clearly less toxic to the ratthan either retinyl acetate or retinoic acid. Recently, it hasalso been shown in mice that N-(4-hydroxyphenyl)-retinamide is markedly less toxic than is all-trans-retinoicacid (5). This was particularly so after i.p. administration,indicating that the differences between the 2 retinoids arenot the sole result of differences in absorption. The dataclearly show that N-(4-hydroxyphenyl)retinamide can betargeted to the rat breast, without causing undesirableretinoid accumulation in the liver, and the whole mountsdemonstrate a marked antiprohiferative effect on mammaryepithelium. The ability of N-(4-hydroxyphenyl)retinamide toprevent mammary cancer in the rat does not appear to bemediated by an alteration of ovarian function, since ratswere found to have normal estrous cycles, as evaluated bycytology of vaginal smears, after 4 months of feeding of thisretinoid.
The ultimate practical use of retinoids for prevention ofcancer in men and women at high risk for development ofcancer will depend on both the efficacy and the safety ofthe specific retinoid which would be used in a given clinicaltrial. We have stressed the concept that modification of thepolar-terminal group of the retinoid molecule offers a usefulway to modify activity, toxicity, metabolism, and tissuedistribution of this class of compounds (22). The N-(4-hydroxyphenyl)amide derivative of retinoic acid is one suchmodification; it is clear that alteration of the terminal grouphas conveyed some highly useful properties on the retinoidmolecule. Further pharmacological studies on N-(4-hydroxyphenyl)retinamide, or one of its derivatives, with the goalof developing a practical agent for prevention of development of breast cancer in women at high risk should bepursued.
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11. Mantel, N. Chi-square tests with one degree of freedom : Extensions ofthe Mantel-Haenszel procedure. J. Am. Stat. Assoc. , 58: 690-700, 1963.
12. Mantel, N. Evaluation of survival data and two new rank order statisticsarising in its consideration. Cancer Chemother. Rep., 50: 163-170, 1966.
13. McDowell, E. M., and Trump, B. F. Histologic fixative suitable fordiagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100:405-414, 1976.
14. Moon, R. C., Grubbs, C. J., and Sporn, M. B. Inhibition of 7,12-dimethylbenz(a)anthracene-mnduced mammary carcinogenesis by retinylacetate. Cancer Res., 36: 2626-2630, 1976.
15. Moon, A. C., Grubbs, C. J., Spom, M. B., and Goodman, D. G. Retinylacetate inhibits mammary carcinogenesis induced by N-methyl-N-nitrosourea.Nature,267:620-621, 1977.
16. Muenter, M. D., Perry, H. 0., and Ludwig, J. Chronic vitamin A intoxication in adults. Hepatic, neurologic and dermatologic complications.Am.J. Med.50: 129-136, 1971.
17. Newton, D. L.. Frolik, C. A., Roberts, A. B., Smith, J. M., Spom, M. B.,NUrrenbach,A., and Paust,J. Biologicalactivityand metabolismof theretinoid axerophthene (vitamin A hydrocarbon). Cancer Res., 38: 1734—1738, 1978.
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andSaffiotti,U.ActivityofvitaminAanaloguesincellculturesof mouseepidermis and organ cultures of hamster trachea. Nature, 253: 47-50,1975.
21. Sporn, M. B., Dunlop, N. M., Newton, D. L., and Henderson, W. R.Relationshipsbetweenstructure and activityof retinoids. Nature,263:110-113,1976.
22. Spom, [email protected]., Newton,D. L., Smith,J. M., Acton, N., Jacobson,A. R.,Brossi, A. Retinoids and cancer prevention: The Importance of theTerminal Group of the Retinoid Molecule in Modifying Activity andToxicity. In: A. C. Griffin and C. R. Shaw (ads.), Carcinogens: Identification and Mechanism of Action, New York: Raven Press, in press, 1979.
N-(4-Hydroxyphenyl)retinamide and Breast Cancer
A B
BREAST.TREATMENTWITHRETINYLACETATE
0.9
0.8
0.7
0.6 BREAST.CONTROLDIET
0.5
0.4
0.3
0.2
0.1
0.0
11D
0.9
0.8
0.7
0.6 LIVER.CONTROLDIET
0.5
00:;2•:4:1A@jl!j!@j!@
32 36 40 44 48 52 56 00 64
Uz
32 36 40 44 48 52 @6 00 64TIME.MINUTES
Chart 4. Analysis of retinoids in breast and liver by high-pressure liquid chromatography after 6 months of feeding of retinoids. Female Sprague-Dawleyrats were treated as described in Table 6. Samples of breast and liver tissue were taken and ground with 5 parts of anhydrous sodium sulfate. The driedtI%ue powders were then extracted with ether (10 ml for breast samples, 20 ml for liver samples), and 4-mI aliquots of the ethereal extracts were taken. Theetherwasremovedwith nitrogen,andthe extractwasredissolvedin 400pJof chloroformto which 500 @siof acetonitrileweresubsequentlyadded.Aliquotsof theseredissolvedextractswerethenappliedto Spherieorboctadecylsilanecolumns;for brent extracts,50-pJsampleswereappliedto thecolumns;forliverextracts,10-@&Isampleswereused.Gradientelutionof columnswasperformedasshownin Chart3.Chromatogramsareshownfromrepresentativesingle animals. The original amounts of tissue samples taken were as follows: A, 0.4 g; B, 0.8 g; C, I .06 g; D, E, and F, 1.0 g. Absorbance was measured ateither325or 350nm.A, —, 325nm, valuesmuftipliedby 25 (0.04absorbanceunit full scale);- - - -, 350nm, valuesmultipliedby 6.25(0.16absorbanceunit full scale); B, 325 nm, and C, 350 nm, values muftiplied by 6.25 (0.16 AUFS); D, E, and F, 325 nm. When sample shown in F was monitored at 350 nm, nopeakswereseenfrom 32to 60mm.
APRIL1979 1345
ACKNOWLEDGMENTS
We thank Dr. Gavin Hildick-Smith for his helpful coordination of thiscollaborative project and Margaret B. Mose for her help in preparation of themanuscript.
REFERENCES
1. Ames, S. A., Risely, H. A., and Harris, P. L. Simplified procedure forextraction and determination of vitamin A in liver. Anal. Chem. , 8: 1378-1381, 1954.
2. Banerjee,M. R.,Wood,B. G., Lin, F.K.,andCrump,L. R.Organcultureof whole mammary gland of the mouse. Tissue Culture Assoc. J., 2: 457-462,1976.
3. Clamon, G. H., Spom, M. B., Smith, J. M., and Henderson, W. R. Effectof a-retinyl acetate on growth of hamsters fed vitamin A-deficient diets.J. Nutr., 105: 215-219, 1975.
4. Clamon, G. H., Sporn, M. B., Smith, J. M., and Saffiotti, U. a- and @-Retinyl acetate reverse metaplasias of vitamin A deficiency in hamstertrachea in organ culture. Nature, 250: 64-66, 1974.
5. Dollar, G. R., Hixeon, E. J., Arthur, M. W., and Denine, E. P. Comparativesubacute toxicity of N-ethyl retinamide,N-(2-hydroxyethyl)retmnamide,and N-(4-hydroxyphenyl)retinamsie in mice. Pharmacologist, 20: 248,1978.
6. Dugan, R. E., Frigerio, N. A., and Siebert, J. M. Colorimetric determination of vitamin A and its derivatives with trifluoroacetic acid. Anal.Chem., 36: 114-117, 1964.
7. Frolik, C. A., Tavela, T. E., and Spom, M. B. Separation of the naturalretinoids by high pressure liquid chromatography. J. Lipid Res., 19: 32-37, 1978.
8. Grubbs, C. J., Moon, A. C., Spom, M. B., and Newton, D. L. Inhibitionof mammary cancer by retinyl methyl ether. Cancer Res. , 37: 599-602,1977.
9. Gullino, P. M., Pettigrew,H. M., and Grantham,F. H. N-Nitrosomethylurea as mammary gland carcinogen in rats. J. NatI. Cancer Inst., 54:401-414, 1975.
Figs. 1 to 3. Representative areas of mammary gland whole mounts from rats given injections of 0.8% NaCI solution iv. at 50 and 57 days old, fed specialdiets, and sacrificed 182 days after the first injection.
Fig. 1. Control diet. Note the marked end bud proliferation (arrows). Alum carmine, x 40.Fig. 2. Retinyl acetate (328 mg/kg of diet). A marked decrease in end-bud proliferation is noted. Compare to Fig. 1. Alum carmine, x 40.Fig. 3. N-(4-Hydroxyphenyl)retinamide (782 mg/kg of diet). An almost total absence of end-bud proliferation is noted. Compare to Figure 1. Alum carmine,
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