[CANCER RESEARCH 40, 3413-3425, October 1980] Structure-Activity Relationships of Retinoids in Hamster Trachea! Organ Culture Dianne L. Newton, William R. Henderson, and Michael B. Sporn1 Laboratory of Chemoprevention, National Cancer Institute. Bethesda, Maryland 20205 ABSTRACT activity of new retinoids, the concentration of which may be as low as 10~'°to 10~" M during the assay procedure. Thus, it Structure-activity relationships are summarized for 87 reti- is possible to measure biological activity with less than 1 mg of noids, using reversal of keratinization in the hamster trachéal a new retinoid; with radioactively labeled retinoid metabolites, organ culture system to measure biological activity. Classes of assays have been performed with only a few jug of material. compounds evaluated include all-frans-retinoic acid and its The length of assay is relatively brief, and test results may be esters, ring-modified analogs of all-frans-retinoic acid and its evaluated within 1 month after beginning the assay. esters, side-chain-modified analogs of all-frans-retinoic acid This article provides data on 87 retinoids, from a total set of and its esters, analogs in which both ring and side chain have over 30,000 hamster tracheas which have been used for as been modified, all-frar/s-retinol and derivatives, all-frans-retinyl says at the National Cancer Institute. Each trachea has come amine derivatives, all-frar/s-retinal derivatives, all-frans-retinoic from a separate animal which had been raised on a vitamin A- acid amides, 13-c/s-retinoic acid and derivatives, and 5,6- deficient diet. From each hamster, the trachea was removed epoxyretinoids. The activity of many synthetic amide deriva under sterile conditions and maintained in organ culture in vitro tives of all-frar/s- or 13-c/s-retinoic acid approaches that of the for 3 to 10 days, as described below. Data are shown for parent compounds. No metabolite of all-frans- or 13-c/s-reti retinoids in which the ring, side-chain, and polar terminal group noic acid has yet been identified which has greater activity of the basic molecule have been modified. than the parent compounds in this assay. New synthetic deriv atives with a gem-dimethyl group at position 4 in the cyclo- MATERIALS AND METHODS hexenyl ring and two aromatic rings in the side chain have activity equal to or greater than that of all-frar/s- or 13-c/s- Standard Assay Procedure. The standard assay procedure retinoic acid, with some activity detectable in the 1CT11 M measures the ability of retinoids to reverse keratinization in a range. defined in vitro system (6, 19). Tracheas from hamsters that were in very early stages of vitamin A deficiency (5) were placed in organ culture. At the time of culture, the animals were INTRODUCTION 29 to 31 days old (they had been weaned at 21 days) and were This article summarizes data obtained during the past 7 still gaining some weight, with an average weight of 47 to 52 g. Their trachéalepithelium was generally low columnar or cu- years in a collaborative testing program which has evaluated the biological activity of new synthetic retinoids, using the boidal, with only occasional patches of squamous metaplasia. hamster trachéalorgan culture assay developed in the labo Each trachea was opened from the larynx to the carina along the membranous dorsal wall and cultured in a serum-free ratories of the Lung Cancer Branch and Laboratory of Chem medium (CMRL-1066 supplemented with crystalline bovine oprevention of the National Cancer Institute. This testing has involved a collaborative effort on the part of the United States insulin, 1.0/ig/ml; hydrocortisone hemisuccinate, 0.1 ftg/ml; government; the pharmaceutical industry in the United States, glutamine, 2 rriM; penicillin, 100 units/ml; and streptomycin, Switzerland, and Germany; and the academic community and 100 fig/ml). Cultures were gassed with 50% oxygen, 45% research institutes in America. The eventual goal is to develop nitrogen, and 5% CO?. The culture dishes were rocked at 35.5 a safe and effective pharmacological agent for prevention of to 36.0 degrees to allow the tracheas contact with both gas common forms of cancer in men and women, one which would and medium. All tracheas were maintained in medium contain be used as a chronic prophylactic measure to inhibit the ing no retinoid for the first 3 days. At the end of 3 days, some development of invasive cancer, much in the same manner in tracheas were harvested; almost all of these tracheas had which fluoride is used for prevention of dental caries. significant squamous metaplasia; in a set of several thousand Since the trachéalorgan culture assay measures the intrinsic such cultures approximately 60% had keratinized lesions. The ability of retinoids to control epithelial cell differentiation (6, remaining tracheas were then divided into different groups which were then treated with either: (a) retinoid dissolved in 31), it is believed to have significant predictive value for the spectrograde DMSO2 (final concentration of DMSO in culture potential use of a new retinoid for prevention of epithelial medium was never greater than 0.1%) or (b) an equivalent cancer. Obviously, any in vitro test has dangerous liabilities for prediction of in vivo activity; in spite of these limitations, the amount of DMSO alone. Culture medium was changed 3 times trachéalorgan culture assay is a most valuable procedure for a week, and all of the remaining tracheas were harvested at the end of 10 days in culture. Tracheas were fixed in 10% initial evaluation of the biological activity of a new retinoid. It is buffered formalin and embedded in paraffin. Cross-sections of an extremely sensitive assay and is used routinely to evaluate 5 jum were made through the midportion, stained with hema- ' To whom requests for reprints should be addressed. Received April 24. 1980; accepted July 2. 1980 2 The abbreviation used is: DMSO, dimethyl sulfoxide OCTOBER 1980 3413 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1980 American Association for Cancer Research. D. L. Newton et al. toxylin and eosin, and then scored with a microscope for the ides as inhibitors of carcinogenesis. Chart 10 shows 2 new presence of keratin and keratohyaline granules, both of which structures, in which both the ring and the side chain of the were found in approximately 90% of control cultures (in a set retinoid molecule have been modified. In these retinoids, a of several thousand such cultures) that had received no retinoid gem-dimethyl group has been inserted at position 4 of the for the entire 10-day culture period. Analogs were scored as cyclohexenyl ring and 2 aromatic rings have been introduced "inactive" if both keratin and keratohyaline granules were into the side chain; these new structures are intensely active. seen; they were scored as "active1 if neither keratin nor In view of the suggestion that 5,6-epoxyretinoic acid might be keratohyaline granules were seen. an activated form of retinoic acid (16, 29), this compound, as Retinoids. Retinoids were received in sealed ampuls, under well as 3 related analogs (5, 6-epoxyretinoic acid methyl ester, argon or nitrogen. These sealed ampuls were stored at -20° 5,6-epoxyretinyl acetate, and the 5,6-epoxy analog of retinyli- or below. Once opened, ampuls were stored in a vapor-phase dene dimedone), were tested (Chart 11). All of these epoxides nitrogen freezer. Stock solutions of retinoids were made in are significantly less active in the trachéalorgan culture assay DMSO, at 0.1 to 1.0 mg/ml, and vials were stored in a vapor- than were their respective parent compounds. phase nitrogen freezer. After thawing, contents of a given vial The trachéalorgan culture assay is highly specific for retinoid were used only once and then discarded. All work with retinoids structures. A wide variety of other terpenoid and related struc was performed under subdued light or in rooms with "gold" tures have been tested and been found to be inactive (data not fluorescent lamps. shown); these compounds include juvenile hormones I, II, and III; mycophenolic acid, and abscisic acid. Although /?-ionone RESULTS has not been tested, it is apparent that the side chain is of Charts 1 to 11 show the activity of 87 retinoids in the hamster major importance for biological activity, since the C,5 and C17 trachéalorgan culture assay. Dose-response curves (19) were analogs of retinoic acid (Chart 3) are almost totally inactive, made for each retinoid, and the values derived for the dose and each of the 4 isomerie dihydroretinoic acid analogs (Chart effective in suppressing keratinization in one-half of the cul 3) is markedly less active than is all-frans-retinoic acid or its tures have been tabulated. All-frans-retinoic acid [50% effec methyl and ethyl esters. Similarly, the Ci8 ketone (Chart 8) was tive dose, 3 x 10~n M in a total of 1853 cultures (Chart 1)] totally inactive at 1 x 10~8 M. has been used as the reference substance. Most of the analogs in which the ring (Chart 2) or the side chain (Chart 3) has been DISCUSSION modified are significantly less active in the trachéal organ culture assay. Retinol, retinyl acetate, and retinyl ethers (Chart The data shown in Charts 1 to 11 indicate that a very wide 4) are all less active than retinole acid, as are retinyl amine range of modifications in the ring, side chain, or polar terminus derivatives (Chart 5). A large number of retinal derivatives have of the retinoid molecule can be made and still allow expression been made, some of which have been useful in various biolog of biological activity.