novel dehydroepiandrosterone analogues with enhanced ...reduced side effects in mice and rats1...

(CANCER RESEARCH 48. 4817-4822, September 1, 1988)

Novel Dehydroepiandrosterone Analogues with Enhanced Biological Activity andReduced Side Effects in Mice and Rats1

Arthur G. Schwartz,2 Marvin L. Lewbart, and Laura L. Pashko

Fels Research Institute and Department of Microbiology, Temple University Medical School, Philadelphia 19140 Â¡A.G. S., L. L. P.], and Steroid Laboratory, Crozer-Chester Medical Center, Chester, Pennsylvania 19013 [M. L. L.]

ABSTRACT

Treatment of laboratory mice and rats with the adrenal steroid, dehy-

droepiandrosterone (DHEA), produces antiobesity and broad spectrumtumor chemopreventive activity. Certain side effects are associated withDHEA administration which could limit its usefulness as a drug. DHEAcan be metabolized into both testosterone and estrone and also increasesliver weight and liver catalase activity. We have developed two syntheticsteroids, 16a-fluoro-5-androsten-17-one and 16a-fluoro-5a-androstan-17-one, which, unlike DHEA, do not stimulate uterine weight in sexually

immature female rats or seminal vesicle weight in castrated male rats,nor stimulate liver weight and liver catalase activity in mice. 16a-Fluoro-5-androsten-17-one is also about three times as potent as DHEA as an

antiobesity agent and is more active when administered p.o. in inhibiting[3H]-7,12-dimethylbenz(a)-anthracene binding to skin DNA and tetra-decanoylphorbol-13-acetate stimulation of epidermal |3H]th) irridine in

corporation in the mouse, two effects believed to be important in thetumor preventive action of DHEA. 16a-Fluoro-5a-androstan-17-one isas active as 16a-fluoro-5-androsten-17-one in inhibiting [3H]-7,12-di-

methylbenz(a)anthracene binding to skin DNA and tetradecanoylphor-bol-13-acetate stimulation in epidermal |3H]thymidine incorporation but,

on the contrary, is not more active than DHEA as an antiobesity drug.Compounds such as 16a-fluoro-5-androsten-17-one and 16a-fluoro-5a-androstan-17-one, which lack specific side-effects of DHEA treatment

and demonstrate greater potency, may have therapeutic application asdrugs for humans.

INTRODUCTION

DHEA3 and DHEA-sulfate are abundantly produced adrenal

steroids in the human (1, 2). Although referred to as adrenalandrogens, these steroids have little, if any, intrinsic androgenicactivity, and it is only through metabolism to steroids such astestosterone that they exert such action. DHEA-sulfate is aprecursor for estrogen biosynthesis in the pregnant humanfemale (3). Otherwise the biological significance of these steroids in the male and nonpregnant female has never been clear.

The plasma levels of DHEA and DHEA-sulfate remain lowuntil around the age of 7, increase during adrenarche, peakbetween the ages of 15 and 25, and then decrease steadilythroughout life to levels of 10-20% of their maximal values (4).A large body of evidence accumulated over the past 25 yearspoints to the existence of subnormal plasma levels of DHEAand DHEA-sulfate in women with breast cancer (5-8). Ofparticular interest is the prospective study of Bulbrook et al. (9)in which 24-h urine specimens were collected from approximately 5000 women on the island of Guernsey with no apparent

Received 12/17/87; revised 5/4/88; accepted 5/25/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by Grant CA-38574 from the National CancerInstitute, Cancer Prevention Grant S1G-6 from the American Cancer Society, theAdvanced Technology Center of Southeastern Pennsylvania, and the Samuel S.Fels Fund.

2To whom requests for reprints should be addressed, at Fels Research Institute,

Temple University Medical School, 3420 N. Broad St., Philadelphia, PA 19140.3The abbreviations used are: DHEA, dehydroepiandrosterone; DMBA, 7,12-

dimethylbenz(a)anthracene; TPA, tetradecanoylphorbol-13-acetate; G6PDH,glucose-6-phosphate dehydrogenase, TCA, trichloroacetic acid; NMR, nuclearmagnetic resonance; EIMS, electron impact mass spectroscopy.

breast cancer. These women were followed for 9 years, at theend of which time 27 women had developed breast cancer. Theexcretion of various urinary steroids in the women who haddeveloped breast cancer was compared with that of 187 carefullymatched controls from the same population who had not developed cancer, and it was found that the excretion of andros-

terone and etiocholanolone (two principle metabolites ofDHEA) was lower in the women who had developed breasttumors.

Barrett-Connor et al. (10) recently reported the results of aprospective study which examined the relationship of DHEA-sulfate levels to subsequent 12-year mortality in a population-based cohort of 242 men, 50-79 years old. After adjusting forage, systolic blood pressure, serum cholesterol level, obesity,fasting plasma glucose level, cigarette smoking status, andpersonal history of heart disease, they found that the DHEA-sulfate level was independently and inversely related to deathfrom any cause and death from cardiovascular disease in particular in men over age 50.

Long-term DHEA treatment of mice and rats produces cancer preventive and possible age-retarding effects (11,12), whichmay be related to the above human epidemiolÃ³gica! findings.Our laboratory has found that DHEA has tumor preventiveactivity in the mouse against spontaneous breast cancer (13)and chemically induced tumors of the lung (14), colon (15), andskin (16, 17). Moore et al. (18) and Garcea et al. (19, 20) havereported similar activity in the rat against chemically inducedthyroid tumors and preneoplastic foci in the liver. Yen et al.(21) and others (13, 22, 23) have found that DHEA producesboth an antiobesity and ant Â¡diaboliceffect in laboratory miceand rats. Casazza et al. (24), however, failed to detect anantiobesity action of DHEA in rats treated with the steroid for16 days. Rats in this latter study were meal fed for 3 h per day,whereas in the prior work an ad libitum regimen was used.

DHEA, when injected i.p. or topically applied to the skin ofmice, inhibits both [3H]DMBA binding to skin DNA (16) andTPA stimulation of epidermal [3H]thymidine incorporation

(25). DHEA is a potent uncompetitive inhibitor of mammalianG6PDH (26, 27), and there is evidence that the inhibition ofboth of the above actions of DMBA and TPA results from alowering of the NADPH and possibly ribose-5-phosphate cellular pools as a consequence of G6PDH inhibition (11, 28).Gordon et al. (29) found that DHEA and 16a-bromo-epian-

drosterone, a more potent inhibitor of G6PDH than DHEA,inhibited the conversion of 3T3 preadipocytes to adipocytesand similarly concluded that inhibition of G6PDH is veryprobably central to the antimitotic and anticarcinogenic actionof DHEA (29). Topical application of DHEA also inhibits theformation of papillomas in mice in the two-stage skin tumori-

genesis system at both the initiation and promotion step andalso inhibits DMBA-induced papillomas and carcinomas in thecomplete carcinogenesis model (16, 17).

Although DHEA has therapeutic promise as an antiobesity,antidiabetic, and cancer chemopreventive agent, certain sideeffects have been observed following long-term administration

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NOVEL DHEA ANALOGUES

0

Fig. 1. Scheme for steroid synthesis; 16a-fluoro-S-androsten-17-one (8354); 1oa-fluoro-Sa-andros-tan-17-one(SJ5<5).

i' r' ' f

DHEA, RÂ«H2, rt'F3/5-Iodo DHEA,

RÂ«H

3, R"F (8354)

5, R-H

4, RÂ«F (8356)

6, R'H

to rodents. DHEA is metabolized to 4-androstene-3,17-dione,which may be further converted into testosterone through theaction of 17/8-hydroxysteroid dehydrogenase, or metabolizedthrough a series of enzymatic reactions to estrone and 17ÃŸ-estradiol. Treatment of young female rats with DHEA causesuterine enlargement (30), and we have also observed a markedelevation of plasma testosterone levels in male C57BL/6 micetreated for prolonged periods with DHEA.4

In addition to its potential hormonal action through metabolism into sex steroids, DHEA treatment at therapeutic dosesproduces hepatomegaly (31). Moore et al. (18) found that whileDHEA administration to rats inhibited the formation of dihy-droxy-di-n-propylnitrosamine-induced clear cell (glycogen stor

ing) preneoplastic foci in the liver, steroid treatment enhancedthe appearance of basophilic foci. Basophilic foci are alsostimulated by compounds such as clofibrate and diethylhexyl-phthalate (32, 33), which induce both hepatomegaly and liverperoxisome proliferation (34).

We report now that two synthetic steroids, 16a-fluoro-5-androsten-17-one and 16a-fluoro-5a-androstan-17-one(Fig. 1)do not demonstrate the estrogenic and androgenic activity ofDHEA, nor do they induce hepatomegaly and liver catalaseactivity (a marker for peroxisomes) (33). 16a-Fluoro-5-andros-ten-17-one and 16a-fluoro-5a-androstan-17-one, when administered p.o, are more active than DHEA as inhibitors of [3H]-

DMBA binding to skin DNA and of TPA stimulation of epidermal [3H]thymidine incorporation. 16a-Fluoro-5-androsten-17-one is also more active than DHEA as an antiobesity agent,whereas 16a-fluoro-5a-androstan-17-one does not demonstratesuch enhanced activity.

MATERIALS AND METHODS

Synthesis and Characterization of 16a-Fluoro-5-androsten-17-one,16a-Fluoro-5a-androstan-17-one, 5-Androsten-17-one, and 5cr-Andros-tan-17-one. Melting points were obtained with a Fisher-Johns apparatusand are uncorrected. Optical activity was determined in chloroformwith a Zeiss 0.005 degree photoelectric polarimeter. Infrared spectrawere obtained by using a zinc selenide MIR crystal in a Perkin-ElmerModel 681 spectrophotometer. NMR spectra were obtained on a VarianXL 200 spectrometer. Mass spectra were obtained with a Finnigan4000 spectrometer by using 70-eV electron impact with an ion sourceat 250Â°C.Microanalyses were carried out by Galbraith Laboratories,

Inc., Knoxville, TN.Preparation of the novel compounds, 16a-fluoro-5-androsten-17-one

and 16a-fluoro-5a-androstan-17-one, starting with the known, 16a-fluoro-3/3-hydroxy-5-androsten-17-one (35) (compound 1), was as follows (Fig. 1). lodination of compound 1 by the method of Corey and

4 Unpublished observation.

Anderson (36) afforded 3/3-iodo-16a-fluoro-5-androsten-17-one (compound 2). Reductive deiodination with zinc dust in ethanol gave 16a-fluoro-5-androsten-17-one (compound 3). HydrogÃ©nationof compound3 in ethanol with 5% palladium on carbon as catalyst furnished 16Â«fluoro-5a-androstan-17-one (compound 4).

For comparative purposes the known 5-androsten-17-one (compound 5) and 5a-androstan-17-one (compound 6) were prepared byiodination/reductive deiodination of DHEA/3/3-iodo-5-androsten-17-one and catalytic hydrogÃ©nationas outlined above.

3/3-Iodo-16a-fluoro-5-androsten-17-one (Compound 2) from Compound 1. To a solution of pyridine (0.4 ml) and o-phenylenephospho-rochloridite (0.6 ml) in anhydrous tetrahydrofuran (10 ml) at ()"(.'was

added 1.53 g (5 mmole) of 16<*-fluoro-3,8-hydroxy-5-androsten-17-onein 10 ml of tetrahydrofuran. After stirring for 2 h at room temperaturethe precipitated pyridinium chloride was filtered off and washed withtetrahydrofuran. The filtrate was concentrated to dryness in a vacuum,and the crude phosphite ester was treated in mÃ©thylÃ¨nechloride (25ml) with elemental iodine (1.27 g). After stirring the mixture at roomtemperature for 3 h, the solution was washed successively with l NNaOH (15 ml) and water (15 ml). Crystallization from mÃ©thylÃ¨nechloride-methanol gave 1.85 g (92.5%) of needles, m.p. 165-167Â°Cdec.For the analytical sample: m.p. 171-172.5Â°C dec.; [a]D + 46.7Â°;IR1752 and 1003 cm-' (16a-fluoro-17-one); 'H NMR (CDC13 &4.033

(complex multiplet, 3Â«H),2.706 (complex doublet, 4H), 5.371 (doubletof triplets, 6H), 1.073 (singlet, 19 CH3), 0.933 (singlet, 18 CH3); EIMSm/z 288.8 (M+ - 127).

C,,H26IFO

Calculated: C 54.81, H 6.30, F 4.50,1 30.48Found: C 54.73, H 6.17, F 4.48,1 30.69.

16a-Fluoro-5-androsten-17-one (Compound 3) from Compound 2. Asolution of 3/3-iodo-16a-fluoro-5-androsten-17-one (1.0 g) in ethanol(200 ml) was subjected to stirred reflux with zinc dust (10.0 g) for 4 h.After removal of the insoluble fraction by nitration, the filtrate residuewas recrystallized from mÃ©thylÃ¨nechloride-methanol as prismaticneedles (700 mg, 96.5%), m.p. 173-176Â°C; [a]D +10.5"; IR 1752 and1000 cm-' (16Â«-fluoro-17-one); 'H NMR (CDC13) a 5.295 (doublet of

triplets, 6H), 1.022 (singlet, 19 CH3), 0.938 (singlet, 18 CH3); EIMSm/z290.1(M+).

C,,H27FO

Calculated: C 78.58, H 9.37, F 6.54Found: C 78.75, H 9.13, F 6.31

16a-FIuoro-5a-androstan-17-one (Compound 4) from Compound 3. Asolution of 16a-fluoro-5-androsten-17-one (3.0 g) in ethanol (650 ml)was added to 660 mg of 5% palladium on carbon and the mixture wasshaken in a hydrogen atmosphere at 40 psi gauge for 90 min. Thecatalyst was tillered off and the fÃltrateresidue was recrystallized frommethanol, affording 2.61 g (86.4%) of needles, m.p. 145-148Â°C. Forthe analytical sample; m.p. 151-152Â°C;[a]D +12.2Â°;IR 1752 and 1003cm-' (16a-fluoro-17-one); 'H NMR (CDC13) a 0.805 (singlet, 19 CH3),

4818




0.909 (singlet, 18 CH3); EIMS m/z 292.2 (NT).

C,9H29FO

Calculated: C 78.03, H 10.00, F 6.50Found: C 77.78, H 10.12, F 6.47.

S-Androsten-17-one (Compound 5). This compound was prepared inoverall yield of 63% from DHEA as in the preparation of compound 3from compound 1; m.p. 110-111Â°C, [a]D -20.1Â°, IR 1738 cm"1 (17-one); EIMS m/z 272.2 (NT). Reported (37) m.p. 105-107'C.

C,,H28O

Calculated: C 83.77, H 10.36Found: C 83.77, H 10.27.

5a-Androstan-17-one (Compound 6 from Compound 5). Catalytic hydrogÃ©nationof 5-androsten-17-one as in the preparation of compound4 from compound 3 provided the saturated ketone in 92. 1% yield; m.p.123-123.5Â°C; [a]D +94.0Â°;IR 1738 cm-' (17-one); EIMS m/z 274.3(M+). Reported (38) m.p. 121-122Â°C.

Calculated:Found:

C 83.15, H 11.02C 83.29, H 11.00.

Antiobesity Test. Female BALB/cJ mice (6 to 8 weeks old) wereobtained from The Jackson Laboratory, Bar Harbor, ME. The animalswere housed six per cage in 11.5- x 7- x 5-inch plastic cages with 12hours of alternating light at 70 Â±2Â°Fand 50 Â±5% relative humidity.

Food was prepared from powdered Purina 5015 chow (17.5% proteinand 11% fat) to which steroid or clofibrate was added (0.25%, w/w)and mixed well. DHEA was obtained from Diosynth, Chicago, IL, andclofibrate from Sigma Chemical Co., St. Louis, MO. Water was thenadded (89 ml/208 g of food), and the dough-like mixture was addeddaily to each cage of mice at noon. Mice were treated for 17 days withthe various diets.

Mice ate most of their food over a 24-h period, although some foodwas scattered throughout the bedding and occasionally larger pieceswere left. In measuring food consumption, a correction was made forthe food lost in the bedding. The bedding was collected after 1 week oftreatment, dried in an oven and weighed, and the original beddingweight was subtracted from this. Correction was also made for theweight of feces in the bedding. This was determined by placing mice incages with elevated wire floors and absorbent paper on the cage bottom,and collecting weekly feces from each cage, which were dried andweighed. Food-restricted mice were given daily rations of approximately60% of the amount of food eaten per day by the mice fed ad libitum.

Mice were weighed between 8:00 and 10:00 a.m. on a Sartoriusbalance using an animal-weighing program in which the unstable display observed with animals in constant motion is avoided by a programmable and automatic mean weight calculator.

Estrogenic Activity. A test similar to that used by Knudsen andMahesh (30), which demonstrates estrogenic action of DHEA, wasused. Female CD rats were obtained from Charles River Laboratories,Wilmington, MA, at 22 days of age and housed six per cage in wirebottomed metal cages. Five days later rats were given injections s.c. (60mg/kg) of 0.4 ml of a solution of steroid in 40% ethanol and 60%propylene glycol, or with vehicle alone, daily for 3 days. On the fourthday, the rats were sacrificed and the uteri were removed and weighed.

Androgenic Activity. The castrated rat assay (39) was used for determination of androgenic activity. Male CD rats, 19 days old, wereobtained from Charles River Laboratories (Kingston, NY) and werehoused five per cage in plastic cages on hardwood bedding. The ratswere anesthetized with Nembutal (50 mg/ml, 0.1 ml administered i.p./100 g of body weight) and castrated. On the day of castration and dailyfor 11 successive days, rats were given injections s.c. of 0.4 ml of asolution of steroid in 40% ethanol and 60% propylene glycol. Controlrats were given injections of vehicle without steroid. On the 12th daythe rats were sacrificed, the seminal vesicles were removed, stripped

free of the coagulating glands, compressed to expel the fluid, andweighed.

Liver Weight and Catalase Activity. Female BALB/cJ mice (6-8weeks old) were treated with 0.25% steroid (w/w), 0.25% clofibrate (w/w), or with control diet for 3 weeks. Mice were sacrificed, the liverswere perfused with cold 0.9% NaCI solution, removed, and weighed.Livers were homogenized in 0.007 M potassium phosphate buffer, pH7.0, (5%, w/v), and centrifuged at 10,000 x g for 30 min. The super-natants were used for catatase activity measurements according to theprocedure of Luck (40). Protein was determined according to Lowry etal. (41), and activity was expressed as units per mg of protein. Theactivity was linearly proportional to protein content over the rangemeasured.

Determination of A, Values of Steroids against G6PDH. Steroidinhibition of G6PDH activity was determined according to the procedure of Oertel and Rebelein (42), using purified bovine adrenal G6PDH(Sigma Chemical Co.). Steroids were dissolved in dioxane before testing. The reaction mixture consisted of 3.0 ml of 0.05 M Tris-HCl and0.01 MMgClz buffer (pH 7.6), 0.1 ml of 0.01 MNADP (Sigma) solution,and 0.02 ml of dioxane or steroid in dioxane. The reaction was run at25Â°Cand was initiated by the addition of 0.05 ml of a 0.031 M glucose

6-phosphate (Sigma) solution. Activity was measured as milliunits fromchanges in absorbance at 340 nm on a Gilford Response recordingspectrophotometer. The inhibitor constant (KÂ¡)for each steroid wasdetermined by the method of Dixon (43).

|3H|DMBA Binding to Skin DNA. Male A/J mice, 5 weeks old, were

obtained from The Jackson Laboratory, Bar Harbor, ME, and housedsix per cage in 11.5- x 7- x 5-inch plastic cages. Mice were shaved 2 to3 days before steroid treatment. Only those showing no hair growthwere used. Steroids were suspended in sesame oil and were administeredthrough p.o. intubation (0.2 ml, 200 mg/kg). [3H]DMBA (100 Â¿iCi,100

nmol, in 0.2 ml acetone) was applied to the shaved skin l h after steroidadministration. ['HjDMBA was purchased from Amersham Corp.,

Arlington Heights, IL, and purified within 3 weeks of use on silica gelthin layer chromatography plates (Brinkman Instruments, Inc., West-bury, NY) with a developing solvent of benzene:ethanol 9:1 and storedat â€”20Â°Cin acetone.

The DMBA was applied in a darkened room, and the mice were keptin the dark until sacrifice 12 h later so as to minimize the loss ofDMBA-DNA adducts due to light exposure (44). DNA was isolated bya modification of the Kirby-Cook phenol procedure as described byKinoshita and Gelboin (45). The skin was excised and placed in cold0.25 M sucrose-0.05 MTris-HCl buffer (pH 7.5), and the epidermis and

dermis were scraped off with a surgical scalpel. The scrapings from twoseparately treated mice in a single treatment group were pooled andhomogenized in 5 ml of 6% sodium p-aminosalicylic acid in a tight-fitting Potter-Elvejhem homogenizer. A 10% sodium dodecyl sulfatesolution (0.5 ml) was added, and the mixture was shaken at roomtemperature for 1 h. This was extracted with an equal volume of aphenol:m-cresol:8-hydroxyquinoline:water mixture (500:70:0.5:55, w/w).

DNA was precipitated from the aqueous layer by the addition of anequal volume of cold 2-ethoxyethanol, collected on a glass rod, anddissolved in 0.01 M potassium phosphate buffer (pH 7.0). The DNAsolution was treated with 40 Mg/ml RNase at 37'C for 30 min, and

then deproteinized with five washings of chloroform:isoamyl alcohol(24:1). The DNA was precipitated by addition of 70% ethanol containing 2% sodium acetate, washed with ethanol, then with ether, and dried.The DNA was hydrolyzed in 5% TCA at 90Â°Cfor 15 min. An aliquot

of the hydrolysate (0.5 ml) was counted on an Intertechnique scintillation counter and assayed for DNA by the diphenylamine reaction (46).

[3H|Thymidine Incorporation into Mouse Epidermis. Male ICR mice,

7 weeks old, were obtained from Skin and Cancer Hospital, Philadelphia, PA, and housed six per cage in 11.5- x 7- x 5-inch plastic cages.Mice were shaved on the back 1-2 days before TPA application, andonly mice showing no hair regrowth were used. Animals were intubatedp.o. with steroid suspended in sesame oil (0.2 ml, 400 or 100 mg/kg).One h after steroid administration, TPA (10 Mgin 0.2 ml of acetone)or acetone vehicle was applied topically to the shaved skin. TPA wasobtained from Sigma Chemical Co. Twenty h later mice were given

4819




injections i.p. of 60 Â¿iCiof [3H]thymidine (2 Ci/mmol; Amersham) 20

min before sacrifice. The animals were sacrificed by cervical dislocationand the residual hair was removed with a depilatory agent. Epidermalscrapings were prepared according to the procedure of Hennings et al.(47), homogenized in distilled water at 4Â°C,and the macromolecules

were precipitated with 0.4 N TCA. Following six washes with 0.2 NTCA at 4Â°Cand two washes with absolute ethanol at room temperature,the nucleic acids were hydrolyzed with 0.5 N TCA at 90Â°Cfor 5 min.

The hydrolysates (0.2-ml aliquots) were counted in an Intertechnique

scintillation counter and assayed for DNA by the diphenylamine reaction.

Statistical Methods. One-way analysis of variance was used to compare groups; t tests based on the mean square error from the analysisof variance were used to compare the treatment groups to the controlgroup when the overall F test was significant at the P < 0.01 level (48).

RESULTS

Both 16a-fluoro-5-androsten-l 7-one and 16a-fluoro-5fv-an-drostan-17-one are more active than DHEA as inhibitors ofbovine adrenal G6PDH (Table 1). Their greater activity isapparently due both to the elimination of the 30-hydroxyl group(5-androsten-l 7-one versus DHEA) as well as to the additionof a 16a-fluoro group (16a-fluoro-5-androsten-l 7-one versus 5-androsten-17-one and 16a-fluoro-5a-androstan-l 7-one versus5a-androstan-l 7-one). As shown in Tables 2 and 3,16a-fluoro-5-androsten-l 7-one and 16a-fluoro-5a-androstan-l 7-one,when administered p.o. to mice, are more active than DHEAin blocking the binding of [3H]DMBA to skin DNA and ininhibiting the TPA stimulation in epidermal [3H]thymidine

incorporation.16a-Fluoro-5-androsten-l 7-one also shows greater activity

than DHEA in reducing the rate of weight gain in BALB/cJmice (Fig. 2). Interestingly, 16a-fluoro-5a-androstan-17-one,which is somewhat more active than 16a-fluoro-5-androsten-17-one in inhibiting G6PDH and shows comparable activity ininhibiting [3H]DMBA binding to DNA and TPA stimulationin [3H]thymidine incorporation, is not as active as 16a-fluoro-5-androsten-l 7-one in reducing weight gain.

16a-Fluoro-5-androsten-l 7-one and 16a-fluoro-5a-andros-tan-17-one, unlike DHEA, do not produce uterine enlargementin female rats (Table 4) nor increase the weight of seminal

Table 1 Inhibition of gIucose-6-phosphate dehydrogenase by steroids

Inhibitor constants were determined by using purified bovine adrenal G6PDH.

Steroid

DHEA16Â«-Fluoro-5-androsten-l 7-one16a-Fluoro-5o-androstan-17-one5-Androsten-l 7-oneSa-Androsten-17-one

18.700.510.131.980.32

Table 2 Effect of DHEA, 16a-fluoro-5-androsten-l 7-one, or I6a-fluoro-5ct-androstan-17-one on [ÃŒH]DMBAbinding to skin DNA ofA/J mice

Male A/J mice were intubated p.o. with steroid at 200 mg/kg, or with sesameoil vehicle, and l h later [3H]DMBA was topically applied to the skin. Twelve hlater the mice were killed, and the amount of [3H]DMBA bound per ^g of skinDNA was determined as described in "Materials and Methods." Each value is

the mean Â±SD for three separate determinations.

TreatmentExperiment

1ControlDHEA16n-fluoro-5-androsten- 17-one

Experiment 2ControlDHEA16a-fluoro-5a-androstan- 17-oneDMBA(mol/Mg

DNAx3.1

Â±0.2(n =3.1 Â±0.51.0Â±0.0022.7

Â±0.3 (n =2.6 Â±0.0050.7 Â±0.210")3)

3)

Table 3 Effect of Â¡6a-fluoro-5-androsten-l 7-one, 16a-fluoro-5a-androstan-Â¡7-one, and DHEA on TPA-stimulatedf'HJthymidine incorporation in ICR

mouse epidermisMale ICR mice were intubated p.o. with the indicated dose of steroid, or with

sesame oil vehicle, I h before topical TPA application. Twenty h later the micewere given injections i.p. of [3H]thymidine and 20 min later they were sacrificedand the amount of ['I l|tli\ nudine incorporated per jig epidermal DNA wasdetermined as described in "Materials and Methods." Each value is the mean Â±

SD for three separately treated mice except as indicated.

Treatment

cpm [3H]thymi-

Experiment 1Control (minus steroid and TPA) 69.1 Â±8.9 (n = 2)TPA 119 Â±6.0DHEA (400 mg/kg) 68.5 Â±7.6DHEA (400 mg/kg) + TPA 63.0 Â±5.116a-fluoro-5-androsten-l 7-one (400 mg/kg) 44.7 Â±8.9 (n = 2)l Ã¶a-fiuoro-S-androsten-17-one (400 mg/kg) 20.6 Â±1.2Â°

+ TPADHEA (100 mg/kg) + TPA 122 Â±11.116a-fluoro-5-androsten-17-one (100 mg/kg) 43.8 Â±1.9Â°

+ TPAExperiment 2

Control 72.2 Â±2.7TPA 193 Â±9.2DHEA (400 mg/kg) 68.0 Â±0.5DHEA (400 mg/kg) + TPA 73.0 Â±11.016a-fluoro-5o-androstan-l 7-one (400 mg/kg) 49.2 Â±3.716a-fluoro-5a-androstan-17-one (400 mg/kg) 24.9 Â±2.9*

+ TPADHEA (100 mg/kg) + TPA 197 Â±3.31oa-fluoro-Sa-androstan-17-one ( 100 mg/kg) 46.6 Â±2.7*

+ TPA" Significantly less than corresponding group treated with DHEA, P < 0.001.* Significantly less than corresponding group treated with DHEA, P < 0.001.

21-

^ 20-

KO

lu

19-

18-

17-

16-

C.-0 DHEA

l137 9 II

DAYS OF TREATMENT

i15

i17

Fig. 2. Effect of steroids and caloric restriction on weight gain of BALB/cJmice. Six- to 8-week-old BALB/cJ mice were treated with nonsteroid diet adlibitum (control), nonsteroid diet at reduced levels (C.R.), or steroid diet (0.25%,w/w) ad libitum. Points, mean weights of mice (n = 6), with SD of <10% of themean. The cumulative food consumptions for 2 weeks, in g/mouse, were: control,46.8; DHEA, 38.9; 16a-fluoro-5-androsten-l 7-one (83S4), 41.8; 16a-fluoro-5a-androstan-17-one (8356), 41.8; and food restricted, 30.7. The final body weightsin the 16a-fluoro-5-androsten-17-one-treated and food-restricted groups weresignificantly less than the control body weights, P< 0.001.

vesicles in male rats (Table 5). Both DHEA and clofibrateproduce hepatomegaly and stimulate liver catalase activity inmice, and neither of these effects are seen with 16a-fluoro-5-androsten-17-one or 16a-fluoro-5a-androstan-l 7-one (Table6).

DISCUSSION

These studies demonstrate that specific side effects of DHEAtreatment, i.e.. estrogenic and androgenic action and induction

4820




Table 4 Effect of DHEA, l6a-fluoro-5-androsten-17-one, or 16a-fluoro-5a-androstan-l 7-one on uterine weight of sexually immature CD rats

Rats were injected daily for 3 days at the indicated dose as described in"Materials and Methods." Uterine weight (mg) is given as the mean Â±SD.

TreatmentExperiment

tNo steroidDHEA (60 mg/kg)16a-fluoro-5-androsten-17-one (60 mg/kg)

Experiment 2No steroidDHEA (60 mg/kg)16Â«-fluoro-5a-androstan-l 7-one (60 mg/kg)Mean

uterine wt(mg)176Â±22(n

= 6)339 Â±63 (n = 5)Â°171 Â±21(Â«=5)160

Â±17(n = 5)436 Â±37 (n = 4)Â°174 Â±32 (n = 6)

" Significantly greater than nonsteroid-treated group, P < 0.001.

TableS Effect of DHEA, 16a-fluoro-5-androsten-l 7-one, or 16a-fluoro-5a-androstan-17-one on seminal vesicle weight in castrated CD rats

Castrated male CD rats were injected daily for 12 days at the indicated steroiddose as described in "Materials and Methods." Seminal vesicle weight (mg) is

shown as the mean Â±SD.

TreatmentMean seminalvesicle wt (mg)

No steroid 5.1 Â±1.1 (n = 13)DHEA (30 mg/kg) 11.6 Â±3.6 (n = 11)Â°DHEA (60 mg/kg) 12.8 Â±2.4 (n = 7)Â°16a-fluoro-5-androsten-l 7-one (30 mg/kg) 4.6 Â±0.5 (n - 10)16a-fluoro-5-androsten-l 7-one (60 mg/kg) 5.8 Â±1.1 (n = 9)16tÂ»-fluoro-5n-androstan-l 7-one (30 mg/kg) 5.5 Â±0.8 (n = 9)16a-fluoro-5o-androstan-l 7-one (60 mg/kg) 6.6 Â±1.1 (n = 10)

" Significantly greater than nonsteroid-treated group, /' < 0.001.

of hepatomegaly and liver catatase activity, are not seen withbiologically active doses of compound 16a-fluoro-5-androsten-17-one and 16a-fluoro-5a-androstan-l 7-one. In order to bemetabolized into estrone, the 30-hydroxyl group of DHEA mustbe oxidized to a 3-oxo group and the A5"6double bond shiftedto the A4~5position, forming 4-androstene-3,17-dione, which

undergoes further metabolic conversion into estrone. Replacement of the 3/3-hydroxyl group in DHEA with a hydrogen atomas in 16a-fluoro-5-androsten-l 7-one should prevent metabolism into 4-androstene-3,17-dione, and this presumably accounts for the lack of estrogenic activity of this compound. Thesimilar absence of a 3/3-hydroxyl group in 16a-fluoro-5a-an-drostan-17-one, as well as the absence of a 5-6 double bond,very likely accounts for its lack of estrogenic action. The 16a-fluoro substitution in 16a-fluoro-5-androsten-l 7-one and 16a-fluoro-5a-androstan-l 7-one may contribute to the reduced an-drogenic activity of the synthetic steroids. The addition of a16a-fluoro group and 16/3-methyl group into either testosteroneor dihydrotestosterone greatly reduces their androgenic potency(35).

The addition of a 16a-fluoroatom in 16a-fluoro-5-androsten-17-one and 16a-fluoro-5o-androstan-17-one enhances G6PDHinhibitory activity in vitro when compared to the correspondingnon fluorina: ed compound (Table 1). The inhibition of [3H]-

DMBA binding to skin DNA and TPA stimulation in epidermal

[3H]thymidine incorporation by DHEA and related analogues

very probably is a result of G6PDH inhibition (11, 25, 28), andthe greater activity of 16a-fluoro-5-androsten-l 7-one and 16a-fluoro-5a-androstan-l 7-one in these assays is probably partlyattributable to the greater G6PDH inhibitory capacity of thesecompounds. Their greater activity Â¡nvivo could also be due toa slower rate of metabolic degradation, since the absence of anoxygen function at position 3 in 16a-fluoro-5-androsten-17-one and 16a-fluoro-5Â«-androstan-l 7-one should prevent me

tabolism into androsterone and etiocholanolone, two primarydegradative products of DHEA.

The food consumptions of mice treated with DHEA, 16Â«-fluoro-5-androsten-l 7-one, or 16a-fluoro-5a-androstan-l 7-one

are comparable and slightly less than that of control mice (Fig.2). Although mice treated with 16a-fluoro-5-androsten-l 7-oneconsume approximately 36% more food than do restricted mice,the steroid-treated mice show similar, or even slightly reduced,mean body weights. This is consistent with previous observations which indicate that much of the antiweight effect ofDHEA treatment is the result of a reduced efficiency of foodutilization in treated animals (12, 31).

Although 16a-fluoro-5Â«-androstan-l 7-one is as active as16Â«-fluoro-5-androsten-l 7-one in inhibiting [3H]DMBA binding to DNA and TPA stimulation of ['Hjthymidine incorpora

tion and is a more potent G6PDH inhibitor, it does not havethe enhanced potency of 16a-fluoro-5-androsten-l 7-one in reducing weight gain. We have similarly found that the ring Bsaturated derivative of DHEA, 3/3-hydroxy-5a-androstan-17-one, is not as effective as DHEA in reducing weight but hasenhanced activity as an inhibitor of G6PDH and Â¡nblockingTPA stimulation in epidermal [3H]thymidine incorporation.4

This dissociation between G6PDH inhibitory activity and an-tiobesity action, as observed by others (49), suggests thatG6PDH inhibition is not the primary mechanism for the an-tiobesity action of these steroids.

We have suggested that the tumor preventive activity ofDHEA may result from G6PDH inhibition as well or from itsantiobesity effect (11), since reducing weight gain through foodrestriction is known to inhibit tumor formation (50). Morerecent evidence suggests that food restriction of mice alsosuppresses epidermal G6PDH activity and inhibits both bindingof [3H]DMBA to skin DNA and TPA stimulation in epidermal[3H]thymidine incorporation, suggesting a similarity in the

mechanism of antitumor action of DHEA and underfeeding(51).

The results reported here with 16a-fluoro-5-androsten-17-one and 16a-fluoro-5a-androstan-l 7-one indicate that it is pos

sible to eliminate certain undesirable side effects of DHEAtreatment and increase therapeutic potency. Such analoguesmay find application as drugs for human use.

Table 6 Effect of I oa-fluoro-S-androsten-17-one, 16a-fluoro-5a-androstan-I7-one, DHEA and clofibrate on hepatomegaly and catalase activity in BALB/cJ miceFemale BALB/cJ mice were treated with control food or food containing either DHEA, 16a-fluoro-5-androsten-l 7-one, 16a-fluoro-5a-androstan-17-one, or

clofibrate (0.25%, w/w). Food consumption was determined weekly for 3 weeks and mice were weighed as described in "Materials and Methods." At the end of 3

weeks, the mice were sacrificed, the livers were removed and weighed, and catalase activity was determined.

TreatmentControl

DHEA16a-fluoro-5-androsten- 17-one16a-fluoro-5a-androstan- 17-oneClofibrateFood restrictedMean

body wtafter 3 wk of

treatment (g) Â±SD21.1

Â±0.8(n = 6)21.3 Â±1.318.0 Â±1.320.7 Â±1.221.2 Â±0.518.0 Â±0.8Total

foodconsumed for

3 wk (g/mouse)74.1

63.863.965.674.145.4Mean

liver wt/bodywt (g/g) Â±SD0.056

Â±0.004 (n = 6)0.072 Â±0.005Â°

0.059 Â±0.0080.058 Â±0.0030.079 Â±0.008Â°

0.052 Â±0.002Mean

livercatalase Â±SD

(units/mgprotein)31.1

Â±6.7(n =97.8 Â±2.4Â°

29.2 Â±2.632.2 Â±6.092.2 Â±8.9Â°

35.1 Â±3.53)' Significantly greater than control, P < 0.001.

4821




ACKNOWLEDGMENTS

The authors thank Denise Fairman and Tanya Howard for theirexcellent technical assistance, and Dr. Walter Boyko, Chemistry Department, Villanova University, and Dr. Doyle Daves, Chemistry Department, Lehigh University, for performing NMR and mass spectros-copy analysis. We thank Dr. Marcia Polansky, Department of Statistics,School of Business and Management, Temple University, for statisticalanalysis of data.

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1988;48:4817-4822. Cancer Res Arthur G. Schwartz, Marvin L. Lewbart and Laura L. Pashko Biological Activity and Reduced Side Effects in Mice and RatsNovel Dehydroepiandrosterone Analogues with Enhanced

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