MILITARY MEDICINE, 167, Suppl. 1:060,2002

Inhibition of Adjuvant-Induced Arthritis by 16a-Fluoro-5-Androsten-17-0ne

Guarantor: Arthur G. Schwartz, PhD Contributors: Arthur G. Schwartz, PhD; Laura L. Pashko, PhD

The adrenal steroid (DHEA) produces tant receptor and that inhibition ofthis enzyme by DHEA me­ -preventive, antiatherosclerotic, antidiabetic, Immuno­ diates some of the biological effects of the steroid. DHEA is a modulating, and anti-inflammatory effects in laboratory ani­ potent uncompetitive inhibitor of G6PDH, the rate-controlling mals. The clinical use of DHEAis limited by its androgenicity. Downloaded from https://academic.oup.com/milmed/article/167/suppl_1/60/4820113 by guest on 01 October 2021 enzyme in thepentose phosphate pathway, 10 and it appearsthat Wehave developed a synthetic congener ofDHEAcalled fluaster­ the antiproliferative and antitumor-promoting effects of the one that, in animal tests, lacks the androgenicity, estrogenicity, and peroxisome-proliferating effects of DHEA but retains the DHEA class of steroids is mediated through inhibition of cancer-preventive, antidiabetic, and anti-inflammatory efficacy. G6PDH.1,11,12 Two important metabolic consequences ofG6PDH This report discusses how fluasterone ameliorates the develop­ inhibition are a lowering ofNADPH-reducing equivalents avail­ ment of joint inflammation in an adjuvant-arthritis model in able forbiosynthetic processes, including ribo- and deoxyribo­ Lewis rats. nucleotide synthesis, and a reduction in ribose-5-phosphate, which is alsorequired for ribo- and deoxyribonucleotide synthe­ Introduction sis. Providing exogenous mixtures ofthe fourdeoxyribonucleo­ sides and ribonucleosides overcomes the inhibitory effect of he adrenal glands in humans produce large quantities of DHEA and related steroids on growth and differentiation in T dehydroepiandrosterone (DHEA) and DHEA-sulfate, and the vitroI3,I4; it alsoovercomes the inhibitory effect ofthesesteroids plasma levels of DHEA-sulfate are the highest of any steroid on and skin neoplasia in viVO. II,I5 These results strongly hormone in the human, yetthe biological role ofDHEA remains suggestthat a major effect of DHEA in these experiments is to an enigma. The plasma concentrations of DHEA and DHEA­ restrict the supplyofpentose phosphate and NADPH. sulfatebegin to rise around the eighthyearoflife (adrenarche), peak duringthe second decade, and thereafter undergo a con­ DHEAAnalog tinuous, age-related decline, which has led to the speculation Thestriking biological effects ofDHEA in laboratory animals that the decline in DHEA levels withagemaycontribute to the suggestthat this steroid, or specific structural analogs, could development ofage-related diseases. become an important classofdrugsin humans. However, DHEA Thestrongestevidence suggesting a preventive role forDHEA has sex-hormonal and peroxisome-proliferating sideeffects that in the development of age-related disease is based on experi­ may limit its use as a drug.":" We developed the synthetic ments in laboratory animals. DHEA treatment suppresses the steroid, 16a-fluoro-5-androsten-17-one (fluasterone), which in development ofexperimentally induced cancersin manydiffer­ 1 mice and rats doesnot demonstrate the androgenic, estrogenic, ent organs in mice and rats. It also ameliorates the develop­ and peroxisome-proliferating effects of DHEA while retaining mentofaorticand coronary arteryatherosclerosis in rabbitsfed the antiproliferative, anti-inflammatory, cancer-preventive, and a high-cholesterol dietand in rabbitsfed a high-cholesterol diet antidiabetic effects ofthe native steroid. 18 Fluasterone is alsoa who also underwent a balloon-catheter-induced aortic injury2,3 4 more potent G6PDH inhibitor than is DHEA: TheKi offluaster­ or a heterotrophic heart transplant. DHEA inhibits develop­ oneis 0.5 JLM vs. 18.7 JLM for DHEA.I9 Fluasterone is currently ment of atherosclerosis without significantly lowering choles­ in phaselill trialsfor the treatmentoftype2 as well as terol levels, indicating an inhibitory effect on atherosclerosis for the treatmentofrheumatoid arthritis. independent oflipid lowering. Topical application of the tumor promoter TPA (12-0-tetra­ In addition to its cancer preventive and antiatherosclerotic decanoylphorbol-13-acetate) to mouse skin produces acute in­ actions, DHEA also produces antl-lnflammatory.v" immuno­ flammatory and hyperplastic effects. Fluasterone applied topi­ modulating.' and neurological'" effects in laboratory animals. cally just before application of TPA abolishes both the Glucose-6-Phosphate Dehydrogenase Inhibition inflammatory and hyperplastic effects of the tumor promoter. Fluasterone treatmentinhibitsthe development ofcollagen-in­ There are no convincing data that DHEA exertsits biological ducedarthritis in DBA/1 mice (J. Schiffenbauer et al., personal effects throughinteraction withoneofthe classical steroid-type communication) and, as shown here,alsoinhibitspawswelling receptors, which have a high affinity for steroid ligands and in adjuvantarthritis. regulate the expression of specific genes by binding to DNA regulatory elements. There is, however, considerable evidence that glucose-6-phosphate dehydrogenase (G6PDH) is an Impor- Methods

Fels Institute for Cancer Research and Molecular Biology, Temple University Animals School ofMedicine, 3307 North Broad Street, Philadelphia, PA 19140. This manuscript wasreceived for review February 2001. The revised manuscript Female Lewis rats, 36 to 38 days old, were obtained from wasaccepted for publication in November 2001. Charles River Laboratories. Ratswere housedthree per cage in Reprint &Copyright ©byAssociation ofMilitary Surgeons ofU.S., 2002. plasticcages in the Central Animal Facility, Allied Health Build-

Military Medicine, Vol. 167, Supplement 1 60 Inhibition of Adjuvant-Induced Arthritis by 16a-Fluoro-5-Androsten-17-0ne 61 ing, Temple University. Therats had ad libitum accesstoPurina 5001 Chow and acidified tap water. 7

Inductionand Assessment ofAdjuvant Arthritis (/) ~ Arthritis was induced by the injection of 1 mgofMycobacte­ 0- 'l:' rium butyricumin 0.1 mL ofheavy mineral oilintothe intrader­ c: :2 mal cervical periscapular region with a 23-gauge needle while '5 6 the rats were under lightanesthesia. Caliper measurements of Gi (i) ankle-joint width were done on day 0 and day 7 and at 2- to E m 3-dayintervals thereafter. Treatment withfluasterone was ini­ :0 (1) tiatedonday8,whenarthritissymptoms beganto appear. Each C»co ~ group contained approximately 10rats. ~ Downloaded from https://academic.oup.com/milmed/article/167/suppl_1/60/4820113 by guest on 01 October 2021 5 Treatmentwith Fluasterone

Oral Micronized fluasterone wassuspended in a sesameoilvehicle 6 8 10 12 14 16 18 20 22 and homogenized in a glass scintillation vial using a Tekmar Days Tissumizer. Amagnetic stirringbar was added to the vial, and Fig. 1. The effect of 400 mglkg fluasterone on paw swelling in female Lewis rats. the sesame oilsuspensionwas stirred and heated (~40°C) for Values shown are mean 2: SE. Significantly less than control: *,p < 0.01; **, approximately Ilf2 hoursbefore intubation. Fluasterone-sesame p < 0.001. oil suspensionswere prepared every 3 daysand whennot used were storedat 4°C. Rats were treated daily with an oral dose of50, 200, or 400 8 Control mg/kg. Eachrat received approximately 0.5 mL offluasterone­ sesameoil or sesameoilalone. Atthe 50-mg/kg dose, fluaster­ 50 mg/kg onewas completely in solution, whereas at the 200- and 400­ ~ fluasterone 0- 7 rug/kg doses, the fluasterone was only partlyin solution. 'l:' c: :c Intraperitoneal Injection '0 ~ An aqueous proprietary solution offluasterone wasused.The 6 200 mg/kg E m fluasterone concentration offluasterone in thesolution was3.4mg/rnl, The :a (1) solution wasfiltered througha 22-nmsterile Nalgene filter into C» ~ sterile leakproof bottles. ~ Ratswere injected intraperitoneally daily withsterile fluaster­ « 5 onesolution at a doseof15or 2.5 mg/kg. Control rats received intraperitoneal injections ofsterile solution withoutfluasterone.

Results o 2 4 6 8 10 12 14 16 18 20 Days Oral Treatment Fig. 2. Effect of 200 mglkg fluasterone or 50 mglkg fluasterone on hindlimb Asshown in Figures 1and 2, orally administered fluasterone diameters. Values shown are mean 2: SE. Significantly less than control: *,p < 0.05; at a doseof400or 200mg/kg produced substantialand signif­ **,P < 0.01. icant reduction in joint inflammation, whereas the 50-mg/kg doseproduced only a slighteffect. orally. Labrie et al." also found that subcutaneously injected DHEA was approximately 30 times as bioavailable as orally InjectedTreatment administered DHEA in reversing the inhibitory effect oforchiec­ The injectable formulation at a dose of 15 mg/kg also pro­ tomy on ventral prostategrowth in rats. Themuch greaterbio­ ducedsubstantial and significant reduction in joint inflamma­ availablllty ofinjected vs.orally administered DHEA maybe due tion (Fig. 3), whereas at a doseof2.5 rug/kg it had no apparent to poor absorption of the orally administered drug or to first­ efficacy (Fig. 4). pass liver converting it into less active or inactive metabolites. Discussion Themechanism bywhich the DHEA steroids exerttheiranti­ inflammatory effect is not clear, but results in this laboratory These experiments demonstrate that fluasterone, when ad­ suggest that this effect may be mediated, at least in part, ministered orally orbyintraperitoneal injection, inhibitsinflam­ through G6PDH inhibition and lowering ofthe NADPH cellular mation (as measured by ankle diameter) in Lewis rats in the pool. adjuvant-arthritis model. Fluasterone is approximately 20times Theleukocyte NADPH-oxidase, which is found in neutrophils more potent when injected intraperitoneally than when given and macrophages, catalyzes the one-electron reduction ofoxy-

Military Medicine, Vol. 167, Supplement 1 62 Inhibition of Adjuvant-Induced Arthritis by 16a-Fluoro-5-Androsten-17-One

in mouse skinbymeasuring an increase in vascularpermeabil­ ity, wefound that topically applied fluasterone completely abol­ ished the inflammatory effect of TPA. If fluasterone exerts its Control anti-inflammatory effect by suppressing NADPH levels and the activity ofthe NADPH-oxidase, provision of NADPH should re­ verse the inhibitory effect of fluasterone. Because NADPH, an anionic dinucleotide, does not penetratecells, weused a prep­ 6 arationofcationic liposomes, which arehighly effective in trans­ Fluasterone fecting DNA into cells, as a means of facilitating NADPH en­ solution, 15 mg/kg tranceintocells." Intradermal injections ofa mixture ofNADPH administered. and cationic liposomes completely reversed the anti-inflamma­ intraperitoneally

toryeffect offluasterone, whereas injections ofeitheran NADPH Downloaded from https://academic.oup.com/milmed/article/167/suppl_1/60/4820113 by guest on 01 October 2021 solution or liposomes alone had no effect. In contrast, the anti­ inflammatory effect ofcorticosterone, the endogenous glucocor­ ticoid in mice, was not significantly affected by injections of NADPH liposomes. s-l---=-:;;::...::=-==-.---.F-r----r---r----,.--., DHEA and its various synthetic congeners may represent a 6 8 10 12 14 16 18 20 new classofanti-inflammatory steroids that donot have the side Days effects of the glucocorttcotds" and that produce their anti-in­ Fig. 3. Effect of intraperitoneal administration of a fluasterone solution. Values flammatory actionbywhat appearsto be a uniquemechanism. shown are mean ± SEt Statistically significant from control: *,p < 0.05; **,p < 0.01

6 References

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