An Orally Bioavailable Synthetic Analog of an Active Dehydroepiandrosterone Metabolite Reduces Established Disease in Rodent Models of Rheumatoid Arthritis

0022-3565/09/3293-1100–1109$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 145086/3474987 JPET 329:1100–1109, 2009 Printed in U.S.A. An Orally Bioavailable Synthetic Analog of an Active Dehydroepiandrosterone Metabolite Reduces Established Disease in Rodent Models of Rheumatoid Arthritis Halina Offner, Gary S. Firestein, David L. Boyle, Raymond Pieters, James M. Frincke, Armando Garsd, Steven K. White, Christopher L. Reading, and Dominick L. Auci Department of Neurology, Oregon Health and Science University, Portland, Oregon (H.O.); Institute for Risk Assessment Sciences-Immunotoxicology, Utrecht University, Utrecht, The Netherlands (R.P.); Division of Rheumatology, Allergy and Immunology, University of California at San Diego School of Medicine, La Jolla, California (D.L.B., G.S.F.); and Hollis-Eden Pharmaceuticals Inc., San Diego, California (J.M.F., A.G., S.K.W., C.L.R., D.L.A.) Downloaded from Received August 20, 2008; accepted March 17, 2009 ABSTRACT Dehydroepiandrosterone (DHEA) treatment provides diverse anti- even when treatments were delayed until 7 days after the onset jpet.aspetjournals.org inflammatory benefits in rodent models of diseases, including of arthritis. Furthermore, dose-dependent benefit was observed rheumatoid arthritis (RA), but only limited benefits to patients. In in the DBA mouse model of collagen antibody-induced arthritis, rodents, DHEA is metabolized to (among others) androstene- as well as reductions in IL-6 and matrix metalloproteinase-3 3␤,7␤,17␤-triol (AET), which retains potent anti-inflammatory mRNA levels in joints at the peak of disease and at the end of activity. 17␣-Ethynyl-5-androstene-3␤,7␤,17␤-triol (HE3286) is the study. HE3286, in contrast to dexamethasone, was not a novel, metabolically stabilized, orally bioavailable derivative of immune-suppressive in several classic animal models of im- AET. In the DBA mouse model of collagen-induced arthritis mune function. Instead, HE3286 treatment was associated with (CIA), once-daily oral treatments (gavage) with HE3286 (40 reduced nuclear factor-␬B activation and in our previous stud- at ASPET Journals on January 16, 2020 mg/kg), beginning at onset of disease, significantly decreased ies, with increased regulatory T cells. We hypothesize that disease. Benefit was associated with reduction in joint inflam- HE3286 may represent a novel, perhaps first-in-class, anti- mation, erosion, and synovial proliferation as measured by inflammatory agent and may more fully translate the benefits of histological analysis and mRNA of proinflammatory cytokines, DHEA, heretofore largely limited to rodents, into treatments for including tumor necrosis factor-␣, interleukin (IL)-6, IL-1␤, and human diseases, including autoimmune disorders such as RA. IL-23. Significant benefit was also observed in the CIA model Dehydroepiandrosterone (DHEA) is the precursor of an- could provide benefit. DHEA showed benefit in animal mod- drogens, estrogens, and other immune-regulating hormones els of RA, including the DBA mouse model of collagen-in- without androgenic or estrogenic activity. DHEA levels in duced arthritis (CIA) (Williams et al., 1997; Kobayashi et al., blood decline sharply after age 40, paralleling declines in 2003; Ro¨ntzsch et al., 2004). However, in carefully controlled responsiveness to foreign antigens; increased reactivity to clinical trials, DHEA treatment produced only modest bene- self; and susceptibility to cancer, metabolic disorders, infec- fits and unwanted side effects (Giltay et al., 1999; van Vol- tious, and autoimmune diseases (Berr et al., 1996). Low lenhoven, 2000). levels of DHEA in patients with rheumatoid arthritis (RA) The inability to translate the benefits of DHEA from ro- have been reported (Wilder, 1996; Cutolo, 2000; Kanik et al., dents to patients remained an enigma. Rodents have little 2000), along with suggestions that replacement therapy endogenous DHEA relative to humans, because of low levels of cytochrome P450 17␣-hydroxylase (Wolf and Kirschbaum, Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. 1999). Marwah et al. (2002) revealed the surprisingly com- doi:10.1124/jpet.108.145086. plex metabolism of DHEA in rodents, in which a multitude of ABBREVIATIONS: DHEA, dehydroepiandrosterone; RA, rheumatoid arthritis; CIA, collagen-induced arthritis; AET, androstene-3␤,7␤,17␤-triol; HE3286, 17␣-ethynyl-5-androstene-3␤,7␤,17␤-triol; CAIA, collagen antibody-induced arthritis; NF-␬B, nuclear factor-␬B; HERF, Hollis-Eden Research Formulation; DTH, delayed type hypersensitivity; PLN, popliteal lymph node; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; BSA, bovine serum albumin; bCII bovine type II collagen; PCR, polymerase chain reaction; F; forward; R, reverse; IFN, interferon; TNF, tumor necrosis factor; IL, interleukin; MMP, matrix metalloproteinase; DEX, dexamethasone; TNP-OVA, trinitrophenyl-ovalbumin; OVA, ovalbumin; HE2500, 5-androstene-16␣-fluoro-17-one (fluasterone); Th, T-helper; A.U., activation units; IQR, interquartile range. 1100 HE3286 Treats Arthritis in Rodents 1101 highly oxygenated metabolites were identified. We hypothe- (w/v) in water. HE3286 was dissolved in vehicle and administered by sized that these oxygenated DHEA metabolites were respon- oral gavage or by subcutaneous or intraperitoneal injection. Control sible for activities attributed to DHEA in rodents. Andro- animals were treated with an equal volume of HERF202. stene-3␤,7␤,17␤-triol (AET) was described previously Animal Care (Butenandt et al., 1938; Reynolds, 1966) as a more highly oxygenated natural DHEA metabolite found in humans (Hill Animals were purchased and housed in accordance with respective et al., 2005) that provided anti-inflammatory benefit in ani- institutional guidelines and requirements of the relevant regulatory ␬ mal models of autoimmunity and trauma (Offner et al., 2002; agencies. NF- B and delayed type hypersensitivity (DTH) studies Auci et al., 2003; Marcu et al., 2006). However, metabolic were performed according to regulations at the La Jolla Institute for Molecular Medicine (La Jolla, CA). CIA studies were performed by H. instability and poor oral bioavailability of AET suggested Offner at Oregon Health and Science University (Portland, OR), as limited pharmaceutical usefulness of this natural hormone described in a previous study (Offner et al., 2004). CAIA studies were (Hollis-Eden Pharmaceuticals Inc., unpublished observa- performed by D. Boyle at University of California at San Diego (La tions). Therefore, analogs of AET were prepared and Jolla, CA) as reported previously (Simelyte et al., 2005). Popliteal screened for improved pharmaceutical properties. The lead lymph node (PLN) assay was performed by R. Pieters at Institute for candidate, HE3286 was composed of the core structure AET Risk Assessment Sciences-Immunotoxicology, Utrecht University with the addition of an ethynyl group at the 17␣-position, (Utrecht, The Netherlands), as reported previously (Pieters and Al- which stabilized the molecule to metabolism. Similar modi- bers, 1999). fications failed at retaining biological activity, whereas oth- Downloaded from Measurements of NF-␬B Activation in Vivo ers resulted in new drugs, such as 17-ethynyl estradiol, an orally bioavailable and pharmaceutically improved version of Animals and Treatments. ICR mice (Charles River Laborato- estradiol (Ranney, 1977). No one could predict whether ries, Inc., Wilmington, MA) were treated with HE3286 (40 mg/kg) by HE3286 would be useful in the treatment of autoimmune intraperitoneal injection or with vehicle alone 0.5 h after, or 24 h disease. We probed the potential of oral HE3286 for utility in before and once again immediately before mice were challenged with 500 ␮g of LPS i.p. (time 0). At 0.2, 1.5, 2 and 2.5 h after the LPS the treatment of RA and found surprisingly broad-based and jpet.aspetjournals.org challenge, animals were sacrificed, and NF-␬B activation in spleen potent anti-inflammatory activity. was evaluated. There are various models of RA in the rodent, and each Spleen Cell Lysate Preparation. Spleens were removed from embraces elements of the pathophysiology relevant to the each mouse and chilled in ice-cold complete Dulbecco’s modified human disease. CIA is an autoimmune-mediated polyarthri- Eagle’s medium (Invitrogen, Carlsbad, CA), cut into three pieces, tis sharing important similarities with RA (Holmdahl et al., and ground between glass slides. Cells were transferred to 15-ml 2002). Mice with CIA display synovitis and erosion of carti- tubes and passed through a cell strainer. Cells were spun for 5 min at 350g. Media were aspirated, and cells were resuspended in 1 ml of lage and bone. The autoimmune response in the CIA model is at ASPET Journals on January 16, 2020 characterized by both the stimulation of collagen-specific T lysing solution to remove red blood cells (approximately 1 min at cells and the production of high titers of specific antibody. room temperature). Complete Dulbecco’s modified Eagle’s medium (12 ml) was then added, and cells were spun at 350g for 5 min. Media The intense synovitis, easily observable histologically, corre- were aspirated, and the cells were resuspended in 10 ml of ice-cold sponds precisely with the clinical onset of arthritis. Because PBS. Cells were spun again for 5 min at 350g. PBS was aspirated, of the pathological similarities between CIA

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