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 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 , 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 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 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 , 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 and RA, the CIA and 1 ml of cold PBS was added. Cells were transferred to Eppendorf model has been the subject of extensive investigation and is tubes on ice. Cells were spun for 10 min in a microcentrifuge, and the a common model in which many putative antiarthritic agents PBS was aspirated. Five times the pellet volume of whole cell lysis are tested. In our previous studies, we reported that HE3286- buffer (20 mM HEPES, pH 7.4, 20% glycerol, 1% Nonidet P40, 1 mM treated mice displayed a significant decrease in CIA disease MgCl2, and 0.5 mM EDTA) was added, and then the tube was score and increased regulatory T cells compared with ani- vortexed and kept on ice for 30 min. Finally, the tube was spun in a mals treated with vehicle alone (Auci et al., 2007). In the microcentrifuge for 20 min at 4°C. The supernatant fluid was trans- Ϫ collagen antibody-induced arthritis (CAIA) model, the typical ferred to an Eppendorf tube and kept at 80°C. Evaluation of NF-␬B Activation. Protein concentration for disease course involves a short period of acute disease fol- spleen cell lysates was measured by using a DC protein assay kit lowed by gradual resolution in joint inflammation (Nanda- (Bio-Rad Laboratories, Hercules, CA). To normalize the samples, 1.2 kumar et al., 2003). The CAIA model permits an analysis of ␮ ␬ optical density750 units (1.7–3 l) was added to each well, and NF- B the contribution of non-T- and B-cell compartments to the enzyme-linked immunosorbent assay was performed using the pathogenesis of experimental RA. Here, we report the results NF-␬B Family Transcription Factor Assay kit (Active Motif, Carls- of our studies testing HE3286 in these models of RA and in bad, CA). This kit is specific for detection of activated NF-␬B in whole classic in vitro and in vivo models of immune function. We cell lysates. Experimental samples and controls were run in dupli- tested the ability of HE3286 to limit NF-␬B activation and to cate. Raji nuclear extract (2 ␮l) was used as a positive control. provide benefit in both CIA and CAIA models. We also eval- Collagen-Induced Arthritis uated its effect on both cellular and antibody-mediated im- mune function. Clinical benefit in RA may be achieved by Mice. DBA/1Lac/J male mice (12/group; The Jackson Laboratory, HE3286 with an improved safety profile. Bar Harbor, ME) were treated (gavage) with HE3286 or with vehicle 7 days after disease onset (50 mg/kg). Induction and Evaluation of CIA. Bovine type-2 collagen Materials and Methods (bCII, immunization grade) was purchased from Chondrex, Inc. (Redmond, WA). To induce CIA, 8-week-old mice were immunized Drugs with 100 ␮g of bCII, emulsified 1:1 with 100 ␮l of complete Freund’s The test compound HE3286 and the vehicle HERF202 were pro- adjuvant (Difco, Detroit, MI) intradermally at the base of the tail. vided by Hollis-Eden Pharmaceuticals Inc. (San Diego, CA). Animals were monitored for onset and progression of disease begin- HERF202 contains 30% ␤-cyclodextrin sulfobutyl ether sodium salt ning 4 to 7 weeks after immunization. A mouse was considered at 1102 Offner et al.

Fig. 1. Effect of HE3286 on NF-␬B ac- tivation in vivo. ICR mice (three/group) were treated with HE3286 (40 mg/kg) by intraperitoneal injection in vehicle (HERF202) or with vehicle alone 24 h before and once again immediately be- fore 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 challenge, animals were sacrificed, and NF-␬B activation was evaluated. Data are expressed as NF-␬B activation units (A.U.) on the y-axis and time on the x-axis. Solid lines connect the medians across time. Vertical bars represent the interquar- tile limits. ϩLPS indicates that LPS Downloaded from was present at that point in time. jpet.aspetjournals.org

TABLE 1 onset of disease when at a score of 1. The arthritic severity of mice Effect of a single treatment with HE3286 on LPS-induced NF-␬B was evaluated using a grading system for each paw according to the activation in vivo following scale: 0, no redness or swelling; 1, slight swelling in ankle ICR mice were treated with HE3286 (40 mg/kg) by intraperitoneal injection in or redness in foot; 2, progressed swelling/inflammation and redness vehicle (HERF202) or with vehicle alone, 0.5 h after mice were challenged with 500 from ankle to mid foot; 3, swelling/inflammation of entire foot except ␮g (intraperitoneally) of LPS (time 0). At 1.5 h after the LPS challenge, animals were sacrificed, and NF-␬B activation was evaluated. Data are expressed as mean NF-␬B toes; and 4, swelling and inflammation of entire foot including toes at ASPET Journals on January 16, 2020 A.U. Ϯ S.D. (Offner et al., 2004). Histology. Fixed joints were decalcified in formic acid and pro- Group n NF-␬B A.U. cessed for paraffin embedding. Tissue sections (5 ␮m) were stained HE3286 (40 mg/kg) 2 0.084 Ϯ 0.0001 with hematoxylin and eosin for histopathological analysis as de- Vehicle 2 0.109 Ϯ 0.004 scribed in our previous study (Offner et al., 2004). The histological arthritis scores were determined in an unblinded manner for prolif- AGC CTT GTC CCT TGA), IL-6 (forward, CCA CGG CCT TCC CTA erative and inflammatory changes and graded from 0 to 3 for each CTT C; reverse, TGG GAG TGG TAT CCT CTG TGA A), IL-1␤ limb. For synovial proliferation, grading was as follows: grade 0, no (forward, TTG ACG GAC CCC AAA AGA TG; reverse, TGG ACA proliferation; grade 1, mild proliferation with two to four layers of GCC CAG GTC AAA G), and IL-23 (forward, TGC TGG ATT GCA reactive synoviocytes; grade 2, moderate proliferation with four plus GAG CAG TAA; reverse, GCA TGC AGA GAT TCC GAG AGA). layers of reactive synoviocytes, increased mitotic activity, and mild or absent synovial invasion of adjacent bone and connective tissue; Collagen Antibody-Induced Arthritis and grade 3, severe proliferation and characterized by invasion and effacement of joint space and adjacent cartilage, bone, and connec- Mice. DBA/1Lac/J male mice (five/group; The Jackson Labora- tive tissue. For articular inflammation, grading was as follows: grade tory) were treated daily with either 0.1 ml of vehicle or with HE3286 0, no inflammation; grade 1, mild inflammation with one aggregate (20, 40, or 80 mg/kg), beginning on day 1 or beginning on day 5 (40 or minimal diffuse leukocyte infiltrates; grade 2, moderate inflam- mg/kg). Certain groups of animals were sacrificed on day 7 (approx- mation with two or more aggregates of leukocytes; and grade 3, imate peak of disease), and joints were taken for PCR analysis of severe inflammation with significant coalescence to diffuse infil- mRNA. Other groups were treated to day 14. trates of leukocytes. Induction and Evaluation of CAIA. To induce arthritis, mice Cytokine and mRNA Detection. Levels of cytokines in spleno- were administered 1 mg of an anti-bCII antibody cocktail (Chondrex, cyte culture supernatants were measured as in our previous studies Inc.) intravenously on day Ϫ2. On day 0, animals were treated (Sinha et al., 2007). Total RNA was isolated from spleens using the (intraperitoneally) with LPS (12.5 ␮g), as described in a previous RNeasy mini kit protocol (QIAGEN, Valencia, CA) and then con- study (Simelyte et al., 2005). The arthritic severity of mice was verted to cDNA using oligo(dT), random hexamers and SuperScript evaluated in a blinded manner using a semiquantitative clinical II RT enzyme (Invitrogen). Real-time PCR was performed as in our scoring system for each paw: 0, no signs of arthritis; 1, swelling previous study (Polanczyk et al., 2005) using QuantiTect SYBR and/or redness of the paw or one digit; 2, two joints involved; 3, more Green PCR master mix (QIAGEN) and primers (Applied Biosystems, than two joints involved, and 4, severe arthritis of the entire paw and Foster City, CA). Reactions were conducted on the ABI Prism 7000 digits. sequence detection system (Applied Biosystems) to detect the follow- mRNA Detection in Joints. The right ankle joint was collected, ing genes: L32 (forward, GGA AAC CCA GAG GCA TTG AC; reverse, dissected to remove extra-articular tissue, and snap-frozen in liquid TCA GGA TCT GGC CCT TGA AC), IFN-␥ (forward, TGC TGA TGG nitrogen. The specimens were pulverized, weighed, and stored at GAG GAG ATG TCT; reverse, TGC TGT CTG GCC TGC TGT TA), Ϫ80°C. Reverse transcription-PCR was performed as described in a TNF-␣ (forward, CAG CCG ATG GGT TGT ACC TT; reverse, GGC previous study (Simelyte et al., 2005) to determine IL-6 and matrix HE3286 Treats Arthritis in Rodents 1103

metalloproteinase (MMP)-3 mRNA expression. Predeveloped se- quence detection reagents (Applied Biosystems) specific for IL-6 and MMP-3 were used at 0.8 ␮l per 20 ␮l of reaction mixture. Each PCR reaction mixture also included 1ϫ TaqMan Universal Master Mix (Boyle et al., 2003). DTH Responses. CD1 mice (Charles River Laboratories, Inc.) were used for these studies. On the day before the start of the experiment, male mice (eight/group) were shaved on their right flank. On day 0, mice were painted (sensitized) on the shaved flank with 50 ␮l of 2.5% oxazolone solution in 95% ethanol (Sigma-Aldrich, Zwijndrect, The Netherlands). Mice were then, beginning on the same day, treated with dexamethasone (DEX; 3 mg/kg i.p.), HE3286 (40 mg/kg, by gavage), or with vehicle (HERF202, by gavage) alone, once daily for 4 days. Twenty-four hours after the final treatment, mice were challenged on the right dorsal ear surface with 25 ␮lof 0.25% oxazolone solution. Both the right and left pinnae thickness Fig. 2. CIA joint histology in DBA mice after treatment with HE3286. were measured at the indicated time points using a micrometer Fixed joints were decalcified in formic acid and processed for paraffin caliper. Data are expressed as left ear minus right ear thickness. embedding. Tissue sections (5 ␮m) were stained with hematoxylin and

eosin for histopathological analysis. The histological arthritis scores were Reporter Antigen Popliteal Lymph Node Assay Downloaded from determined in an unblinded manner for proliferative and inflammatory changes (see Materials and Methods for details). SPS, synovial prolifer- Mice. Nonspecific pathogen-free female BALB/c mice (6–12 weeks ation score; AIS, articular inflammation score. old) were obtained from the Utrecht University breeding facility (Gemeenschappelyk Dier Laboratorium, Utrecht, The Netherlands) and randomly assigned to specific treatment groups.

TABLE 2 Histological analysis of joint tissue from vehicle- and HE3286-treated animals jpet.aspetjournals.org Two hind limbs from three to four mice were submitted in paraformaldehyde. Limbs were decalcified in HCl and sectioned in half longitudinally through the stifle and tibiotarsal joints. Five-micrometer sections were processed and stained with hematoxylin and eosin.

Stiffle Joint Tibiotarsal Joint and Tarsal Joints Dose SPS AIS Notes SPS AIS Notes 40 mg/kg 0 0 Very few mononuclear cells 2 3 Tibiotarsal joint was unremarkable. Tarsal joints had moderate synovial proliferation into articular spaces. Multifocal neutrophilic and at ASPET Journals on January 16, 2020 mononuclear inflammatory cells are present within the synovium and adjacent stroma. 40 mg/kg 0 0 Very few mononuclear cells 1 1 Mild synovial proliferation is present and scattered mononuclear cells in stroma. 0 mg/kg 3 2 Marked synovial proliferation and growth into 3 3 Marked synovial proliferation and in growth into adjacent bone and cartilage. Erosion and loss of adjacent bone and cartilage, and filling in the articular cartilage are present. Scattered articular spaces. Marked erosion and loss of mononuclear and neutrophilic inflammatory cells articular cartilage are present. Multifocal are present within the synovium and adjacent neutrophilic and mononuclear inflammatory stroma. cells are present within the synovium and adjacent stroma. 0 mg/kg 2 1 Moderate synovial proliferation is present and 0 0 Section through tibiotarsal joint was incomplete. scattered mononuclear cells in stroma and However. tarsal joints were overall synovial space contains a focal area of unremarkable. neutrophils. 0 mg/kg 3 2 Marked synovial proliferation and growth into 3 3 Marked synovial proliferation and in growth into adjacent bone and cartilage. Erosion and loss of adjacent bone and cartilage, and filling in the articular cartilage are present. Scattered articular spaces. Marked erosion and loss of mononuclear and neutrophilic inflammatory cells articular cartilage are present. Multifocal are present within the synovium and adjacent neutrophilic and mononuclear inflammatory stroma. cells are present within the synovium and adjacent stroma with associated necrosis. 0 mg/kg 3 2 Marked synovial proliferation and growth into 3 3 Marked synovial proliferation and in growth into adjacent bone and cartilage. Erosion and loss of adjacent bone and cartilage, and filling in the articular cartilage are present. Scattered articular spaces. Marked erosion and loss of mononuclear and neutrophilic inflammatory cells articular cartilage are present. Multifocal are present within the synovium and adjacent neutrophilic and mononuclear inflammatory stroma. cells are present within the synovium and adjacent stroma with associated necrosis. 40 mg/kg N.D. N.D. Processing artifact prevented adequate evaluation. N.D. N.D. Processing artifact prevented adequate evaluation. 40 mg/kg 3 3 Marked synovial proliferation and in growth into 0 0 Section through tibiotarsal joint was incomplete. adjacent bone and cartilage. Erosion and loss of However, tarsal joints were overall articular cartilage are present. Diffuse unremarkable. mononuclear and neutrophilic inflammatory cells are present within the synovium and adjacent stroma. The articular spaces contain large numbers of neutrophils and fibrin. N.D., not done. 1104 Offner et al. Downloaded from jpet.aspetjournals.org

Fig. 3. Proinflammatory cytokine mRNA detected in splenocytes taken from HE3286-treated CIA mice at the end of study (day 22 after onset). Splenocytes were pooled, frozen, subsequently thawed, and evaluated for expression of cytokine mRNA by the RNase protection assay. Data are expressed as relative units on the y-axis and either drug or vehicle on the x-axis. Bottom and top boxes indicate the first and third quartiles, ϫ 6 ␮ respectively. The line connects the medians of the two samples. For IL-17 measurements, splenocytes (4 10 ) were stimulated with bCII (25 g/ml) at ASPET Journals on January 16, 2020 for 48 h. IL-17 levels in supernatants were determined using the 10-Plex Luminex kit (Bio-Rad Laboratories).

Chemicals and Reagents. Chemicals were obtained from Assay. Naive mice (five/group) were injected subcutaneously into Sigma-Aldrich unless stated otherwise. Saline (0.9%; B. Braun Mel- the right hind footpad with 50 ␮l of a freshly prepared sensitizing sungen, Melsungen, Germany) and citrate buffer (0.1 M citric acid dose (50 ␮g) of TNP-OVA. Mice were treated (daily for 5 days) with and 0.1 M disodium phospate, pH 6.0) were used to dilute DEX (5 ␮g i.p.), with HE3286 (4 or 40 mg/kg given by oral gavage) in test chemicals. Trinitrophenol-ovalbumin (TNP-OVA) was prepared HERF202 (100 ␮l) vehicle, or with vehicle alone, or with both as described previously (Pieters and Albers, 1999). HE3286 and DEX. The 5-␮g dose of DEX was chosen, based on

Fig. 4. Effect of HE3286 treatment on well estab- lished CIA. DBA male mice (12/group) were immu- nized with 100 ␮g of bCII/100 ␮g of complete Freund’s adjuvant (100 ␮l) on day 0. Mice were then treated (by gavage) with HE3286 (50 mg/kg) daily, beginning 7 days after disease onset (day 1). Data are expressed as median CIA disease score per paw. The median duration of treatment in the vehicle- treated group was 22.0 days compared with 20.5 days in the HE3286-treated group. Bars represent interquartile ranges. Solid line connects medians. p Ͻ 0.001 versus vehicle on day 28. Arrow indicates start of treatment. HE3286 Treats Arthritis in Rodents 1105

Fig. 5. HE3286 effects on CAIA. DBA male mice (five/group) were administered 1 mg of an anti-bCII antibody cocktail intravenously on day Ϫ2. On day 0, animals were treated (intraperitoneally) with LPS (12.5 ␮g). Ani- mals were treated daily with either 0.1 ml of vehicle or with HE3286 (20, 40, or 80 mg/kg) beginning on day 1, or with 40 mg/kg or ve- hicle beginning on day 5. Data are expressed as the average clinical score of the group (Ϯ1 ,ء .S.D.) on the y-axis and time on the x-axis dosed on days 5 through 14. The bottom and top of each vertical line represent the 25th and 75th percentile, respectively. Plotted functions connect group medians. Arrow in- dicates start of treatment. Downloaded from

previous studies in this assay system that defined the minimally daily arthritic severity scores Ϯ S.D. Differences were evaluated by effective immune suppressive dose. Five days later, mice were killed Student’s t test. jpet.aspetjournals.org by cervical dislocation, and the PLN was excised and separated from adherent fatty tissue. PLNs were isolated in ice-cold PBS/1% BSA; and single-cell suspensions were prepared, washed (350g at 4°C), Results resuspended in 1 ml of PBS/1% BSA, and counted using a Coulter counter (Beckman Coulter, Inc., Fullerton, CA). HE3286 seemed well tolerated at these doses, with respect to frank toxicity and body weight. Proliferation Studies ␬

Effect of HE3286 on NF- B Activation in Vivo. When at ASPET Journals on January 16, 2020 Spleens were obtained from C57BL/6 mice by aseptic techniques. mice were pretreated (Ϫ24 and 0 h) with HE3286 (40 mg/kg The spleens were teased and the suspension filtered to obtain single i.p.) and challenged (intraperitoneally) with 500 ␮g of LPS, 6 cells. Cells (10 mononuclear cells/ml RPMI 1640 medium, 10% fetal levels of activated NF-␬B observed in spleens at all time ␮ bovine serum, and 50 M 2-mercaptoethanol) were incubated in points (0.2, 1.5, 2, and 2.5 h) after challenge were reduced 96-well plates, 0.2 ml per well, for 3 days with no stimulus, or with compared with spleens from animals treated with vehicle approximately 5 ␮g/ml concanavalin A, 2 ␮g/ml phytohemagglutinin, or 1:100 dilution of pokeweed mitogen (Invitrogen). Parallel cultures alone (Fig. 1). The overall profiles of these groups, in terms of ␬ Ͻ contained HE3286 at 1000, 100, and 10 nM. Compound was dis- activated NF- B, were significantly different (p 0.001). solved in dimethyl sulfoxide at 10 mg/ml and then diluted to the Similar results at the 1.5-h time point were obtained with desired concentration with culture medium, leading to a maximal protocols using a single dose (40 mg/kg) of HE3286 given to final solution containing 0.01% dimethyl sulfoxide. DEX at 40 nM animals at 0.5 h after LPS challenge (Table 1). served as the positive control. Cells were incubated overnight with Effect of HE3286 in CIA When Treatment Begins at 3 approximately 1 ␮Ci/ml [ H]thymidine, and proliferation was deter- Disease Onset. In our previous studies, we found that when mined by incorporation of label into cell DNA after filtration, wash- treatment with HE3286 began at disease onset, within 13 ing, and liquid scintillography. Five individual mice were used, days of treatment, HE3286-treated mice displayed a signifi- with each spleen cell preparation incubated in triplicate for each cant (p Ͻ 0.04) decrease in CIA score compared with animals condition. treated with vehicle alone from approximately 13 days after Statistical Analysis onset to approximately 18 days after onset (Auci et al., 2007). For NF-␬B profiles, overall profile homogeneity was assessed by To seek the minimally effective dose, the study was re- means of the Wilcoxon-Mann-Whitney test stratified by time. The peated and more extensive histological evaluation was per- level of significance is set to ␣ϭ0.05, two-sided. Resulting p values formed. The 40-mg/kg dose group showed benefit as deter- were adjusted for multiple comparisons (Westfall et al., 1999; Cytel mined by a strong trend toward reduced cumulative disease Software Corporation, 2005). CIA scores and cytokine levels were index scores (the sum of the daily arthritic severity scores; modeled via the linear mixed model, with treatment, time from CIA 94.5 Ϯ 37 in the HE3286-treated group versus 164 Ϯ 90 for onset, and the interaction between time from onset and treatment as the vehicle-treated group; p ϭ 0.06). Lower doses of HE3286 independent variables. To facilitate direct interpretation, CIA scores (10 or 4 mg/kg) were not effective (cumulative disease index were analyzed as measured, in a scale of 1 to 4. Dose-response trends scores of 172 Ϯ 73 and 138 Ϯ 59; p ϭ 0.54 and 0.87, respec- at each time point were tested for the significance of the slope through a simple linear regression through the means model and tively). Joint tissue samples taken from mice at the end of the formally by use of the exact nonparametric Jonckheere-Terpstra test study revealed expected histopathological changes, including (Cytel Software Corporation, 2005). Statistical analysis was per- marked synovial proliferation, erosion, and loss of articular formed on the SAS system, version 9.1 (SAS Institute, Cary, NC). cartilage in joints from three of four of the vehicle-treated Cumulative disease index scores were calculated as the sum of the animals analyzed. In agreement with our previous study, 1106 Offner et al. intact smooth tissue, fewer mononuclear cell infiltrates and less proliferation were observed in most joints from animals (n ϭ 3) treated with 40 mg/kg HE3286 (Fig. 2; Table 2) (Auci et al., 2007). Effect of HE3286 Treatment on NF-␬B-Associated mRNAs and Cytokines in Spleen. Treatment with HE3286 (40 mg/kg) was also associated with changes in ex vivo cytokine mRNA expression by splenocytes taken at end of the study. Levels of mRNA for IFN-␥, IL-1␤, IL-23, IL-6, and TNF-␣ seemed to be decreased in spleens taken and pooled at the end of the study (Fig. 3). However, the pooling of samples led to an effective sample size of three replicates and did not allow for effective statistical analysis of these observations. IL-17 levels were decreased by almost 50% in cell cultures from the animals treated with the highest dose of HE3286 compared with vehicle-treated controls [vehicle median (IQR), 17.5 (16.6, 17.7) versus HE3286 median (IQR),

9.8 (9.7, 10.2)]. Decreases were also observed in TNF-␣, IL- Downloaded from 13, IFN-␥, and IL-2. Levels of IL-5, IL-6, IL-1␤, and IL-10 were unchanged (data not shown). Effect of HE3286 in CIA When Treatment Begins in Well Established Disease. We next determined whether treatment with HE3286 could also provide benefit in this CIA

model when treatments were delayed until disease was well jpet.aspetjournals.org established. CIA, as judged by disease score, was well estab- lished in mice by 7 days after onset (Fig. 4), in which the average disease score per paw was approximately 1.3 of a possible score of 4. Within 12 days of the start of treatment, mice treated with HE3286 had CIA scores that trended lower compared with vehicle-treated mice (p Ͻ 0.001). Disease

progressed in severity in the vehicle-treated group reaching at ASPET Journals on January 16, 2020 maximal per-paw score of 4 by 23 days after CIA onset. In contrast, disease did not seem to progress in the HE3286- treated group, in that the average disease score never rose significantly above 1.6 for the duration of the study. Effect of HE3286 in CAIA. In CAIA, daily treatment with HE3286 produced clear benefit in a dose-dependent manner (Fig. 5). CAIA began to develop by day 3 in most of the vehicle-treated and HE3286-treated animals, in which the Fig. 6. HE3286 Effects on IL-6 and MMP-3 mRNA levels in hind paw overall, average disease score was 0.5 to 0.75. Disease peaked joints CAIA. DBA male mice (five/group) were administered 1 mg of an anti-bCII antibody cocktail intravenously on day Ϫ2. On day 0, animals in all groups on days 5 to 7. Remarkably, arthritis in the were treated (intraperitoneally) with LPS (12.5 ␮g). Animals were high-dose group (80 mg/kg) was virtually absent throughout treated daily with either 0.1 ml of vehicle or with HE3286 (20, 40, or 80 the course of the study. The benefit provided by HE3286 mg/kg) beginning on day 1 or on day 5 (40 mg/kg). Certain groups of animals were sacrificed on day 7 (approximate peak of disease), and joints treatment was dose-dependent, because the 40-mg/kg dose were taken for PCR analysis of mRNA. Other groups were treated to day produced benefit that was greater than the 20-mg/kg dose, 14. Levels of IL-6 (top) or MMP-3 (bottom) mRNA were determined in but less than the 80-mg/kg dose (Jonckheere-Terpstra p Ͻ these animals, and in animals treated to the end of the study (day 14) by using TaqMan PCR. Data are expressed as relative units Ϯ S.D. on the 0.001). All animals lost weight as a result of disease induc- y-axis and time/dose group on the x-axis. tion. However, all HE3286-treated animals returned to base- line weight by the end of the study, in contrast to vehicle- either vehicle or with saline (PBS) exhibited strong DTH treated animals, which did not (data not shown). In one responses between 2 and 72 h after challenge with oxazolone group of animals, treatment with HE3286 (40 mg/kg) was solution (Fig. 7). Treatment with HE3286 (40 mg/kg) did not only given on days 5–14. HE3286 treatment provided this seem to effect the generation of immunological memory in group of animals with significant (p Ͻ 0.05) benefit beginning this model, because ear swelling was similar in the HE3286- on day 9. HE3286 treatment was associated with dose-depen- treated animals and those treated with saline or vehicle. In dent decreases (compared with vehicle administration) in contrast, mice treated with DEX had greatly reduced ear IL-6 (Fig. 6, top) and MMP-3 (Fig. 6, bottom) mRNA in joints swelling, compared with either the HE3286-treated, saline- of animals taken at peak of disease (day 7) and at the end of or vehicle-treated groups, indicating that DEX powerfully the study (day 14). This study was repeated using the 40- suppressed the DTH memory response. HE3286 did not mg/kg dose of HE3286 with similar results. However, lower markedly interfere with DEX-mediated suppression of DTH doses (10 and 1 mg/kg) were not effective (data not shown). memory responses. Effect of HE3286 in Classic Immune Assays, Includ- Corroborating results were obtained in other in vivo mod- ing the Murine DTH and PLN Assays. Mice treated with els, including the murine ovalbumin (OVA) immunization HE3286 Treats Arthritis in Rodents 1107

Fig. 7. Effect of HE3286 on murine DTH responses in vivo. CD1 male mice (eight/group) were shaved on their right flank on the day prior (day Ϫ1) to the start of the experiment. On day 0, mice were painted (sensitized) on the shaved flank with 50 ␮l of 2.5% oxazolone solution in 95% ethanol. Mice were then (beginning on the same day) treated with dexametha- sone (3 mg/kg i.p.), HE3286 (40 mg/kg, by gavage), or vehicle alone once daily for 4 days. Twenty-four hours after the final treatment, mice were chal- lenged on the right dorsal ear surface with 25 ␮l of 0.25% oxazolone solution. Both the right and left pinnae thick- ness were measured at the indicated time points after challenge using a mi- crometer caliper. Data are expressed as ⌬ thickness, right ear minus left Downloaded from ear on the y-axis and time on the x-axis. Solid lines connect the medians across time. Vertical bars represent the interquartile limits. jpet.aspetjournals.org

model and the PLN assay, an assay typically used to gauge Discussion the immune toxic potential of drugs and pollutants (Pieters, 2001; Pieters et al., 2002). In the PLN assay, mice are in- Oral HE3286 treatment provided significant benefit in both the CIA and CAIA models of rheumatoid arthritis, even jected into the footpad with a sensitizing dose of hapten- when treatment was delayed until disease was well estab- carrier conjugate (TNP-OVA). HE3286 (40 mg/kg) was given lished. HE3286, in contrast to dexamethasone, was not pro- orally for 5 days, and PLN was harvested. HE3286 did not at ASPET Journals on January 16, 2020 foundly suppressive in classic models of cellular or antibody- decrease cell numbers or cytokine production in the PLN, in mediated immunity. Treatment was associated with contrast to DEX treatments, which profoundly decreased all reductions in activated NF-␬B, proinflammatory cytokines these parameters (Table 3). Studies in the PLN assay were mRNAs, and MMP-3. Across multiple studies, including repeated twice with similar results. In the murine OVA im- those reported here and previously (Auci et al., 2007), we munization model, where mice were immunized on days 0, 7, hypothesize a mechanism based on reduced NF-␬B activation and 21, HE3286 was found to only slightly (Ͻ25%) decrease and increased regulatory T-cell levels. serum levels of OVA-specific antibody on day 30. In our previous studies, treatment of CIA with HE3286 HE3286 was also judged as not immune-suppressive in was associated with significant reduction in disease score several in vitro models using both mouse and human cells. and with dramatic improvement in tissue integrity as judged ␮ HE3286, at concentrations as high as 1 M, did not exert any by histological analysis (Auci et al., 2007). This suggested a profound suppressive effects in the mitogen-induced lympho- generalized tissue-sparing activity of the drug in the joints of cyte proliferation assay (Table 4) or mixed lymphocyte re- treated animals and correlated well with observations of sponse assay (data not shown). This was again in contrast to reduced disease score. These observations parallel the activ- DEX, which demonstrated profound immune suppressive ac- ity reported for oral DHEA in this model (Ro¨ntzsch et al., tivity in both these models. 2004), albeit at much higher doses (400 mg/kg). HE3286 provided similar benefit with a single daily administration of TABLE 3 1/10 the effective dose reported for DHEA and thus seems to Effect of HE3286 in reporter antigen PLN assay be far more potent. The HE3286-mediated benefit we ob- BALB/c mice were injected in the right hind foot pad with 50 ␮l of a sensitizing dose served in CIA was also similar to that reported for anti- of TNP-OVA. HE3286 (100 ␮l in HERF202 by gavage) or dexamethasone (5 ␮gin100 TNF-␣ treatment in this model (Joosten et al., 1999). HE2500 ␮l injected intraperitoneally) was given daily on consecutive days starting immedi- ately after sensitization with TNP-OVA. Five consecutive days after injection of is another synthetic steroid that we reported previously to be TNP-OVA, mice were killed, and PLN was removed. Single-cell suspensions were effective in this same CIA model (Offner et al., 2004). prepared, resuspended in 1 ml of PBS/1% BSA and counted. Data in PLN assay are median (IQR) of lymphocyte cell counts expressed as millions of cells per node. HE2500 retains certain immune-regulating properties, is nontoxic, and is practically devoid of androgenic or estrogenic Group n Median (IQR) side effects. However, far higher doses of HE2500 were re- Vehicle 5 8.1 (7.7, 8.6) quired for efficacy, both in our studies in CIA and in the rat HE3286 (4 mg/kg) 5 11.0 (9.5, 11.9) HE3286 (40 mg/kg) 5 9.5 (8.5, 9.6) model of adjuvant induced arthritis, as reported by others DEX ϩ HE3286 (4 mg/kg) 5 3.9 (3.3, 4.7) (Schwartz and Pashko, 2002). Furthermore, in contrast to DEX ϩ HE3286 (40 mg/kg) 5 2.1 (2.1, 2.6) HE3286, HE2500 was only effective in CIA when given by DEX 5 2.7 (2.5, 3.2) the subcutaneous route. 1108 Offner et al.

TABLE 4 Effect of HE3286 in mitogen-induced proliferation assays Splenocytes (106 cells/ml) were incubated for 3 days with no stimulus, or with concanavalin A (5 ␮g/ml), phytohemagglutinin (2 ␮g/ml), or pokeweed mitogen (1:100) Ϯ HE3286 (1000, 100, and 10 nM). Dexamethasone (40 nM) served as the positive control. Cells were incubated overnight with 1 ␮Ci/ml ͓3H͔thymidine, and proliferation was determined by liquid scintillography. Five individual mice were used, with each spleen cell preparation incubated in triplicate for each condition. Data are expressed as median (IQR) cpm per group for various stimulants.

Stimulant Group No Stimulant Phytohemagglutinin Pokeweed Mitogen Concanavalin A

Med/IQR cpm DEX (40 nM) 112 (85, 172) 4488 (3363, 5926) 94 (83, 195) 102 (59, 149) Vehicle 527 (283, 697) 9066 (8757, 12,312) 421 (278, 711) 147 (129, 198) HE3286 (10 nM) 1756 (796, 2419) 10,905 (10,298, 12,442) 1437 (914, 2150) 145 (124, 170) HE3286 (100 nM) 566 (513, 1230) 11,603 (11,368, 11,702) 1741 (819, 2895) 186 (185, 214) HE3286 (1000 nM) 647 (563, 1386) 11,632 (10,077, 11,887) 2677 (1579, 4557) 140 (74, 166) Med/IQR, median (Q1, Q3); median (IQR) cpm per group for various stimulants.

In the present study, we also found that treatment with affected the expression of certain NF-␬B target genes whose HE3286 beginning at CIA onset was associated with de- functions in vivo are known to play a role in the pathophys-

creases in IL-23 mRNA in spleen. IL-23 is essential for T-cell- iology of inflammation. These include TNF-␣, IL-1␤, and Downloaded from mediated autoimmune disease and promotes inflammation IL-6. Our observations suggest that the mRNAs to these via IL-17 and IL-6 (Yen et al., 2006). Furthermore, IL-23 same cytokines may have been suppressed in HE3286- promotes the expansion of Th17 cells (Cooke, 2006), which treated mice. Inhibition of the NF-␬B pathway by HE3286 is share reciprocal developmental pathways with regulatory T currently under investigation in our laboratories. Although cells (Bettelli et al., 2006). Indeed, we found decreased IL-17 our in vivo studies on NF-␬B activation suggest inhibition of ␬ in splenocyte cultures from HE3286-treated animals. This the NF- B pathway may be involved in the activity observed jpet.aspetjournals.org suggests that the HE3286-mediated expansion of regulatory in models of RA, they do not address the relative potency of T cells we observed in our previous study (Auci et al., 2007) HE3286 with respect to other treatment approaches. may relate to a suppression of IL-23 production and Th17 The precise mechanism(s) by which HE3286 mediates cells. It would be of great interest to measure regulatory T these effects remain obscure. Although HE3286, in its anti- cells and/or Th17 cells in the joint tissue of treated and inflammatory activity, and in its ability to expand regulatory untreated animals during the course of CIA, and this is the T cells, resembles glucocorticoids, HE3286 was not found to

subject of continued investigations in our laboratories. be immune-suppressive in any of the classic models of im- at ASPET Journals on January 16, 2020 HE3286 was still effective in the late-onset version of the mune function. Even with extended dosing (over 4 days) in CIA model, when treatment was delayed until disease was DTH, HE3286 did not suppress generation of immunological well established. In these experiments, disease in the memory. Furthermore, HE3286 does not directly interact HE3286-treated animals seems to have been arrested in that (either via binding or transactivation) with any of the known it failed to progress much beyond levels observed at the nuclear hormone receptors, including the glucocorticoid or beginning of treatment, whereas disease continued to sex steroid receptors (T. Wang, S. Villegas, Y. Huang, S. K. progress in the vehicle-treated animals. The late-onset ver- White, C. Ahlem, M. Lu, J. M. Olefsky, C. Reading, J. sion of the CIA model more closely resembles established RA Frincke, and J. R. Flores-Riveros, submitted for publication). disease as it usually presents in clinical situations. Although these remarkable observations do not suggest a HE3286 also provided significant benefit in both onset and mechanism of action or a molecular target for HE3286, they late-onset versions of the CAIA model, in which the contri- do suggest a highly attractive safety profile. In fact, HE3286 bution of T- and B-cell compartments to the pathogenesis of seems to be bone-sparing in both in vitro and in vivo models experimental RA is minimized. It is interesting to note that (Hollis-Eden Pharmaceuticals Inc., unpublished observa- methotrexate, a current mainstay in the treatment of RA, is tions). These observations do not rule out the possibility that not effective in this model (Lange et al., 2005). The HE3286- a metabolite or metabolites of HE3286 may contribute to mediated benefit in CAIA was associated with dramatic re- anti-inflammatory or other activities. With respect to poten- ductions in IL-6 and MMP-3 mRNA levels in joints, mea- tial targets, these may include surface receptors, nuclear sured both at the peak of disease and at the end of the study. hormone receptors, or steroidogenic enzymes such as CYP7B MMP-3 is thought to be an important driver of disease in or CYP11B (Lathe, 2002). Such possibilities are currently human RA (Ichikawa et al., 1998). The observation that under investigation in our laboratories. HE3286 provides benefit in both the CAIA and CIA models HE3286 demonstrated improved metabolic stability com- probably relates to the multifaceted mechanism of action of pared with AET in studies using human microsomes. (Hollis- this compound and bodes well for the potential success of Eden Pharmaceuticals Inc., unpublished observations). In HE3286 in the treatment of human RA. fact, the addition of 17␣-ethynyl group effectively blocks me- We found that HE3286 treatment was associated with tabolism at this position. HE3286 demonstrates 25% oral reductions in activation of NF-␬B. Over time, evidence has bioavailability in rodents and primates. Recent observations accumulated implicating NF-␬B as a mediator of autoimmu- in ongoing phase I/II trials suggest a longer half-life in hu- nity and a potential therapeutic target for treating autoim- mans compared with rodents (8 versus 1.5 h, respectively) mune diseases (Dejardin, 2006). If an inhibition of the NF-␬B and demonstrate that putative efficacious blood levels are pathway by HE3286 was consequential to the activity ob- easily attainable with chronic dosing at doses as low as 10 served in models of RA, then it would be expected to have mg/day. This would be on the same order of potency as HE3286 Treats Arthritis in Rodents 1109 commonly prescribed glucocorticoids (Hollis-Eden Pharma- (2000) Adrenocorticotropin, glucocorticoid, and secretion in patients with new onset synovitis/rheumatoid arthritis: relations with indices of inflamma- ceuticals Inc., unpublished observations). Together, these ob- tion. J Clin Endocrinol Metab 85:1461–1466. servations suggest that the anti-inflammatory activity of Kobayashi Y, Tagawa N, Muraoka K, Okamoto Y, and Nishida M (2003) Participa- tion of endogenous dehydroepiandrosterone and its sulfate in the pathology of HE3286 observed in rodents may translate to humans. collagen-induced arthritis in mice. Biol Pharm Bull 26:1596–1599. 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