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LGD-5552, an Antiinflammatory Glucocorticoid Receptor Ligand with Reduced Side Effects, in Vivo

Discovery Research, Ligand Pharmaceuticals, San Diego, California 92121

Address all correspondence and requests for reprints to: Jeffrey N. Miner, Ardea Biosciences, 4939 Directors Place, San Diego, California 92121. E-mail: jminer{at}ardeabio.com.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
STEROIDAL GLUCOCORTICOIDS ARE among the most effective antiinflammatory agents known for rheumatoid arthritis, multiple sclerosis, and other inflammatory diseases. Unfortunately, long-term use of these molecules results in significant mortality and morbidity in patients due to the impact of their side effects (1). Both the beneficial and detrimental effects of steroids are mediated by the glucocorticoid receptor, a hormone-dependent transcription factor capable of regulating numerous physiological pathways. The powerful antiinflammatory activity of steroids is likely due to direct effects on lymphocytes as well as suppression of a host of inflammatory mediators. The side effects include increased incidence of diabetes due to increased glucose production and decreased insulin sensitivity, higher risk of fracture due to osteoporosis mediated by detrimental effects on bone homeostasis, and decreased wound healing due to effects on cell types mediating this process as well as decreased ability to respond to stress through suppression of the hypothalamic-pituitary-adrenal axis and the resulting adrenal atrophy (2). It would be beneficial to patients if molecules could be found that separate the antiinflammatory effects of steroids from some of the side effects. We term these molecules selective glucocorticoid receptor modulators (SGRMs) for molecules that exhibit selectivity for inflammation vs. side effects as described (2, 3). The group from Schering have used the term SeGRA for selective glucocorticoid receptor agonist (4). Herrlich (5) proposed the hypothesis that separating the activation and repression functions of glucocorticoid receptor (GR) might be sufficient to produce a selective GR modulator. This hypothesis represented a superb starting point and has resulted in the identification several potentially useful compounds, a few of which exhibit some separation between activation and repression functions in vitro (6) but not in all tissues (7) and others that have demonstrated in vivo antiinflammatory efficacy with reduced side effects (8, 9).

We synthesized the compound LGD-5552 [(5Z)-5-[(2-fluoro-3-methylphenyl)methylene]2,5-dihydro-10-methoxy-2,2,4-trimethyl-1H-(1)benzopyrano[3,4-f]quinolin-9-ol] after an extensive high throughput screening effort using a GR-dependent cotransfection assay and subsequent medicinal chemistry targeting of SGRMs (10, 11). This paper describes the characteristics of LGD-5552, a C5-benzylidene compound.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro binding
Extracts from SF-9 moth cells infected with recombinant baculovirus expressing the indicated receptor were used in labeled hormone binding assays. Growth and purification of recombinant human (h) GR baculovirus followed the protocol as described (8). The extract and binding assay buffer consisted of 25 mM sodium phosphate, 10 mM potassium fluoride, 10 mM sodium molybdate, 10% glycerol, 1.5 mM EDTA, 2 mM dithiothreitol, 2 mM {3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate}, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) at room temperature. Intracellular receptors produced in this fashion exhibit reproducible interaction with known ligands at the published affinity. These preparations were subjected to extensive quality control experiments before the assays, covering receptor response, specificity, size, and reference ligand affinity. Receptor assays were performed with a final volume of 250 µl containing from 50–75 µg of extract protein, plus 1–2 nM [3-H]dexamethasone at 84 Ci/mmol and varying concentrations of competing ligand (0–10^–5 M). Assays were set up using a 96-well minitube system and incubations were carried out at 4 C for 18 h. Equilibrium under these conditions of buffer and temperature was achieved by 6–8 h. Nonspecific binding was defined as that binding remaining in the presence of 1000 nM unlabeled dexamethasone. At the end of the incubation period, 200 µl of 6.25% hydroxyapatite was added in wash buffer (binding buffer in the absence of dithiothreitol and phenylmethylsulfonyl fluoride). Specific ligand binding to receptor was determined by a hydroxyapatite-binding assay. Hydroxyapatite absorbs the receptor-ligand complex, allowing for the separation of bound from free radiolabeled ligand. The mixture was vortexed and incubated for 10 min at 4 C, centrifuged, and the supernatant removed. The hydroxyapatite pellet was washed two times in wash buffer. The amount of receptor-ligand complex was determined by liquid scintillation counting of the hydroxyapatite pellet after the addition of 0.5 mM EcoScint A scintillation cocktail from National Diagnostics (Atlanta, GA).

After correcting for nonspecific binding, IC₅₀ values were determined. The IC₅₀ value is defined as the concentration of competing ligand required to reduce specific binding by 50%; the IC₅₀ values were determined graphically from a log-logit plot of the data. Dissociation constant (Kd) values for the analogs were calculated by application of the Cheng-Prussof equation. Steroid standards are included in each assay, and resulting Kd values are determined by use of a modified Cheng-Prussoff equation (12).

Mineralocorticoid receptor (MR), androgen receptor (AR), progesterone receptor (PR), and estrogen receptor- expression in the baculovirus system and binding assays were conducted similarly except that labeled ligands were aldosterone (1–2 nM ³-H aldosterone; Amersham, Piscataway, NJ; TRK 434, specific activity 60 Ci/mmol), dihydrotestosterone (DHT; 1–2 nM ³-H DHT at 130 Ci/mmol), progesterone (2–3 nM ³-H progesterone, 93 Ci/mmol; Sigma, St. Louis, MO), and estradiol (2–3 nM ³-H estradiol, 114 Ci/mmol; NEN Life Science Products, Boston, MA), respectively. Each binding assay point is done in duplicate and each full experiment is repeated three or more times.

Plasmids
pRSV-hGRnx and MMTV-luciferase were obtained from Ron Evans (Salk Institute, La Jolla, CA). A reporter construct containing 600 bp of the E-selectin promoter region fused to the luciferase gene (E-sel/luc) was used in repression assays together with an expression vector encoding human glucocorticoid receptor driven by the Rous sarcoma virus enhancer (RSV)-hGR, cotransfected with a β-galactosidase (β-Gal) expression vector as a control.

Compounds and formulations
Dimethyl sulfoxide (Sigma); RNeasy minicolumns, RNA isolation and purification kit, and RNase-Free DNase clean-up kit were obtained from QIAGEN (Valencia, CA). Superscript first-strand synthesis system for RT-PCR kit was obtained from Invitrogen (Carlsbad, CA). Real-time PCR master mix was obtained from Applied Biosystems International (Foster City, CA). LGD-5552 was synthesized at Ligand Pharmaceuticals, Inc., and prednisolone was purchased from (Steraloids, Newport, RI). Both LGD-5552 and prednisolone were formulated for animal studies in 50% olive oil and 0.9% carboxymethyl cellulose in water.

Transfection
DMEM and Eagle MEM were obtained from BioWhittaker (Walkersville, MD). All fetal calf serum was purchased from Hyclone (Logan, UT). CV-1 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were seeded 48 h before transfection in 96-well microtiter plates. Cells were transiently transfected by the calcium phosphate coprecipitation procedure (13) with a reporter plasmid, MMTV-LUC, containing the mouse mammary tumor virus (MMTV) long terminal repeat linked to luciferase, a β-Gal expression plasmid, pCMV-β-Gal, coding for the constitutive expression of Escherichia coli β-Gal, an expression vector constitutively expressing the selected receptor under the control of the RSV promoter and filler DNA (pGEM).

HepG2 cells were from American Type Culture Collection, grown in DMEM (BioWhittaker) containing 10% (vol/vol) fetal calf serum (Hyclone), 2 mM L-glutamine, and 55 µg/ml gentamicin. Unless otherwise noted, 5 µg/ml of a human GR-expression plasmid vector (RSV-hGR), 5 µg/ml MMTV-LUC reporter plasmid, 2.5 µg/ml of pRSV-β-Gal as a control for transfection efficiency, and 7.5 µg of filler DNA (pGEM4) at a final concentration of 20 µg/ml were precipitated and then added to the cells. The medium was changed 16 h later to contain 5% charcoal-stripped fetal calf serum and steroid ligands with or without test compounds (10 µM) for 24 h. Cells were then lysed and assayed as described (14). The E-selectin transfection assay is similar except that 5 µg/ml E-sel/luc reporter plasmid was added instead of MMTV-luciferase. The medium was changed 16 h after transfection to contain 10% charcoal-stripped fetal calf serum, TNF (10 ng/ml), IL-1β (1 ng/ml), and test compounds (10 nM to 10 µM) with or without 0.32 nM dexamethasone for 24 h. Cells were then lysed and assayed as described above.

Test animal housing and care
Animals were housed either five per cage (immature rats) or two to three per cage (mature rats) under controlled lighting conditions on a 12-h cycle, with lights on at 0600 h. Rat chow (Harlan Teklad 8604; San Diego, CA) and water were available ad libitum. The experimental protocol was approved by the Institutional Animal Care and Use Committee, and the animals were maintained in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

Adjuvant-induced arthritis model
Lewis rats were used for the adjuvant-induced arthritis model. Fourteen days after measurement of baseline hindpaw volume and the injection of Freund’s adjuvant intradermally into the base of the tail, rats were culled and randomized into treatment groups. Rats were orally dosed for 14 d with either vehicle or 10 or 30 mg/kg of prednisolone and LGD-5552. Under the dosing paradigm, the compounds are stable for at least 2 wk. Daily right hindpaw volumes were measured by water plethysmography. Data are paw volumes (milliliters) of the right paw for 10–15 rats/group. Rats from the adjuvant-induced arthritis (AIA) study were euthanized 4 h after the last dose and blood samples were collected. Blood was allowed to clot and serum collected and stored at –70 C until they were assayed for monocyte chemoattractant protein (MCP)-1 levels. Ankle joints were isolated and pulverized under liquid nitrogen using a 6850 freezer mill (Spex Inc., Metuchen, NJ). Pulverized samples were then homogenized and sonicated on ice in 2 ml of protein extraction buffer (PBS, 0.05% Tween 20, protease inhibitors cocktail mix) followed by centrifugation. The supernatant was then filtered through 0.45 µm filter, and the samples were stored at –80 C until they were assayed for rat MCP-1 and IL-10 levels. The MCP-1 and IL-10 ELISA kits (Pierce Endogen, Rockford, IL) are solid-phase sandwich ELISAs, in which serum samples are diluted 1:25 (MCP-1) and 1:2 (IL-10) and then incubated in an antirat MCP-1 or antirat IL-10 precoated 96-well strip plates. After a series of washes, biotinylated antibody reagent is added followed by streptavidin-horseradish peroxidase. A substrate is then added followed by stop solution. Sample absorbance is determined at 450 nm, and those readings are corrected by the absorbance of the same samples measured at 550 nm.

Total RNA from the ankle joints was isolated after pulverization under liquid nitrogen using a 6850 freezer Mill (Metuchen, NJ) in Trizol. Six micrograms of total RNA from these joints were used to prepare the target for hybridization of Affymetrix microarray chips according to standard Affymetrix protocols. The labeled target was subsequently hybridized to U34A rat expression arrays (containing 8799 sequences) in an Affymetrix 640 hybridization oven. After washing and staining in the Affymetrix fluidic station, the array was scanned with an Affymetrix GeneArray 2500 scanner. Affymetrix MAS 5 software was used to generate signal intensity and to designate present and absent calls.

Experimentally induced autoimmune encephalitis (EAE) model
EAE was induced by immunizing the rats with an emulsion containing 200 µg of guinea pig myelin basic protein peptide 69–87, and complete Freund’s adjuvant containing 400 µg of heat killed Mycobacterium tuberculosis. One hundred microliters of the emulsion were injected sc into each hind footpad of the rats (200 µl total volume). Rats were monitored daily for the clinical signs of EAE by a blinded investigator and disease severity scored according to an established disability index: 0, no disease; 0.5, tail weakness; 1, tail paralysis; 1.5, tail paralysis and wobbly gait; 2, hind limb weakness; 2.5, hemiplegia (one limb paralyzed); 3, paraplegia; 3.5, paraplegia with forelimb weakness; and 4, quadriplegia. Rats were divided into three groups. One group was treated with vehicle (n = 7), a second group was treated with prednisolone (n = 8), and a third group was treated with LGD-5552 (n = 8). Drugs were suspended in a solution of 50% olive oil and 50% carboxymethyl cellulose (0.9%), and 30 mg/kg of each drug were delivered to the rats by gavage daily starting on the day of immunization and continuing for 19 d.

Hypertension
Male Wistar-Kyoto rats (100–149 g) from Harlan (Indianapolis, IN) were acclimated for a week before the start of the experiment and divided into cohort A and cohort B. During the acclimation period, all of the rats were orally dosed daily with vehicle [50:50 Bertolli Classico olive oil (Englewood Cliffs, NJ) and carboxymethyl cellulose (0.9%; CMC; Sigma)]. Blood pressure (BP) was determined for each cohort on alternative days during the acclimation period, with at least four BP measurement before the start of the experiment. After a week of acclimation, animals (body weight 170 g) were sorted into treatment groups based on the mean of their last two BP measurements during the acclimation period. The treatments were vehicle; prednisolone (Steraloids) at 1, 3, 10, and 30 mg/kg/d; and LGD-5552 at 1, 3, 10, and 30 mg/kg/d. For each treatment, n = 10, BP was determined on d 1, 3, 5, 7, 9, 11, and 13 at 5 h after dosing. The dosing was staggered to make sure each animal’s BP was determined as close to 5 h after dosing as possible. BP was always taken according to the numerical order of the animal identification in both the acclimation and treatment periods, and dosing was done in the same order each day.

Carrageenan paw edema assay
After an overnight fast, hindpaw volumes of 180-g male Sprague Dawley rats were measured by water plethysmography, followed by oral dosing with LGD-5552 or prednisolone 1 h before injecting 100 µl of 1% carrageenan into the plantar region of the right hindpaw. Four hours after the carrageenan challenge, right hindpaws volumes were remeasured to evaluate changes in volume due to inflammation; uninjected left hindpaws served as internal negative controls. In addition, a group of animals in which saline was injected instead of carrageenan, were used as the no-edema controls and represented 100% efficacy. Data are expressed as changes in paw volumes and, for comparison purposes, recalculated in terms of relative efficacy using 30 mg/kg prednisolone as 100% efficacy.

In vivo side effect assays
Immature (19 d old) male Sprague Dawley rats from Harlan were acclimated for 2–3 d after arrival. Before the first dose, the rats were sorted into groups such that no statistically significant differences in mean body weights were observed. Rats began receiving daily treatment the morning of d 1. The rats were dosed daily for a consecutive 7 d via oral gavage (4 ml/kg). Body lengths were measured on the day before the first dose (all studies) and d 7 (for 7 d treatment studies). Prednisolone was used as a positive control. Approximately 24 h after the last dose, rats were killed by decapitation and trunk blood was collected into vacutainer tubes (no. 367661 and no. 366512; Becton Dickinson, Franklin Lakes, NJ). The spleen, thymus, brain, and adrenal glands were then isolated, blotted dry, and weighed individually. Organ weights were expressed relative to the weight of the brain (milligrams organ weight per gram of brain weight). Quantitation of testosterone, corticosterone, IGF-I, osteocalcin, triglycerides, and/or cholesterol was performed on serum samples for each study (data not shown). Tibia length was determined using a digital caliper as the distance between the tips of the medial condyle and intermediate ridge between the distal articular surfaces.

For serum, whole blood was collected into vacutainer tubes (no. 367661 and no. 366512; Becton Dickinson). Blood samples were allowed to clot 4 C for approximately 2 h. Serum was separated by centrifugation (2500 rpm, 30 min; Sorvall RC 3C Plus centrifuge, rotor H2000B) and collected. Aliquots were stored at –80 C.

For plasma, approximately 300 µl whole blood were collected into EDTA-containing microtainer tubes (no. 365973; Becton Dickinson). The tubes were centrifuged at 1000 x g for 5 min at 4 C to separate the plasma. Plasma samples were stored at –20 C.

For the preparation of LGD-5552, prednisolone, or dexamethasone dose formulations, the appropriate amount of test compound for the highest concentration to be gavaged was weighed. The compound was initially suspended in olive oil at a 2x concentration. Then the formulation was diluted with an equal volume of 0.49% CMC in water. The final vehicle consisted of 50% olive oil, 0.45% CMC, and 49.55% water. Bertolli Classico 100% pure olive oil (lot L205BR) was used for formulations. CMC (lot 64H1176) was purchased from Sigma-Aldrich (St. Louis, MO). Isoflurane was purchased from Halocarbon Products Corp. (River Edge, NJ).

Data analysis was performed using one-way ANOVA using JMP version 5.1.2 (SAS Institute, Cary, NC). If the data were not normally distributed or had unequal variances, analysis was performed on transformed data as determined by Box-Cox transformation (9). In cases in which data were analyzed after logarithmic transformation, the results are expressed as the geometric means ± SEM. The Dunnett’s test was performed to determine significant (P < 0.05) differences against vehicle controls.

Data were fitted to a modified four parameter logistic equation to obtain an estimate of the ED₅₀. The model estimates ED₅₀s in a logarithmic scale because log ED₅₀ is a more robust estimate of the potency. The model also provides an SE for the estimate that is used to calculate 95% confidence limits of the estimated potencies. The following equation exemplifies the basic, reparametrized four-parameter logistic equation used in estimating potencies in these studies: Y = (A – D)/[1 + eB ·(logC-logx)] + D, where A is the maximum, D is the minimum, B is the slope, C is either the ED₅₀, depending on the direction of the response, and x is the dose of the compound used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LGD-5552 has a molecular weight of 443.515 (C₂₈H₂₆FNO₃) and binds GR with an affinity of 2 nM (Fig. 1). It is a nonsteroidal compound comparable in size with prednisolone but differs in ring saturation as well as substituents. Similarly, prednisolone, a known steroidal antiinflammatory GR ligand has a molecular weight of 358.43 and an affinity for GR of 5 nM (Fig. 1).

View larger version (22K): [in this window] [in a new window]   FIG. 1. The benzylidene LGD-5552 exhibits selective binding to hGR, antagonizes prednisolone-induced transcriptional activation of the MMTV promoter, and shows agonistic properties in repressing IL1β-TNF-induced activation of the E-selectin and IL-6 promoters. No significant cross-reactivity with hAR and hPR is observed in cotransfection assays, whereas the compound is a potent hMR blocker. The structure of LGD-5552 (C28H26FNO3; molecular weight of 443.5) and that of prednisolone and dexamethasone for comparison purposes are shown above a table with the activity of both steroidal glucocorticoids and LGD-5552. The binding affinity for hGR is shown as Kd in nanomolar concentration in the first column. Activation assays are conducted in CV-1 cells by cotransfecting the MMTV promoter in a reporter construct and a hGR expression vector. These assays were run in agonist, by just adding the compounds mentioned in the table, or antagonist mode by adding LGD-5552 to XX nM prednisolone. In the repression assays, HepG2 cells transfected with a reporter construct containing either the E-selectin or IL-6 promoter as well as a hGR expression vector are challenged with 1 ng/ml IL1β-TNF. The assay evaluates the ability of the compounds to reduce cytokine-induced activation of the reporter gene. Cross-reactivity assays are conducted by transfecting the respective receptor expression vector and MMTV-luciferase reporter plasmid in agonist and antagonist modes. In the antagonist mode, DHT, progesterone, and aldosterone are used at 1 nM, respectively. For the activation and repression assays, efficacies are calculated as percent of the dexamethasone response, which is set arbitrarily to 100%. In the agonist mode cross-reactivity assays, efficacies are calculated as percent of the specific standard for the assay: DHT, progesterone, or aldosterone for the hAR, hPR, and hMR cross-reactivity assays, respectively. In the antagonist mode, efficacy is calculated as the percent reduction of the agonist-induced response. Dex, Dexamethasone; Pred, prednisolone.

In vivo antiinflammatory activity
We also measured the in vivo antiinflammatory activity of LGD-5552 and prednisolone in an adjuvant-induced arthritis model. This is a stringent, therapeutic model of rheumatoid arthritis in which glucocorticoids are known to be active. This is one of the most widely used animal models of chronic inflammation involving complex pathologies in joint, soft tissue, and bone. Pharmacological effects of antiinflammatory agents on joint swelling, synovitis, and periosteal new bone formation can be evaluated via plethysmography and histology as well as by magnetic resonance imaging of the affected joints (17). We induced inflammation in the paws by injection of Freund’s complete adjuvant intradermally into the base of the tail on d 0. Dosing begins after paw inflammation is maximal at d 16. This period during the disease progression is characterized by significant soft tissue injury (edema, synovitis, and joint separation) but is before the onset of destruction in the ankle bones. Figure 2 shows the paw volume of controls and treated animals between d 16 and 30 after adjuvant administration and compares the antiinflammatory activity of LGD-5552 and prednisolone. LGD-5552 and prednisolone are highly effective at inhibiting this inflammatory response at both 10 and 30 mg/kg. The LGD-5552-treated group reached maximal efficacy at d 18, 19, and 20 at the 30 mg/kg dose, and paw volumes were similar to those observed in the no-disease controls (Fig. 2B, open squares). In contrast, paw volumes at d 18, 19, and 20 in the 30 mg/kg prednisolone-treated group were still significantly different from the no-disease group. Whereas these data suggest early onset of efficacy of LGD-5552 in this model, increasing the dose of prednisolone to 60 mg/kg gives equivalent efficacy as 30 mg/kg of LGD-5552 (data not shown).

View larger version (35K): [in this window] [in a new window]   FIG. 2. LGD-5552 is a fully efficacious antiinflammatory agent in the AIA model and, at high doses, achieves full efficacy more rapidly than prednisolone. Paw volumes over time for animals treated orally with vehicle (black circles), prednisolone 0.1 (red down pointing triangles), 1 (green circles), 3 (blue squares), 10 (pink triangles), and 30 (gray diamonds) mg/kg (A) or LGD-5552 1 (B, green circles), 3 (B, blue squares),10 (B, pink triangles), and 30 (B, gray diamonds) mg/kg for 15 d. Joint inflammation in this model is induced by the administration at d 0 of complete Freund’s adjuvant in the base of the tail. Animals injected with saline are used as the no-disease control group (A and B, black squares). The inflammatory process reaches a maximum at 15 d whereupon treatment is begun. The treatment continues for 15 d, and daily paw volumes are measured by plethysmography as an indicator of inflammation. Vehicle-treated rats are indicated by the black circles. Black squares denote paw volumes for normal, nondisease animals and would indicate a complete recovery of the inflammatory process. Red down-pointing triangles, 0.1 mg/kg; green circles, 1 mg/kg; blue squares, 3 mg/kg; pink upward-pointing triangles, 10 mg/kg; gray diamonds, 30 mg/kg. Statistical evaluation of the data were conducted by two-way ANOVA with repeated measures. To identify specific group-to-group differences, the Tukey-Kramer test was used. Asterisks identify groups that exhibited statistically significant (P < 0.05) differences vs. vehicle-treated animals at the same time point and indicate significant activity for the compounds. Crosses denote statistically significant (P < 0.05) differences vs. the normal, no-disease group, and therefore, they indicate groups that have not achieved maximal efficacy.

View larger version (14K): [in this window] [in a new window]   FIG. 3. LGD-5552 differentially modulates inflammation-related genes when compared with prednisolone in a model of rheumatoid arthritis (AIA) in vivo. IL-10 and MCP-1 levels (upper and middle panels) were measured in treated and untreated animals from an AIA experiment similar to that shown in Fig. 2. Animals were euthanized at d 7 of the treatment period, and IL-10 and MCP-1 levels were measured in extracts of the ankle joints by ELISA. Compounds were dosed at 3, 10, and 30 mg/kg for both prednisolone and LGD-5552 for 7 d. In addition, relative COX 2 mRNA levels were determined in ankle joint total RNA. The bottom panel shows relative COX2 gene expression in response to various doses of either LGD-5552 (gray bars) or prednisolone (black bars). Nondiseased and diseased vehicle-treated animals were included as controls (white and hatched bars, respectively). Statistical analysis was conducted on data elevated to the power 0.2 using a one-way ANOVA followed by the Fisher’s test. *, P < 0.05 vs. vehicle; , P < 0.05 vs. no disease.

View larger version (15K): [in this window] [in a new window]   FIG. 4. LGD-5552 treatment blocks EAE. A, Mean disease score for immunized rats treated with vehicle (n = 7; open diamonds), immunized rats treated with prednisolone at 30 mg/kg (n = 8; closed squares), and immunized rats treated with LGD-5552 at 30 mg/kg (n = 8; closed triangles). Treatments were administered by gavage daily starting on the day of immunization and continuing until d 20 after immunization. B, Beneficial effect of LGD-5552 on the incidence of disease in immunized animals. Open bar, Vehicle-treated animals (100% of these animals exhibited signs of disease); gray bar, prednisolone-treated rats (30 mg/kg); black bar, LGD-5552-treated group (30 mg/kg). Numbers in parentheses within the bars illustrate the number of animals presenting signs of encephalitis of the total number of animals in the group. Incidence data were statistically evaluated using two-tailed Fisher’s exact probability test and the absolute P values are indicated on top of the bars.

View larger version (23K): [in this window] [in a new window]   FIG. 5. LGD-5552 is less active than prednisolone in increasing MABP in Wistar-Kyoto rats. Wistar-Kyoto rats were dosed for 13 d with compound, and MABP (mm Hg) was measured every other day. The x-axis describes days of treatment with the MABP response plotted on the y-axis. Vehicle controls (black circles) are shown for both panels. Prednisolone is in dose response from 1 to 30 mg/kg in the left panel and LGD-5552 is in dose response from 1 to 30 mg/kg on the right panel. Green squares indicate the groups treated with 1 mg/kg of the compounds. Blue open-pointing triangles denote the 3 mg/kg groups. Gray diamonds represent the 10 mg/kg-treated groups, whereas the pink diamonds indicate the 30 mg/kg-treated groups. Statistical analysis was conducted by two-way ANOVA with repeated measures followed by the Tukey-Kramer test on raw data. *, P < 0.05 vs. vehicle.

View larger version (12K): [in this window] [in a new window]   FIG. 6. LGD-5552 shows a better selectivity profile than prednisolone when compared in an acute model of inflammation vs. various end points after subacute therapy. The relative efficacy of LGD-5552 vs. 30 mg/kg of prednisolone (defined as 100%) is plotted for the acute inflammation model, the CPE assay, vs. a specific side effect end point determined in a subacute dosing paradigm for these compounds. The first panel compares prednisolone and LGD-5552 responses in the inflammation model to the compounds effects on body weight after 7 d of oral treatment. The other panels compare antiinflammatory activity to effects on bone growth (tibia length), thymus weight, and adrenal weight in the same subacute paradigm. Dose responses are shown for each compound and for each tissue.

LGD-5552 exhibits good oral pharmacokinetics with an area under the curve of 4.63 µg/h·ml and a maximum concentration of 53 µg/ml, compared with prednisolone with an area under the curve of 2.54 µg/h·ml and a maximum concentration of 86 µg/ml in rats dosed for 7 d with 10 mg/kg compound. These data indicate that LGD-5552 exhibits antiinflammatory properties similar to prednisolone with a diminished side effect profile [compare the separation between the solid (antiinflammatory efficacy) and open (side effect profile) symbol dose response curves for the different side effects in Fig. 6].

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A prolonged effort by numerous laboratories has gone into the detection and development of tissue SGRMs for the treatment of inflammatory disease. A number of promising chemotypes have been discovered and are in various stages of characterization (1, 4). These potential SGRMs are steroidal (20) and nonsteroidal (21, 22, 23) and have been demonstrated to have various activities that suggest a separation between antiinflammatory properties and steroidal side effects (2). Unique in vitro activity has been described (2, 6, 21, 22, 24) and in some cases demonstrated antiinflammatory activity in vivo together with reductions in side effects (8, 23), others demonstrate antiinflammatory efficacy but not side effect information (25, 26, 27).

We have characterized a benzylidene, LGD-5552, for use in inflammatory diseases. The compound binds competitively with steroids in the ligand binding domain of GR and, in contrast to steroidal glucocorticoids, is a strong antagonist of dexamethasone-mediated MMTV-luciferase reporter activity. Similarly to steroids, LGD-5552 induces very efficient repression by GR and is capable of inhibiting both E-selectin- and IL-6-promoter induction by the inflammatory mediators: TNF and IL-1. LGD-5552 has some weak antagonist activity on MR (108 nM) and has little or no activity on other steroid receptors such as the hAR and hPR.

Both prednisolone and LGD-5552 were very effective in treating inflammation in the adjuvant-induced arthritis model at both 10 and 30 mg/kg.

We examined the regulation of gene expression in inflamed joints of these animals and detected unique regulation by LGD-5552 at some genes by analysis of mRNA (data not shown). We used an ELISA to measure a number of inflammatory markers in blood from these animals to corroborate the mRNA findings. The antiinflammatory cytokine IL-10 was up-regulated by LGD-5552 when compared with the same dose of prednisolone. This cytokine exerts its antiinflammatory effect in part through strong negative regulation of proinflammatory cytokines expressed in mononuclear phagocytes (28). High levels of IL-10 dampen the response of lipopolysaccharide-induced genes in macrophages; thus, IL-10 increases may contribute to the efficacy seen with LGD-5552. Other cytokines are involved in enhancing the inflammatory response. These include MCP-1 (29), which was strongly down-regulated by LGD-5552 and weakly down-regulated by prednisolone. The Cox2 gene was repressed by both glucocorticoid and LGD-5552. We cannot rule out the possibility that there is posttrancriptional regulation of these markers by these compounds, but we believe it is likely to be primarily an effect on mRNA expression. Thus, both prednisolone and LGD-5552 are fully effective in treating adjuvant-induced arthritis in Lewis rats, producing functional normalization of the paw volumes in a few days after initiation of the treatment, and each ligand exhibits a unique inflammatory marker expression profile.

In addition to arthritis, glucocorticoids are also used to treat patients with multiple sclerosis. We tested prednisolone and LGD-5552 in a rodent model of multiple sclerosis in which glucocorticoids are known to be active, the Lewis rat experimental autoimmune encephalitis or EAE model. Both compounds strongly reduced both disease scores and incidence of disease in this model. Importantly, LGD-5552 appeared to have increased efficacy vs. prednisolone because even at the highest dose level, prednisolone-treated animals still exhibited the signs and symptoms of disease including reduced use of hind limbs, dragging limbs, and generally reduced mobility. In contrast, the LGD-5552-treated animals were indistinguishable from nondiseased animals, exhibiting normal behavior and complete mobility.

The demonstration of full efficacy of LGD-5552 in both arthritis and multiple sclerosis models is an important requirement for a SGRM; however, reductions in side effects vs. steroids remain a critical hurdle. We confirmed that the agonist prednisolone raises MABP significantly within a few days of dosing at all doses tested, even as low as 1 mg/kg·d. Interestingly, LGD-5552 was unable to raise MABP at either 1 or 3 mg/kg and increased it only at the higher doses after more than a week of dosing. This difference is not likely due to the potency or efficacy of LGD-5552 because of the data shown for the AIA and the MS model show similar activities of both compounds. We have examined the response of salt-fed adrenalectomized rats to both steroid and LGD-5552 treatment. Urine output and potassium and sodium levels suggest an intermediate response between a glucocorticoid and mineralocorticoid antagonist response for LGD-5552. It is possible that the antagonist activity we detected on the MR plays a role here. These MABP results suggest that LGD-5552 might exhibit reduced impact on blood pressure in patients, thereby reducing one of the problems associated with glucocorticoid administration.

To examine a broader array of side effects, we used another rat model of side effects in which we treat normal Sprague Dawley rats with vehicle, prednisolone, or LGD-5552 for 1 wk and then measure selected glucocorticoid-responsive endpoints including: body weight, tibia length, thymus weight, and adrenal weight. After administration of prednisolone, each of these end points decreases significantly. LGD-5552 exhibits less activity at all these end points. The SGRM fails to reach full suppression of body weight and tibia length and is much less potent than prednisolone at reducing thymus and adrenal. To obtain a read on efficacy, we used the CPE model, a recognized measure of a localized, acute inflammatory response. Both prednisolone and LGD-5552 are fully efficacious at blocking inflammation in this assay. The data for CPE efficacy plotted alongside each side effect end point clearly illustrate that LGD-5552 demonstrates full efficacy as an antiinflammatory agent and reduces the impact on side effects. Glucocorticoids cause a decrease in muscle mass in rats as well as decreases in appetite, which are likely responsible for the decreased weight (30, 31).

Changes in tibia length are suggestive of glucocorticoid effects on the growth plate of the long bones. Glucocorticoids are known to reduce growth in children (32); fortunately, on ceasing treatment, growth resumes and treated children eventually reach a normal stature. AL438, a nonsteroidal SGRM, appears to separate on this end point (8, 33). Similarly, LGD-5552 appears to exhibit only weak partial activity on this end point, suggesting less impact on growth, a potential benefit in pediatric populations.

The effect of glucocorticoids on rat thymus weight may be related to the longer-term immunosuppressive effects of glucocorticoids. Glucocorticoids are known to reduce the number of white blood cells in blood, and in several experiments, LGD-5552 and prednisolone exhibited similar reductions in lymphocyte count. In contrast, thymus weight is significantly less suppressed by LGD-5552, compared with prednisolone. This may be important because glucocorticoids are known to increase the incidence of infection in steroid-treated patients (34). Decreased immune function and thymic involution may play a role in preventing a sufficient response to infections in patients treated with glucocorticoids.

Glucocorticoids reduce the secretion/production of cortisol by suppression of the hypothalamic-pituitary-adrenal axis and decrease the size of the site of cortisol production, the adrenal gland. Prednisolone strongly suppressed adrenal weight; in contrast, LGD-5552 suppressed adrenal gland weight less potently and was fully active only at the highest dose tested: 30 mg/kg. Although we cannot entirely rule out differential tissue distribution of LGD-5552 as one mechanism of the separation between its strong activity as an antiinflammatory agent and its reductions in side effects, several experiments looking at tissue distribution have revealed nothing that suggested this mechanism was important. Rather, we believe that the compound fundamentally alters the outer receptor structure when bound in the ligand binding pocket, resulting in changes in the ability of the protein to interact with coactivators and corepressors. This changes its effect on gene expression in a gene-specific manner resulting in differential response of specific genes. This notion has precedent because the compound AL-438 (8) exhibits striking selectivity on a number of side effect assays and is fully efficacious as an antiinflammatory agent. This molecule exhibits significantly different interactions with coactivators primordial germ cell-1 among others, which we hypothesize is responsible for its unique activity. AL-438 is not as potent an antiinflammatory agent as LGD-5552, nor is it as separated in measures of activation and repression are used. It does exhibit a beneficial antiinflammatory/side effect profile, however, in glucose and bone. In contrast, LGD-5552 exhibits selectivity on bone growth, BP, and thymus organ weight and remains a powerful antiinflammatory agent. It appears that LGD-5552 acts selectively to suppress inflammation without inducing some of the side effects of glucocorticoids in both acute and chronic models of inflammation and side effects. This class of nonsteroidal GR-dependent antiinflammatory drugs may offer a safer alternative to steroidal glucocorticoids in the treatment of inflammatory disease.

	Acknowledgments

 
We thank Mark Chapman, Louise de Grandpre, Junlian Hu, and Eric Vajda for helpful comments and suggestions on both the experimental strategy and the interpretation of results.

	Footnotes

 
Current address for R.J.A.: Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037.

Current address for B.B.: Department of Pharmacology, Elbion AG, Meissner Strasse 191, 01445 Radebeul, Germany.

Current address for K.B.: Millipore Corp., 28820 Single Oak Drive, Temecula, California 92590.

Current address for S.L.: Metabasis Therapeutics, 11119 North Torrey Pines Road, La Jolla, California 92037.

Current address for M.D.L.: Ardea Biosciences, 4939 Directors Place, San Diego, California 92121. E-mail: leibowitz.mark{at}gmail.com.

Current address for J.R.: Exelixis, 4757 Nexus Centre Drive, San Diego, California 92121.

Current address for H.V.: 1566 Caudor Street, Encinitas, California 92024-1216.

Current address for W.-C.Y.: Oncomed Pharmaceuticals, Inc., 800 Chesapeake Drive, Redwood City, California 94063.

Current address for A.N.-V.: Transmed Institute, 2425 L Street Northwest, Suite 440, Washington, DC, 20037.

Disclosure Statement: All authors except B.B. were employed by Ligand Pharmaceuticals during the course of this research.

First Published Online January 24, 2008

Abbreviations: AIA, adjuvant-induced arthritis; AR, androgen receptor; β-Gal, β-galactosidase; BP, blood pressure; CMC, carboxymethyl cellulose; COX, cyclooxygenase; CPE, carrageenan paw edema; DHT, dihydrotestosterone; EAE, experimentally induced autoimmune encephalitis; GR, glucocorticoid receptor; h, human; Kd, dissociation constant; MABP, mean arterial BP; MBP, myelin basic protein; MCP, monocyte chemoattractant protein; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; PR, progesterone receptor; RSV, Rous sarcoma virus enhancer; SGRM, selective glucocorticoid receptor modulator.

Received October 2, 2007.

Accepted for publication January 17, 2008.

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