A Novel Antiinflammatory Maintains Glucocorticoid Efficacy with Reduced Side Effects
Michael J. Coghlan1,
Peer B. Jacobson,
Ben Lane,
Masaki Nakane,
Chun Wei Lin,
Steven W. Elmore,
Philip R. Kym,
Jay R. Luly2,
George W. Carter,
Russell Turner,
Curtis M. Tyree,
Junlian Hu,
Marc Elgort,
Jon Rosen and
Jeffrey N. Miner
Abbott Laboratories (M.J.C., P.B.J., B.L., M.N., C.W.L., S.W.E., P.R.K., J.R.L., G.W.C.), Abbott Park, Illinois 60064; Ligand Pharmaceuticals, Inc. (C.M.T., J.H., M.E., J.R., J.N.M.), San Diego, California 92121; and Mayo Clinic (R.T.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Jeffrey N. Miner, Department of Molecular and Cellular Biology, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, California 92121. E-mail: jminer{at}ligand.com.
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ABSTRACT
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Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor
coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.
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INTRODUCTION
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GLUCOCORTICOIDS (GCs) are front-line, highly efficacious antiinflammatory agents when used alone or in conjunction with other therapies. Their use, however, is ultimately limited due to severe side effects (1), including diabetes, osteoporosis, lipid redistribution, water retention, and psychosis. Despite these complications, prednisone (prednisolone) and dexamethasone (Dex) are routinely used for a wide range of inflammatory conditions (2). Both the side effects and the antiinflammatory activity of these drugs are dependent on the GC receptor (GR), a member of the steroid receptor subfamily of intracellular receptors (3).
Molecular cloning and dissection of the GR, a ligand-regulated transcription factor that binds cortisol, revealed multiple modes of transcriptional regulation: repression and activation (4, 5). Typically, the GR/ligand complex within the nucleus activates gene expression by directly binding to specific GC response elements (GREs) in the promoter regions of regulated genes. The precise molecular mechanisms by which GR represses transcription are not fully characterized, although the receptor has been shown to directly modulate the activity of transcription factors such as activating protein 1 (AP-1) (6, 7), nuclear factor (NF)
B (8), and NF-IL6 (9), key regulators of genes that encode cytokines and other inflammatory mediators (10).
Genetic experiments with GR mutants that lack the capacity for transcriptional activation have demonstrated that activation and repression are separate and distinct functions of the receptor both in vitro and in vivo (11).
Steroidal GR ligands have been described that appear to dissociate activation and repression in vitro (12). These molecules have been tested in vivo, and they are efficacious antiinflammatory agents; however, they do not exhibit a beneficial side effect profile (13). It is currently unclear whether simply dissociating activation from repression in a ligand will result in a beneficial therapeutic profile.
Positive and negative transcriptional regulation by steroid hormone receptors requires specific accessory proteins (coactivators and corepressors), which interact directly with the receptors and provide a link to the transcriptional machinery (14). The interaction between these coregulators and intracellular receptors is regulated by hormone; some types of interactions are enhanced by hormone, and others are hindered depending on the specific receptor and coregulator involved. These interactions facilitate transcriptional repression and activation by several mechanisms including modulation of histone acetylation at the promoter as well as stabilization of the transcriptional machinery by either direct binding, or by regulating posttranslational modifications (15). One of these regulatory proteins, peroxisomal proliferator-activated receptor
coactivator-1 (PGC-1), has been shown to play a critical role in GC-mediated stimulation of glucose production from the liver. The rate-limiting enzyme in the gluconeogenic pathway (phosphoenol pyruvate carboxy kinase) requires the action of PGC-1 to effectively respond to GCs (16, 17). GCs are important activators of hepatic glucose production and may contribute to the onset and severity of diabetes and metabolic syndrome X. Other coactivators involved in GR activity have been identified. Recent data from Rogatsky and Yamamoto (18) suggest that the GR-interacting protein 1 (GRIP-1) coactivator, a protein previously implicated in GR-mediated transcriptional activation, may play a role in GR mediated transcriptional repression of the inflammatory mediator collagenase (19). Thus, coregulators may play distinct roles in mediating the positive and negative effects of GCs. As a consequence of this regulatory network, a ligand that altered the interaction between the receptor and specific coactivators may exhibit tissue- and/or gene-specific activity. We have examined coactivator and corepressor interactions with GR to understand the tissue specific activity of the receptor as it relates to both antiinflammatory activity as well as side effects. The strong antiinflammatory efficacy of GCs is complex, and likely due to both repression of a large number of proinflammatory cytokines and key inflammatory mediators such as TNF, IL-6, IL-12, and prostaglandin E2, as well as activation of a perhaps smaller number of antiinflammatory genes (IL-10, IL-4, and TGFß; Ref. 20). The side effects of GCs are also associated with both repression and activation of specific genes (21). These include transcriptional activation of enzymes involved in gluconeogenesis, lipid metabolism, and enzymes involved in muscle metabolism such as glutamine synthetase (22) and gelsolin (23). Bone-related side effects are associated with transcriptional repression of genes involved in osteoblast function and bone formation such as osteocalcin (24) and osteoprotegerin in vitro (25) and in the clinic (26). These complexities suggest that it may be necessary to find a gene- or tissue-specific ligand to achieve the desired therapeutic profile. We describe a powerful nonsteroidal antiinflammatory agent in vivo that acts through the GR and exhibits a beneficial therapeutic profile in rodent models of glucose and bone-related side effects. By a mechanism that appears to involve differential coactivator recruitment, this compound exhibits gene-specific regulation, capable of fully regulating only a subset of the genes normally regulated by steroidal GCs.
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RESULTS AND DISCUSSION
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Altered Gene Regulation by a New Class of GR Ligands
Compound Abbott-Ligand 438 (AL-438) was derived by modifying a synthetic progestin scaffold resulting in the discovery of a series of high affinity, GR-selective, ligands (Fig. 1A
; Refs. 27 and 28).

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Figure 1. Structure and Binding Activity of AL-438 vs. Steroidal GCs
A, Abbott-Ligand-438 (AL-438) is structurally distinct from the steroids (prednisolone and Dex). Although AL-438 possesses a tetracyclic core similar to classic GCs, there are several key structural differences including 1) the lack of the C-11 ß-hydroxyl group mandatory for function in steroidal GR ligands; 2) the absence of the C-3 ketone typically found in corticosteroids; 3) the secondary amine function in AL-438; 4) the presence of the methoxy group in AL-438 needed for GR selectivity and functional activity. B, AL-438 and prednisolone bind preferentially to GR. Extracts from SF-9 moth cells infected with recombinant baculovirus expressing the indicated receptor were used in labeled hormone binding assays. The appropriate tritiated steroid was used at 80% of the Kd, followed by titration with prednisolone or AL-438. Competition for binding with the label was used to calculate the inhibition constant (Ki) for test compounds. The data shown are the means of at least three experiments, each run in triplicate. AR, Androgen receptor; ER, estrogen receptor; PR, progesterone receptor.
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Figure 1B
compares the binding affinities of AL-438 and prednisolone for five steroid receptors. AL-438 shares the same intracellular receptor binding profile as prednisolone in that both ligands have some affinity for mineralocorticoid receptor (MR) as well as high affinity for GR. However in MR-dependent reporter gene assays, AL-438 is a very weak antagonist, whereas prednisolone is a full agonist at nanomolar concentrations (data not shown).
Several key inflammatory genes were used to measure the possible antiinflammatory effects of AL-438. GR-mediated repression of the E-selectin promoter in transfected HepG2 cells induced with TNF and IL-1 is shown in Fig. 2A
. The assay for E-Selectin transcriptional repression employs a 600-bp portion of the E-selectin promoter containing both AP-1 and NF
B sites, with no canonical GREs in this segment of DNA. This portion of the E-selectin promoter is cloned upstream of a luciferase (luc) reporter gene and transfected into HepG2 cells together with an expression vector producing human GR (hGR). The E-selectin gene is one of several selectins critical for the movement of inflammatory cells across the vascular wall. In the presence of prednisolone or AL-438, full inhibition of the action of the proinflammatory cytokines TNF and IL-1ß on the E-selectin promoter is achieved (Fig. 2A
). Similarly, AL-438 is both efficacious and potent (100% efficacy and 60 nM potency) at inhibiting IL-1ß-induced endogenous IL-6 expression in GR containing human skin fibroblasts as measured by ELISA (Fig. 2B
). Inhibition of the expression of IL-6 and E-selectin represent potential mechanisms for some aspects of the antiinflammatory effect of GCs, possibly by the down-regulation of NF
B (9). The side effects of GCs may also involve transcriptional down-regulation because repression of genes involved in bone turnover and formation may play a role in GC-induced osteoporosis. Accordingly, we also assessed transcriptional repression of the bone turnover marker osteocalcin in the human osteoblast cell line MG63 (Fig. 2C
). In contrast to TNF/IL-1 induction of the E-selectin gene, compound AL-438 was unable to inhibit osteocalcin as efficiently as prednisolone. Osteocalcin inhibition is considered a marker of the destructive effects of GCs on bone. The mechanism of action of GCs at the osteocalcin gene may differ from that at the E-selectin gene. A role for direct competitive binding by GR to the TATA box of the osteocalcin promoter has been suggested (24), as well as direct GR inhibitory activity on transcriptional activation by the vitamin D receptor (29). In addition, AL-438 exhibits weak (60% efficacy, 60 nM potency) inhibition of osteoprotegerin (25), another bone formation marker strongly inhibited by steroids in MG63 osteoblasts (data not shown). Less inhibitory activity on these bone markers in osteoblasts by AL-438 suggests that the compound may have less impact on bone in vivo than the steroids. AL-438 appears to exhibit gene-specific repression; it is fully capable of repressing E-selectin, and IL-6, but weaker at inhibiting osteocalcin and osteoprotegerin. Next we examined AL-438 activity in transcriptional activation. A standard cotransfection assay measuring transcriptional activation from the TAT3:luc promoter containing three tandem simple GREs from the tyrosine aminotransferase promoter is shown in Fig. 2D
(30). This assay was conducted in HepG2 cells, and the results indicate that AL-438 exhibits similar efficacy, though slightly less potency, compared with prednisolone. In marked contrast, in cultured fibroblasts, the induction of the endogenous aromatase gene, a known direct GR target, demonstrated distinct differences between prednisolone and AL-438. Aromatase is responsible for the conversion of testosterone to estrogen and is possibly associated with certain endocrine side effects of GCs (31). Figure 2E
demonstrates that, at all doses, AL-438 is distinctly less potent and less efficacious than prednisolone at increasing transcription from the aromatase gene. Note that the reduced activity of AL-438 on aromatase is not due to metabolism or differential uptake by these cells because AL-438 if fully efficacious in the IL-6 assay shown in 2B in the same cell background. Furthermore, experiments run in antagonist mode indicate that the compound can inhibit prednisolone-mediated activation of aromatase and mouse mammary tumor virus (MMTV) down to 60% and 20%, respectively. The simplest interpretation of these results is that AL-438 is reaching the receptor under the conditions of the experiment. Similar results are obtained with an MMTV:luc reporter system transfected into HepG2 cells (20% efficacy 60 nM potency; data not shown). Together, these findings indicate that AL-438 exhibits gene-specific regulation, is fully capable of repression and activation in some gene contexts, but is weaker in other gene contexts. We then tested whether this differential in vitro gene regulation translated into any benefit in animal models of inflammation and side effects.

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Figure 2. AL-438 Exhibits Gene Specific Regulatory Activity
A, AL-438 can repress TNF/IL-1ß induced E-selectin promoter activity. Cells transfected with the E-selectin promoter construct and an hGR expression vector are treated with TNF and IL-1ß in the absence or presence of compound. Luc values are normalized to ß-Gal activity measured in the same extracts. This experiment has been repeated 10 times with similar results. B, Efficient transcriptional repression of IL-1ß induced IL-6 expression in human skin fibroblasts by AL-438. Confluent human skin fibroblasts (HSKF1501) were treated with test compounds (10-1010-6 M) for 1 h and then treated with 2 ng/ml IL-1ß. Cells were incubated for 24 h. IL-6 levels in the medium were determined by a sandwich ELISA using antihuman IL-6 antibodies. C, AL-438 is unable to inhibit osteocalcin (OC) as efficiently as prednisolone. RNA from MG63 cells treated with vehicle, 1 µM prednisolone, or 1 µM AL-438 was isolated and examined by Northern blot for osteocalcin and GAPDH; the resultant gel and quantitation are shown. D, AL-438 activates a GRE containing promoter as efficiently as prednisolone. The TAT3:luc promoter containing three GREs from the tyrosine aminotransferase promoter (30 ) was transfected into HepG2 cells together with an expression vector encoding human GR. Luc values are normalized to ß-Gal activity measured in the same extracts. This experiment has been repeated greater than three times with similar results. E, Weak induction of aromatase expression in HSF cells by AL-438. Aromatase enzyme levels are assessed in confluent human dermal skin fibroblasts, and the results shown in graph form. This experiment has been repeated three times with similar results.
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Antiinflammatory Activity of AL-438
AL-438 activity as an acute antiinflammatory agent was measured by performing the carrageenan-induced paw edema assay (CPE). The rat CPE assay is a classical model of acute inflammation, and it has been widely used as a standard assay for antiinflammatory agents, including GCs (32). Rats are dosed orally with vehicle, prednisolone, or AL-438, and 1 h later, carrageenan is injected into the right hindpaw, causing the development of acute edema. The paw volume is measured 3 h later and representative results are shown in Fig. 3A
. AL-438 and prednisolone had comparable potency in rat CPE (ED50s = 11 mg/kg and 3 mg/kg, respectively), and the compounds also had similar efficacies (64% and 77%, respectively) at the highest doses tested (30 mg/kg). Therefore, AL-438 is effective in the acute inflammation assay. Another more chronic model of inflammation is adjuvant-induced arthritis in the rat. This is one of the most widely employed animal models of chronic inflammation involving complex changes in 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 (33). After the injection of Freunds complete adjuvant into the right hindpaws on d 0, rats are culled on d 14 and subsequently dosed for 14 d. Dosing begins when soft tissue injury (edema, synovitis, and joint separation) is maximal but before the onset of changes in the bone. Figure 3B
compares the inhibitory activity of AL-438 and prednisolone on hindpaw edema in the adjuvant arthritis model. AL-438 and prednisolone are equally effective at inhibiting this inflammatory response, though prednisolone is slightly more potent, with oral ED50s of 1 and 9 mg/kg, respectively. This potency difference corresponds well with the slightly less potent activity detected in several in vitro assays including E-selectin (Fig. 2A
) and TAT (Fig. 2D
). It is important to note that edema measurements represent paw volumes from the uninjected left hindpaws because the injected paw typically contains substantial bone and necrotic lesions that complicate the analysis. The steroid and AL-438 appeared to differ in their overall impact on the health of the animals. The grooming behavior, overall activity, and state of health of AL-438-treated rats was equivalent to normal, nonadjuvant-injected rats, which was in marked contrast to the vehicle-treated, adjuvant and even the 30 mg/kg dose of prednisolone-treated animals, all of which were exhibiting signs of stress and disease. Even though the efficacy of AL-438 and prednisolone were equivalent in the paw measurement parameter, these nonquantitative assessments suggested that AL-438 efficacy was actually better than prednisolone. This is likely due to more rapid effects of AL-438 at inhibiting inflammation during the first few days of dosing. Thus, the gene-specific transcriptional regulation profile exhibited by AL-438 did not diminish its efficacy as a potent antiinflammatory agent in both acute and chronic models.

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Figure 3. In vivo Antiinflammatory Activity of AL-438
A, Antiinflammatory activity of AL-438 in the carrageenan paw edema model: after an overnight fast, hindpaw volumes of male Sprague Dawley rats were measured by water plethysmography, followed by oral dosing with AL-438 or prednisolone; uninjected left hindpaws served as internal negative controls. Data are expressed as percentage of inhibition of the difference in hindpaw volumes of carrageenan controls. This experiment has been repeated four times with similar results. B, Full efficacy of AL-438 in the established adjuvant arthritis model: hindpaw edema is prevented by both prednisolone and AL-438. Data are expressed as percentage of inhibition of the change in left hindpaw volume between d 0 and d 30 of adjuvant controls. This experiment has been repeated twice with comparable results.
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Reduced Side Effects of AL-438
Given equivalent antiinflammatory efficacy, we investigated the side effects of AL-438 compared with steroids. Long-term GC use brings about profound changes in fat and glucose metabolism, resulting in obesity, generalized insulin resistance, and diabetes. GC-induced hyperglycemia can be observed immediately after dosing in both rodents and humans (34). These metabolic effects of GCs likely result from a combination of increased hepatic glucose output, altered fatty acid metabolism and increased insulin resistance in muscle and fat (35). When given to fasted, male Sprague Dawley rats (Harlan, Indianapolis, IN), prednisolone induces a moderate hyperglycemic response that is both dose and time dependent. Figure 4
summarizes the effects of prednisolone, AL-438 and RU-486, a potent GR antagonist (inhibition constant = 0.42 nM), on plasma glucose over the first 5 h after a single dose. These results are expressed as total area under the curve during a time course of glucose measurements. In contrast to prednisolone, which results in an acute increase in plasma glucose 26 h after dosing, the glucose levels from AL-438 (and RU-486)-treated animals remain virtually unchanged from vehicle and untreated controls. Figure 4
also demonstrates that predosing rats with AL-438 or RU-486 effectively antagonizes the hyperglycemic response induced by prednisolone. This indicates that although AL-438 is bound to GR in vivo, it does not cause hyperglycemia at equivalent antiinflammatory doses. These in vivo data suggest that AL-438 might have less impact on glucose metabolism than steroids while maintaining good antiinflammatory efficacy.

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Figure 4. AL-438 Does Not Produce Hyperglycemia
Sprague Dawley rats were fasted overnight and dosed orally with prednisolone, AL-438, or RU-486 or vehicle alone. Five-hour plasma glucose levels were measured. Prednisolone-treated rats demonstrated a moderate, acute hyperglycemic response at 10 mg/kg, whereas rats dosed with AL-438 or the GR antagonist, RU-486 (30 mg/kg), showed no significant changes in plasma glucose from vehicle or undosed controls. The dose of AL-438 (30 mg/kg AL-438) was selected based on equivalent efficacy to prednisolone in the CPE model. AL-438 was also tested for antagonist activity in this model. Separate groups of rats were orally dosed with vehicle, RU-486 or AL-438, 30 min before oral administration of a 10 mg/kg prednisolone challenge. Both RU-486 and AL-438 completely antagonized the prednisolone-induced hyperglycemic response under the conditions of this assay.
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Reduced Impact of AL-438 in Bone
The most debilitating side effect of long-term steroid use is osteoporosis and increased risk of fracture and associated morbidity. Because AL-438 appears less able to repress bone formation-related genes in osteoblast lines, we tested bone formation rates in Sprague Dawley rats treated orally with vehicle, prednisolone at 10 mg/kg, or AL-438 at 30 mg/kg for 30 d. These differential doses compensated for the potency differences between prednisolone and AL-438 and were equally efficacious in the CPE and adjuvant arthritis models. Calcein and tetracyclin were injected at specific times during the treatment period to label mineralizing bone with fluorescent markers. Histomorphometric analysis of tibiae from these animals are shown in Fig. 5
(36). Consistent with the effects noted in osteoblast cell culture, AL-438 does not inhibit bone mineral apposition rate (an index of osteoblast activity) in cancellous bone, and it is distinctly weaker than prednisolone at inhibiting bone formation in cortical bone.

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Figure 5. AL-438 Exhibits Reduced Impact on Bone Compared with Prednisolone
Tibiae were isolated from tetracyclin and calcein labeled rats treated with vehicle, 10 mg/kg prednisolone, and 30 mg/kg AL-438 (n = 8 rats/group). Specific histomorphometric measurements were performed on cross-sections of these bones and the results were averaged across each group and compared between treatments. A, Cancellous bone mineral apposition rate is the area bound by the two fluorochrome labels divided by the product of the calcein labeled perimeter length and the labeling interval. This measures osteoblast activity in cancellous bone. B, Cortical bone formation rate is the area bound by the two fluorochrome labels divided by the labeling interval. This measures the product of osteoblast activity and osteoblast number in the cortical compartment. Both measures are significantly reduced by the steroid prednisolone (a, P < 0.05 compared with vehicle; b, P < 0.05 compared with prednisolone).
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Mechanism of Action
How does AL-438 generate this unusual in vivo profile? We noted earlier that, unlike prednisolone, AL-438 exhibits no MR agonist activity in vitro, and is actually a weak MR antagonist. This difference is potentially very important and could result in an improved profile for the hypertensive and cardiovascular effects of prednisolone. It will be of interest to test AL-438 for differences in these parameters. However, in the context of the bone and glucose side effects measured in this manuscript, we do not believe that the lack of MR agonist activity plays an important role in these endpoints. Rather, the in vitro differences between AL-438 and prednisolone suggest a fundamental difference in the way these two ligands alter receptor structure. Thus, we hypothesized that the beneficial profile of AL-438 might be the result of altered interactions between the receptor and selected coactivators. GC regulation of hepatic glucose production has recently been connected directly to the transcriptional coactivator PGC-1 (16, 17), and GRIP-1 appears to play a role in transcriptional repression by GR of proinflammatory genes (19). We therefore tested the ability of AL-438 to induce an interaction with these coactivators compared with the steroids. Our results for two such interactions are shown in Fig. 6
. The prednisolone/GR complex binds each coactivator efficiently; however, under the same conditions, the AL-438/GR complex is unable to efficiently interact with the coactivator PGC-1 while remaining fully capable of binding to GRIP-1. The interactions between GR and PGC-1 are reduced in the presence of AL-438 in both biochemical pull-down experiments using reticulocyte lysate-translated GR as well as in two-hybrid assays in transfected cells, whereas the GRIP interaction is efficient in the presence of either the steroid or AL-438. Efficient interaction with GRIP is consistent with repression of proinflammatory genes by AL-438 (19). Reductions in PGC-1 interaction compared with steroids may help explain reduced hyperglycemia in vivo by AL-438. Thus, AL-438 appears to change the profile of coactivators interacting with the receptor that may in turn result in altered in vivo profiles. Further experimentation will be required to establish whether the lack of PGC-1 interaction induced by this ligand is responsible for this beneficial profile on glucose. Additionally, alterations in the interactions between GR and other coactivators and corepressors may be expected based on these findings. Our initial examination of the profile of AL-438 has revealed that, in contrast to RU-486, which increases interaction with corepressors NCoR and silencing mediator of retinoid and thyroid hormone receptors, this molecule exhibits weak interactions in much the same way that a full agonist does (data not shown).

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Figure 6. Differential Cofactor Interactions Induced by AL-438 and Prednisone
Interactions between GR and two specific coactivators GRIP-1 (18 ) and PGC-1 (42 ) were measured in a two-hybrid assay. Interaction between GR and the coactivator is measured by luc activity upon the addition of GC. AL-438 exhibits essentially the same activity as the steroid at inducing GRIP-1 interactions; however, in contrast, AL-438 is markedly less able to induce GR interaction with PGC-1. B, Altered coactivator interactions by GR when bound to AL-438 (pull-down). The differential interaction detected in cells is also seen in biochemical pull-down experiments using in vitro translated GR, activated with hormone or compound and mixed with GST-PGC-1 or GST-GRIP-1 and washed extensively. Changes in the conformation of the receptor in response to ligand likely affect the interactions with PGC-1.
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Thus, it appears that despite being fully active at inhibiting inflammation, AL-438 exhibits dramatically reduced side effects in at least two critical parameters (glucose metabolism and bone). We hypothesize that this result is due to structural changes in the receptor induced by this novel ligand. These structural changes reduce the ability of the receptor to bind to PGC-1, but not GRIP-1. These changes do not affect the strong antiinflammatory activity in vivo and may explain the beneficial side effect profile of these compounds. This class of molecule may be useful to further understand the distinct roles of transcriptional coactivators in GR function, both in vitro and in vivo, and offers an entirely new approach to the development of a safer, but fully efficacious class of antiinflammatory compounds.
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MATERIALS AND METHODS
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In Vitro Binding
Extracts from Spodoptera frugiperda-9 moth cells infected with recombinant baculovirus expressing the indicated receptor were used in labeled hormone binding assays. Growth and purification of recombinant hGR baculovirus followed the protocol outlined by Summers and Smith (37). 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-cholamidopropyl)-dimethylammonio)-1-propane sulfonate, 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 5075 µg of extract protein, plus 12 nM [3H]-Dex at 84 Ci/mmol and varying concentrations of competing ligand (010-5 M). Assays were set up using a 96-well mini-tube system and incubations were carried out at 4 C for 18 h. Equilibrium under these conditions of buffer and temperature was achieved by 68 h. Nonspecific binding was defined as that binding remaining in the presence of 1000 nM unlabeled Dex. 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 according to the protocol of Wecksler and Norman (38). 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 was removed. The hydroxyapatite pellet was washed 2x 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 (39). Steroid standards are included in each assay, and resulting Kd values are determined by use of a modified Cheng-Prussoff equation (39).
MR, androgen receptor, progesterone receptor, and estrogen receptor-
expression in the baculovirus system and binding assays were conducted similarly except that labeled ligands were aldosterone [12 nM 3H aldosterone from Amersham Pharmacia Biotech (Arlington Heights, IL; specific activity 60 Ci/mmol), dihydrotestosterone (12 nM 3H dihydrotestosterone at 130 Ci/mmol), progesterone (23 nM 3H progesterone, Sigma, St. Louis, MO), 93 Ci/mmol, and estradiol (23 nM 3H estradiol, NEN Life Science Products), and 114 Ci/mmol], respectively. Each binding assay point is done in duplicate and each full experiment is repeated three or more times.
Pull-down assay.
In vitro-translated hGR (TnT, Promega Corp., Madison, WI), activated with hormone, compound, or solvent, was mixed with glutathione-S-transferase (GST)-agarose loaded with Escherichia coli-expressed GST-coactivator fusions [GST-PGC-1 amino acid (aa) 91186 and GST-GRIP-1 aa 730-1121] for 30 min at 4 C. After washing three times in TNEN buffer (2 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.05% Nonidet P-40) at 4 C, samples were eluted and processed for electrophoresis and autoradiography.
Transfection.
HepG2 cells were from American Type Culture Collection, Manassas, VA) were grown in DMEM (BioWhittaker, Inc., Walkersville, MD) containing 10% (vol/vol) fetal calf serum (HyClone Laboratories, Inc., Logan, UT), 2 mM L-glutamine, and 55 µg/ml gentamycin. Cells were transiently transfected using the calcium phosphate coprecipitation method (40). Unless otherwise noted, 5 µg/ml of a Rous sarcoma virus-human GR-expression plasmid vector (RSV:hGR), 5 µg/ml MMTV-LUC reporter plasmid, 2.5 µg/ml of pRSV-ß-Gal (ß-galactosidase) as a control for transfection efficiency, and 7.5 µg of filler DNA (pGEM4) at a final concentration of 20 µg/ml were precipitated 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 (41). The E-selectin transfection assay is similar except that 5 µg/ml E-sel/luc reporter plasmid was added instead of MMTV:Luc. The medium was changed 16 h after transfection to contain 10% charcoal-stripped fetal calf serum, TNF
(10 ng/ml), IL-1ß (1 ng/ml), and test compounds (10 nM to 10 µM) with or without 0.32 nM Dex for 24 h. Cells were then lysed and assayed as described above.
Plasmids.
pRSV:hGRnx and MMTV:Luc were obtained from Ron Evans (The Salk Institute, La Jolla, CA). A reporter construct containing 600 bp of the E-selectin promoter region fused to the luc gene (E-sel/luc) was used in repression assays together with an expression vector encoding human GR driven by the RSV-hGR, cotransfected with a ß-Gal expression vector as a control. The activation assays used the TAT3:luc promoter containing three GREs from the tyrosine aminotransferase promoter (30) and the same transfection conditions. For the mammalian two-hybrid assay, HepG2 cells were transfected with an luc reporter driven by five gal4 binding sites (5xGal4:luc; Ref. 30), VP16 hGR expression vector, constructed as described (30), ß-gal control expression plasmid, and expression vectors for the appropriate coactivator [human PGC-1 (aa 91186; Ref. 42), murine GRIP-1 (aa 322-1121; Ref. 18)] tethered to the Gal4 DNA binding domain. Interaction between GR and the coactivator is measured by luc activity upon the addition of GC. For pull-down assays, the GR in vitro transcription/translation vector T7hGRnxg was used. This plasmid was constructed as described (30) by first inserting the Glu-Glu tag epitope sequence (43) into pRSVhGRnx at the KpnI and SalI sites; hGRnxg was then inserted into the pT7-link expression vector at the NcoI and BamHI sites (44).
Cell-based analysis.
Osteocalcin RNA was measured in MG63 cells treated with vehicle, 1 µM prednisolone, or 1 µM AL-438 for 24 h, and RNA was isolated and examined by Northern blot for osteocalcin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results were normalized to GAPDH expression. IL-6 measurements were conducted by ELISA. Confluent human skin fibroblasts (HSKF1501) were treated with test compounds (10-1010-6 M) in 100 µl of medium and incubated for 1 h. One hundred microliters of 2 ng/ml IL-1ß (Roche Molecular Biochemicals, Indianapolis, IN) in medium were added and the plates were incubated for 24 h. The supernatant was transferred to new plates and IL-6 levels in the medium were determined by a sandwich ELISA using antihuman IL-6 antibodies (Endogen, Inc., Woburn, MA; M-621-B, M-620) and horseradish peroxidase. Aromatase enzyme levels were assessed by an assay that measures the conversion of 3H-androstenedione to estrogens where the 3H2O byproduct of the estrogen synthesis is quantified vs. a GC standard (Dex). Confluent human dermal skin fibroblasts were treated with test compounds (10-1010-5 M) in 10% FBS/DMEM and incubated for 24 h. Ten microliters of 2 µM 1ß-3H androstenedione (NEN Life Science Products) were added, and the cells were incubated at 37 C for an additional 6 h. Two hundred microliters of the culture supernatant were transferred to new plates, treated with TCA, and centrifuged at 3000 rpm for 5 min. One hundred eight microliters of supernatant were extracted with chloroform and the isolated aqueous layer was treated with dextran-coated charcoal before addition of 2 ml of scintillant in a minivial. Radioactivity was counted and the results were quantified vs. Dex.
In Vivo Assays
Inflammation.
The antiinflammatory activity of AL-438 was tested using the carrageenan paw edema model. After an overnight fast, hindpaw volumes of 180-g male Sprague Dawley rats were measured by water plethysmography, followed by oral dosing with AL-438 or prednisolone 2 h before injecting 100 µl of 1% carrageenan into the plantar region of the right hindpaw. Three hours after carrageenan challenge, right hindpaws were remeasured to evaluate changes in edema; uninjected left hindpaws served as internal negative controls. Data are expressed as the percentage inhibition of the difference in hindpaw volumes of carrageenan controls (n = 8 rats/group). AL-438 antiinflammatory activity was also tested in the established adjuvant arthritis model 14 d after measurement of baseline hindpaw volume and the injection of Freunds complete adjuvant, male Lewis rats were culled and randomized into treatment groups. Rats were orally dosed for 14 d, and on d 28, hindpaw volumes were measured by water plethysmography. Data are expressed as percentage inhibition of the change in left hindpaw volume between d 0 and d 30 of adjuvant controls (n = 1015 rats/group).
Glycemic control assay.
Sprague Dawley rats (180 g) were fasted overnight and dosed orally with prednisolone (10 mg/kg), AL-438 (30 mg/kg), or RU-486 (30 mg/kg) or vehicle alone. Plasma glucose levels were measured via tail bleeds over 5 h using a Medisense Precision G glucometer (n = 5 rats/group). The dose of AL-438 (30 mg/kg AL-438; n = 8 rats/group) was selected which showed equivalent efficacy to prednisolone in the CPE model. AL-438 was also tested for antagonist activity in this model. Separate groups of rats were orally dosed with vehicle, RU-486 or AL-438, 30 min before oral administration of a 10-mg/kg prednisolone challenge. Plasma glucose was measured multiple times over 5 h (n = 5 rats/group).
Bone histomorphometry.
Tibiae were isolated from tetracyclin- and calcein-labeled 200-g male Sprague Dawley rats treated for 30 d with vehicle, 10 mg/kg prednisolone, and 30 mg/kg AL-438 (n = 8 rats/group) and processed for histomorphometry as described (36). Histomorphometric measurements were conducted using standard methods (36).
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ACKNOWLEDGMENTS
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We thank Emily Guido, Shin-Shay Tian, Maya Iskandar, Chris Larson, Mike Stallup, Mike Garebedian, Mark Chapman, and Ron Evans for significant contributions to this manuscript. Robin Chedester assisted in manuscript preparation.
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FOOTNOTES
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1 Present address: Eli Lilly & Co., DC 0528, Lilly Corporate Center, Indianapolis, Indiana 46285. 
2 Present address: Oxford Bioscience Partners, 222 Berkeley Street, Suite 1650, Boston, Massachusetts 02116. 
Abbreviations: aa, Amino acid; AL-438, compound Abbott-Ligand 438; CPE, carrageenan-induced paw edema assay; Dex, dexamethasone; ß-Gal, ß-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoid; GR, GC receptor; GRE, GC response element; GRIP-1, GR-interacting protein 1; hGR, human GR; Kd, dissociation constant; luc, luciferase; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; NF, nuclear factor; PGC-1, peroxisomal proliferator-activated receptor
coactivator-1; RSV, Rous sarcoma virus.
Received for publication October 18, 2002.
Accepted for publication December 23, 2002.
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