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ENDOCRINE AND DIABETES

Demonstration of Proof of Mechanism and Pharmacokinetics and Pharmacodynamic Relationship with 4'-Cyano-biphenyl-4-sulfonic Acid (6-Amino-pyridin-2-yl)-amide (PF-915275), an Inhibitor of 11β-Hydroxysteroid Dehydrogenase Type 1, in Cynomolgus Monkeys

Diabetes Biology (B.G.B., A.F., J.H.), Exploratory Biology (J.C.), Department of Pharmacokinetics, Dynamics, and Metabolism (N.H.), Structural and Computational Biology (P.A.R.), Pfizer Global Research and Development, La Jolla, California; Drug Metabolism/DMPK, Covance Laboratories, Madison, Wisconsin (F.T.); and Endocrinology, Division of Medical Sciences, University of Birmingham, Birmingham, United Kingdom (P.M.S.)

Received August 20, 2007; accepted October 2, 2007.

	Abstract

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Glucocorticoids, through activation of the glucocorticoid receptor (GR), regulate hepatic gluconeogenesis. Elevated hepatic expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) play a key role in ligand-induced activation of the GR through the production of cortisol. Evidence from genetically modified mice suggests that inhibition of 11βHSD1 might be a therapeutic approach to treat the metabolic syndrome. We have identified a potent 11βHSD1 inhibitor, 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide (PF-915275), that is selective for the primate and human enzymes. The objective of this study was to demonstrate target inhibition with PF-915275 and to quantify the relationship between target inhibition and drug exposure in monkeys. We characterized the ability of PF-915275 to inhibit the conversion of prednisone, a synthetic cortisone analog that can be distinguished from the endogenous substrate cortisone, enabling a direct measure of substrate to product conversion without the complication of feedback. Adult cynomolgus monkeys were administered either vehicle or various doses of PF-915275 followed by a 10-mg/kg dose of prednisone. Prednisone conversion to prednisolone and the concentrations of PF-915275 were measured by liquid chromatography/tandem mass spectrometry. PF-915275 dose-dependently inhibited 11βHSD1-mediated conversion of prednisone to prednisolone, with a maximum of 87% inhibition at a 3-mg/kg dose. An exposure-response relationship was demonstrated, with an estimated EC₅₀ of 391 nM (total) and 17 nM (free). Insulin levels were also reduced in a dose-related manner. These results should enable the development of a biomarker for evaluating target modulation in humans that will aid in identifying 11βHSD1 inhibitors to treat diabetes and other related metabolic diseases.

Evidence indicating a role for 11βHSD1 as a therapeutic approach to treat type 2 diabetes and related metabolic diseases has been determined in genetically engineered mice (Paterson et al., 2005). Transgenic mice that overexpress 11βHSD1 specifically in adipose tissue develop visceral obesity that is exacerbated on a high-fat diet, along with other symptoms of the metabolic syndrome, including insulin-resistant diabetes, hyperlipidemia, and hypertension (Masuzaki et al., 2001). Conversely, transgenic mice that overexpress 11βHSD2, the enzyme that catalyzes the conversion of the active glucocorticoid cortisol back to inactive cortisone, have improved insulin sensitivity and glucose tolerance, and they are protected from weight gain on a high-fat diet (Kershaw et al., 2005).

The 11βHSD1 knockout mouse is protected from hyperglycemia associated with stress or obesity through reduced hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK), which controls the rate-limiting step of gluconeogenesis and other glucose-mobilizing enzymes such as glucose-6-phosphatase. Because 11βHSD1 is highly expressed in liver relative to other tissues (Liu et al., 2005), inhibition of 11βHSD1 activity provides an opportunity to reduce glucocorticoid levels specifically in the liver and splanchnic circulation (Basu et al., 2004). Beneficial effects of 11βHSD1 inhibition may also occur outside of the liver via improved β-cell function (Davani et al., 2000) and lipid modulation through adipose tissue (Masuzaki et al., 2001).

In humans, elevated cortisol levels, as seen in patients with Cushing's syndrome (Beauregard et al., 2002), cause hyperglycemia, visceral obesity, and hypertension as well as osteoporosis (Cooper et al., 2002) and depression (e Silva, 2005). The nonselective 11βHSD1 inhibitor carbenoxolone increases hepatic insulin sensitivity in humans (Walker et al., 1995). Small-molecule inhibitors of 11βHSD1 have been shown to ameliorate hyperglycemia in rodent models of diabetes and insulin resistance (Alberts et al., 2003; Hermanowski-Vosatka et al., 2005).

The objective of this study was to demonstrate proof of mechanism of in vivo target inhibition with a novel selective and potent 11βHSD1 inhibitor, PF-915275, in primates. Target inhibition was characterized in primates using prednisone, a synthetic cortisone analog, to avoid interference by endogenous cortisone substrate. We demonstrate here dose- and exposure-dependent in vivo inhibition of 11βHSD1-mediated conversion of prednisone to prednisolone in normal cynomolgus monkeys.

	Materials and Methods

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Reagents. Cortisone, cortisol, prednisone, and prednisolone, were obtained from Sigma-Aldrich (St. Louis, MO). PF-915275 or N-(6-aminopyridin-2-yl)-4'-cyanobiphenyl-4-sulfonamide) was synthesized in Pfizer Laboratories (San Diego, CA), and the method and characterization will be published elsewhere.

Biochemical Assay. Cloning, expression, purification, and measurement of human 11βHSD1 activity are as described previously (Castro et al., 2007).

Cell-Based Assays. HEK293 cells stably transfected with human 11βHSD1 or 11βHSD2 cDNA, as described previously (Bujalska et al., 1997), were used to study the specificity of 11βHSD inhibitors.

HEK293T-11βHSD1 Cell Reporter Assay. Human kidney HEK293 cells were stably transfected with human 11βHSD1 gene and a reporter plasmid containing DNA sequences for specific recognition of glucocorticoid-activated glucocorticoid receptors. These sequences were fused to a luciferase reporter gene (Luc), allowing for quantification of 11βHSD1 enzyme modulation. Cells were grown in DMEM Complete containing 1.0 mM nonessential amino acids, 25 mM HEPES, 10 µg of gentamicin, 20 µg of phleomycin D1, 500 µgof Geneticin (G-418; Invitrogen, Carlsbad, CA), L-glutamine, antibiotics, and 10% charcoal/dextran fetal bovine serum. Cells were plated in 384-well flat-bottomed white polystyrene TC-treated microplates, at 20,000 cells/well, 40 µl/well volume, in serum-free DMEM. Plates were incubated at 37°C, 5% CO₂ overnight before addition of inhibitor compounds. Different concentrations of inhibitor compounds were added in 10% (v/v) dimethyl sulfoxide (5 µl/well), followed by addition of 3 µM cortisone (5 µl/well), and cells were incubated at 37°C (5% CO₂) for 6 h. At the end of the incubation, 25 µl/well of SteadyLite HTS was added, and plates were incubated for 10 min at room temperature on a shaker. Plates were then read on top. The concentration of inhibitor compound causing 50% inhibition of light signal was determined. All results were compared with 100% activation control, i.e., cells treated only with cortisone.

HEK293T-11βHSD2 Cell Assay. Human kidney HEK293 cells were stably transfected with human 11βHSD2 gene. Cells were plated at 35,000 cells per well in 100 µl of assay medium (DMEM without phenol red supplemented with 0.1% fetal bovine serum; G-418, and antibiotics) in a 96-well poly-D-lysine-coated plate. Plates were incubated overnight at 37°C, 5% CO₂. Compounds were serially diluted in dimethyl sulfoxide, starting at 10 mM. Five microliters of each dilution was added to a corresponding tube containing 995 µlof 50 ng cortisol/ml in assay medium and mixed via pipetting. Twenty-five microliters of each compound/cortisol dilution was then added to the corresponding wells. Cells were incubated in the presence of 10,000 pg/ml cortisol for 6 h at 37°C. After incubation, supernatants were transferred to a second 96-well plate for quantification of cortisol correlate-enzyme immunoassay (EIA) Cortisol kit (Assay Designs, Ann Arbor, MI). Cortisol concentrations were reported as percentage of inhibition of the 11βHSD2 enzyme.

Monkey and Human Hepatocyte Cortisone/Cortisol Conversion Assay. Freshly plated primary human hepatocytes and cryopreserved suspension primary Cynomolgus monkey hepatocytes were obtained from Lonza Walkersville, Inc. (Walkersville, MD). Inhibition of 11βHSD1 enzyme activity was assessed in these cells by measuring the decrease in cortisol (enzyme product) accumulation in cultures cotreated with cortisone (enzyme substrate) and the potential enzyme inhibitor. Cortisol signal was quantitatively determined in the supernatant of treated cells by means of the Cortisol EIA kit.

Human hepatocytes were purchased in 24-well plates. Upon arrival, they were left to adjust overnight at 37°C, 5% CO₂ before commencement of treatment. Monkey suspension cells were kept frozen in liquid nitrogen. When needed for testing, cells were thawed following the provider's instructions and plated in 96-well microplates at 60,000 cell/well, in hepatocyte basal medium (Lonza Walkersville, Inc.). Treatment commenced by incubating cells for 15 min with various concentrations of inhibitor compounds followed by addition of cortisone (500 nM final concentration) for an additional 30 min. At the end of the incubation, supernatants were analyzed for cortisol content.

Animal Care and Pharmacokinetic and Pharmacodynamic Study Protocol in Normal Cynomolgus Monkeys. The primate study described herein was conducted at Covance Laboratories (Alice, TX) in accordance with applicable Covance Standard Operating Procedures and at the discretion of the laboratory animal veterinarian. All procedures in the protocol and study specific procedures were in compliance with the Animal Welfare Act Regulations (9 CFR 3) and approved by the Covance Laboratories Institutional Animal Care and Use Committee. Adult male cynomolgus monkeys (2–5 kg; n = 6) were individually housed in cages during this study, and all animals were fasted overnight through approximately 4-h postdose (until immediately after prednisone administration) to pass the drug absorption phase (T_max = 2.3–3.3 h postdose; N. Hosea, unpublished data). PF-915275 was formulated as a solution in ethanol/polyethylene glycol 400/water [10:40:50, v/v/v] and prednisone as a suspension in 0.5% methylcellulose. The oral dose (PF-915275, vehicle, and prednisone) was administered via nasogastric intubations. As shown in Fig. 1, the study protocol used baseline conversion of prednisone to prednisolone as vehicle control in phase 1, followed by doses of PF-915275 ranging from 0.1 to 3 mg/kg in four additional phases. Blood was collected via a femoral vein with syringe and needle followed by immediate transfer into tubes containing K₂EDTA anti-coagulant. Samples were centrifuged to obtain plasma, frozen immediately, and stored at -70°C until analysis by LC/MS/MS methods summarized below.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Biomarker study design used for understanding pharmacokinetics and mechanistic biomarker conversion relationship of PF-915275 in cynomolgus monkeys.

Pharmacokinetics. Pharmacokinetic analysis was performed using WinNonLin (Pharsight, Mountain View, CA) and noncompartmental analysis. The statistical method used for data analysis was repeated measures analysis of variance with Bonferroni's multiple comparison test. From the plasma concentration time profiles, area under the curve (AUC) values were determined using the linear-trapezoidal rule for prednisolone, prednisone and PF-915275 over the time frame of 4.5 to 8 h postadministration of PF-915275 (i.e., 0.5–4 h postprednisone administration). The resulting partial AUC values for prednisolone were normalized to the partial AUC for prednisone for each animal, and the ratio of the AUC values was correlated to partial AUC for PF-915275. These data were modeled using a simple E_max model to estimate an AUC value, which gave a 50% inhibition of prednisone to prednisolone. The resulting partial AUC (4.5–8 h), which gave a 50% reduction in prednisolone/prednisone ratio compared with vehicle, was used to determine the average concentration over that time frame giving a 50% inhibition of 11βHSD1 and used to define EC₅₀ (nanomolar). The extent of plasma protein binding for PF-915275 was determined using equilibrium dialysis with freshly prepared monkey plasma.

Insulin Assay. Monkey plasma insulin levels were measured using Mercodia insulin enzyme-linked immunosorbent assay kit (catalog no. 10-1113-01; Alpco Diagnostics, Salem, NH) per manufacturer's instructions

PEPCK Real-Time PCR. One-step quantitative real-time PCR was carried out on 10 ng of RNeasy (QIAGEN, Valencia, CA) purified total RNA isolated from cultured primary human hepatocytes. Primers and dual labeled probe used were as follows: forward, 5'-AGAGCACATGCTGGTTCTGGGTAT-3'; reverse, 5'-GTGCGTCAAACTTCATCCAGGCAA-3'; and probe, 5'5-carboxyfluorescein-AACCAACCCTGAGGGTGAGAAGAAGT-5-carboxytetramethylrhodamine-3'. Reactions were run on a 7900 real-time PCR System (Applied Biosystems, Foster City, CA). Reactions were performed in triplicate. Absolute quantitation was achieved by comparing to a PEPCK standard curve constructed using human Universal Reference RNA standard (Stratagene, La Jolla, CA). The standard curves had r² values of at least 0.99. Additionally, glyceraldehyde-3-phosphate dehydrogenase expression was used to confirm equal sample loading.

	Results

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Potency and in Vitro Pharmacology of 11βHSD1 Inhibitor PF-915275. In humans, 11βHSD1 catalyzes the conversion of inactive cortisone into active cortisol. When assayed for its ability to inhibit purified recombinant 11βHSD1 in a biochemical assay, PF-915275 proved to be a specific and potent inhibitor of the human ortholog, with a K_i of 2.3 nM, and with minimal inhibitory activity toward the mouse enzyme (K_i of 750 nM; Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Comparison of PF-915275 inhibition of 11bHSD1 activity in various biochemical and cell-based assays Each value represents the mean obtained from at least two independent assays. 11βHSD1-HEK293-LUC cells were incubated with PF-915275 with or without 300 nM cortisone for 6 h. Cell lysate luciferase activity was measured. Hepatocytes were pretreated with PF-915275 for 15 to 30 min before the addition of 500 nM E. Primary adipocytes were incubated with 300 nM E. Human hepatocytes were then incubated overnight, whereas monkey hepatocytes were incubated for a total of 1 h before assessing E-to-F conversion in the supernatant of treated cells.

Several cell-based assays were used to address PF-915275 inhibition of 11βHSD1. A human kidney cell line (HEK293-LUC) was generated into which the human 11βHSD1 gene was stably transfected together with a luciferase reporter gene containing glucocorticoid receptor response elements (GRE). Because cortisol, but not cortisone, activates GRE, the luciferase signal is proportional to the degree of conversion of cortisone (E) to cortisol (F) and inhibition of 11βHSD1 activities decreases the luciferase signal following cortisone administration. Table 1 summarizes the data from both in vitro- and cell-based assays. As shown in Table 1, PF-915275 is a potent inhibitor of 11βHSD1 (in vitro HEK293 EC₅₀ = 15 nM) in this human 11βHSD1 overexpressed cell line when coincubated in the presence of 300 nM enzyme substrate. Consistent with the species differences between human and rodent observed in biochemical assays, PF-915275 was a poor inhibitor of 11βHSD1 in rat FAO hepatoma cells, with an EC₅₀ of 14,500 nM. PF-915275 demonstrated species-dependent potency for inhibiting cellular conversion of E to F in dog, monkey, and human in primary hepatocytes, with activity in human hepatocytes > monkey hepatocytes > dog hepatocytes (Table 1; Fig. 2). PF-915275 was also tested for selectivity against human 11βHSD2 using HEK293 cells stably transfected with 11βHSD2. As shown in Table 1, PF-915275 did not significantly inhibit 11βHSD2 (only 1.5% inhibition when tested at 10 µM). Thus, PF-915275 is a highly selective inhibitor of 11βHSD1. Figure 2 shows the dose-dependent effect of PF-915275 on conversion of cortisone to cortisol in primary human and monkey hepatocytes, with an EC₅₀ of 20 and 100 nM, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Effect of PF-915275 on conversion of cortisone (E) to cortisol (F) in primary human and monkey hepatocytes. Primary hepatocytes were pretreated for 15 min with PF-915275, with concentrations ranging from 0.1 to 10,000 nM followed by cortisone (500 nM) for 30 min. Cortisol levels were measured by Cortisol EIA kit (Assay Designs) according to the manufacturer's instructions. Values are mean ± S.E.M.

Glucocorticoids regulate the expression of PEPCK through a known glucocorticoid-responsive element in its promoter, thereby stimulating hepatic gluconeogenesis. PEPCK is a GR-induced gene that transcriptionally regulates hepatic gluconeogenesis. Treatment of primary human hepatocytes with PF-915275 confirmed a direct relationship between inhibition of cortisone conversion to cortisol and inhibition of expression of PEPCK (Fig. 3), supporting the hypothesis that inhibition of 11βHSD1 activity will reduce GR-activated hepatic gluconeogenesis.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effect of PF-915275 on conversion of cortisone to cortisol and on PEPCK gene expression in primary human hepatocytes. Primary human hepatocytes were pretreated with PF-915275, with concentrations ranging from 1 to 10,000 nM for 30 min followed by cortisone (500 nM) for 4 h. Cortisol levels were measured by EIA and PEPCK transcription by quantitative reverse transcription-PCR. Values are mean ± S.E.M.

In Vivo 11βHSD1 Activity, as Measured by Biomarker Conversion of Exogenous Prednisone to Prednisolone, in Cynomolgus Monkeys. As described above, PF-915275 does not have rodent enzyme inhibitory activity, and it could not be used to demonstrate biomarker inhibition or efficacy in rodent models. The target inhibition was characterized using PF-915275 in vivo in primates using prednisone to prednisolone conversion in the absence and presence of PF-915275 as a biomarker of 11bHSD1 inhibition.

Primates use cortisone as endogenous substrate for 11βHSD1 conversion to cortisol. Biochemical evidence described above (Fig. 2) suggests that the monkey enzyme converts synthetic substrate prednisone to prednisolone as efficiently as it converts endogenous steroids. In the biochemical assay using the human 11βHSD1 enzyme, the apparent K_m for cortisone (328.2 ± 46.9 nM) and prednisone (199.9 ± 28.0 nM) are similar. The rate constant, k_cat, for cortisone (0.3 min^-1) is somewhat higher than for prednisone (0.035 min^-1), but turnover studies in human hepatocytes demonstrate that both cortisone and prednisone are substrates for 11βHSD1, generating cortisol and prednisolone, respectively. As shown in Fig. 4, inhibition of either cortisone or prednisone turnover with PF-915275 yielded similar EC₅₀ values to that in human hepatocytes (EC₅₀ by PF-915275 was 18 and 13 nM using cortisone and prednisone substrates, respectively). Therefore, the primate study was performed using exogenous prednisone as surrogate-substrate biomarker to avoid interference by the normal feedback of the hypothalamus-pituitary-adrenal-axis of endogenous glucocorticoids.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Comparison of prednisone and cortisone as 11βHSD1 substrates in primary human hepatocytes. Human primary hepatocytes were pretreated for 15 min with PF-915275 (ranging from 0.1 to 10,000 nM) before the addition of 500 nM cortisone or prednisone for another 30 min. Cortisol and prednisolone concentrations were measured by Cortisol EIA kit according to the manufacturer's instructions.

PF-915275 Dose-Dependently Inhibits 11βHSD1 Activity in Cynomolgus Monkeys. As described under Materials and Methods, non-naive adult normal male cynomolgus monkeys were used in this study. The study protocol (Fig. 1) used baseline conversion of prednisone to prednisolone as vehicle control in phase 1, followed by doses of PF-915275 ranging from 0.1 to 3 mg/kg in four additional phases. The steroid and drug levels were monitored over time. Compared with vehicle treatment, there was no significant effect of PF-915275 on endogenous plasma cortisone or cortisol levels when measured 4 h after the drug administration (data not shown).

As shown by baseline measurement in Fig. 5A, 11βHSD1-mediated biomarker conversion could be demonstrated in vivo using exogenous prednisone substrate in primates. As shown in Fig. 5B, PF-915275 dose-dependently inhibited 11βHSD1-mediated conversion of prednisone to prednisolone. A maximum of 87% inhibition was observed, with the highest tested dose of 3 mg/kg. The resulting levels of prednisolone relative to prednisone from 0.5 to 4 h postprednisone administration (4.5–8 h post PF-915275 administration) was correlated to PF-915275 exposure, and this time range was used to determine the EC₅₀ for 11βHSD1 inhibition. The data were amenable to analysis using a simple E_max model since the effect seemed directly related to plasma drug levels over time (i.e., no hysteresis effect over time was discernible). The resulting EC₅₀ in terms of AUC (4.5–8 h) was 479 ng · h/ml. Given the long half-life of PF-915275 in monkey (22 h), the plasma levels of PF-915275 during the biomarker conversion assay were relatively constant. The resulting average total plasma concentration at EC₅₀ was therefore determined to be 137 ng/ml (391 nM total and 17 nM free). Thus, the resulting partial AUC (4.5–8 h), which gave a 50% reduction in the prednisolone/prednisone ratio compared with vehicle, was used to determine the average concentration over that time frame.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of PF-915275 on in vivo conversion of prednisone to prednisolone in cynomolgus monkeys. A, time-dependent prednisone to prednisolone conversion. B, AUC of prednisolone/prednisone ratio for baseline and for indicated doses of PF-915275. C, change in AUC prednisolone/prednisone ratio with respect to PF-915275 AUC. Statistical method used for data analysis was repeated measures analysis of variance with Bonferroni's multiple comparison test. *, p < 0.01; **, p < 0.001 compared with baseline vehicle treatment. Values are mean ± S.E.M., n = 6. Modeling the data with an Emax model indicated gave an EC50 of 479 ng · h/ml over the 4.5 to 8-h interval, giving an average concentration at EC50 of 137 ng/ml.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Effect of PF-915275 on plasma insulin levels in normal cynomolgus monkeys. After an overnight fast, primates were dosed with vehicle or PF-915275. Four hours later, animals were dosed with 10 mg/kg prednisone, and then they resumed feeding. Insulin levels were measured in plasma samples collected 8 h after vehicle or PF-915275 dosing. Values or mean ± S.E.M., n = 6.

	Discussion

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As exemplified in patients with Cushing's syndrome, glucocorticoids play an important role in the pathogenesis of obesity and insulin resistance. Glucocorticoid receptor activation is controlled by the isoforms of 11β-hydroxysteroid dehydrogenase at the prereceptor level. 11βHSD1 plays a pivotal role in modulating glucocorticoid activity in liver and adipose, where it regulates hepatic gluconeogenesis and adipocyte differentiation, respectively (Walker, 2001; Tomlinson et al., 2004). In humans, impaired cortisone to cortisol metabolism in obesity may be a protective mechanism that helps preserve insulin sensitivity and prevents diabetes mellitus (Valsamakis et al., 2004). The nonselective inhibitor carbenoxolone has been shown to modestly improve insulin sensitivity in healthy and type 2 diabetic subjects (Robert et al., 2003), suggesting the potential metabolic benefits of inhibiting 11βHSD1 in the liver. 11βHSD1 inhibitors may represent an attractive approach to treat patients with type 2 diabetes mellitus and obesity. However, clinically useful therapeutic metabolic benefit requires a selective 11βHSD1 inhibitor.

We have identified and characterized PF-915275 as a potent and selective inhibitor of 11βHSD1. When assayed for its potential to inhibit purified recombinant 11βHSD1 in a biochemical assay, PF-915275 proved to be a much more potent inhibitor of the human subtype compared with the mouse enzyme. This was also confirmed when assayed in rat, dog, monkey, and human hepatocytes. The mouse and human 11βHSD1 enzymes display only 79% amino acid identity; hence, there is potential to identify inhibitors selective for the human enzyme with little or no inhibitory activity against rodent enzyme. Compounds with different activities for the human and mouse orthologs have been reported previously (Barf et al., 2002).

The cell assay results described here show that PF-915275 inhibits cortisone or prednisone conversion to active cortisol or prednisolone, respectively, in monkey and human hepatocytes. The inhibitory activity of PF-915275 is similar using either cortisone or prednisone as substrate. This in vitro characterization validates the use of prednisone as an in vivo surrogate biomarker in an animal model or in humans. We also show that PF-915275 inhibits the expression of PEPCK in a dose-dependent manner that is likely to reduce hepatic gluconeogenesis, with similar potency to the in vitro data in primary human hepatocytes.

Modern approaches to drug discovery and development focus on the identification of biomarkers that indicate the activity of novel agents in vivo (Lesko et al., 2000). Mechanistic biomarkers that measure target activity provide the opportunity to assess the effect of novel agents on target activity and to develop quantitative relationships between target modulation and efficacy. Previously, labeled cortisone has been used as a mechanistic biomarker of 11βHSD1 activity in rodent to study novel inhibitors (Hermanowski-Vosatka et al., 2005). The synthetic substrate prednisone has been used as a mechanistic biomarker of 11βHSD1 activity for the nonselective 11βHSD1 inhibitor carbenoxolone in humans (Tomlinson et al., 2007). With the intention to develop a biomarker for 11βHSD1 activity that can be translated to human, we used in this study prednisone as a mechanistic biomarker in nonhuman primates to assess the in vivo activity of a selective 11βHSD1 inhibitor, PF-915275. Use of prednisone as an in vivo surrogate substrate for 11βHSD1 should also avoid interference by endogenous cortisone.

Our results demonstrate 11βHSD1-mediated biomarker conversion using exogenous prednisone in cynomolgus monkeys. We show that PF-915275 inhibited 11βHSD1-mediated conversion of prednisone to prednisolone in a dose- and concentration-dependent manner over the time frame of the biomarker response. Additionally, the extent of 11βHSD1 inhibition directly correlated with circulating plasma concentrations in that there was no discernible lag time or hysteresis in the relationship between drug levels and response. In addition to the demonstration of proof of mechanism via inhibition of biomarker conversion, we also have evidence that PF-915275 lowers plasma insulin levels. These data, along with inhibition of PEPCK expression in human and monkey hepatocytes, suggest possible in vivo diabetes efficacy in primates and humans by PF-915275. Further studies are needed to determine whether 11βHSD1 inhibition leads to amelioration of metabolic diseases in humans.

The growing number of individuals with insulin resistance and type 2 diabetes are a global health concern, and there is a large unmet medical need for more effective and safer therapies. Inhibitors of 11βHSD1 represent a promising but still unproven approach to treat insulin resistance and related metabolic diseases. This study highlights that PF-915275 is an attractive compound for clinical testing, and it suggests that prednisone can be used as a biomarker in the clinic.

	Acknowledgements

 
We acknowledge the assistance provided by John May (statistics), Kathy Ogilvie (advice), Mike Jirousek (advice), Jim Fraser (advice), Bernadette Pascual (technical), Jamie Lenz (technical), John Williams (technical), Sean Riley (technical), Amy LaPaglia (technical), Bill Carley (advice), Robert Chott (technical), Jennifer Vogel (technical), Ricardo Martinez (advice), and Jacques Ermolieff (biochemical assay). We also acknowledge Christine Loh and Stephane Poirier (Research Technology Center, Pfizer, Inc., Cambridge, MA) for initial efforts to generate HEK293T/11βHSD1/GRE-LUC cell line.

	Footnotes

 
B.G.B. and N.H. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.128280.

ABBREVIATIONS: 11βHSD1, 11β-hydroxysteroid dehydrogenase type 1; 11βHSD2, 11β-hydroxysteroid dehydrogenase type 2; PEPCK, phosphoenolpyruvate carboxykinase; PF-915275, 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; EIA, enzyme immunoassay; LC/MS/MS, liquid chromatography/tandem mass spectrometry; AUC, area under the curve; PCR, polymerase chain reaction; GR, glucocorticoid receptor; GRE, glucocorticoid response element(s); E, cortisone; F, cortisol.

¹ Current affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, California.

Address correspondence to: Dr. B. Ganesh Bhat, Diabetes and Metabolism Pharmacology, Genomics Institute of the Novartis Foundation, 10675 John Jay Hopkins Dr., San Diego, CA 92121. E-mail: gbhat{at}gnf.org

	References

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