Selective glucocorticoid receptor modulation inhibits cytokine responses in a canine model of mild endotoxemia

Pharmacological Research 125 (2017) 215–223

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Perspective

Selective glucocorticoid receptor modulation inhibits cytokine responses in a canine model of mild endotoxemia Johann Bartko a,b , Ulla Derhaschnig a,c , Tania Neels a , Gerald H. Nabozny d , Christian Harcken d , Jost Leuschner e , Frerich De Vries f , Bernd Jilma a,∗ a

Department of Clinical Pharmacology, Medical University of Vienna, Austria Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria c Department of Emergency Medicine, Medical University of Vienna, Austria d Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA e LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Germany f Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany b

a r t i c l e

i n f o

Article history: Received 23 June 2017 Received in revised form 7 August 2017 Accepted 12 September 2017 Available online 18 September 2017 Keywords: Glucocorticoids Prednisolone GRMs Endotoxemia Canine Inflammation

a b s t r a c t Selective glucocorticoid receptor modulators (GRMs) promise to reduce adverse events of glucocorticoids while maintaining anti-inflammatory potency. The present study tested the anti-inflammatory activity of two novel non-steroidal GRMs (GRM1: BI 607812 BS, GRM2: BI 653048 BS*H3PO4) in comparison to prednisolone in a canine model of low dose endotoxemia. This study compared the anti-inflammatory and pharmacokinetic profile of escalating daily oral doses of GRM1 (1, 2.5, 5 and 10 mg/kg) and GRM2 (0.1, 0.25 and 1 mg/kg) with prednisolone (0.25 and 0.5 mg/kg) and placebo after intravenous infusion of endotoxin (0.1 ␮g/kg) to Beagle dogs. This was followed by a 14-day evaluation study of safety and pharmacokinetics. Endotoxin challenge increased TNF-␣ ∼2000-fold and interleukin-6 (IL-6) 100-fold. Prednisolone and both GRMs suppressed peak TNF-␣ and IL-6 by 71–82% as compared with placebo. The highest doses of GRM1 and GRM2 reduced the mean body temperature increase by ∼30%. The endotoxin-induced rise in plasma cortisol was strongly suppressed in all treatment groups. Pharmacokinetics of both GRMs were non-linear. Adverse effects of endotoxemia such as vomiting were mitigated by GRM2 and prednisolone, indicating an antiemetic effect. During the 14-day treatment period, the adverse event profile of both GRMs appeared to be similar to prednisolone. Both GRMs had anti-inflammatory effects comparable to prednisolone and showed good safety profiles. Compounds targeting the glucocorticoid receptor selectively may provide an alternative to traditional glucocorticoids in the treatment of inflammatory disease. © 2017 Published by Elsevier Ltd.

1. Introduction Glucocorticoids are potent anti-inflammatory drugs, commonly used in many different acute and chronic diseases. On the downside, several adverse effects limit their therapeutic potential and nearly every organ system can be affected. Undesirable effects on vasculature [1], adipose tissue [2], glucose- [3] or bone metabolism [4] cause considerable morbidity and mortality. Glucocorticoids mediate their anti-inflammatory action predominantly through the glucocorticoid receptor (GR). The GR is a ligand-dependent

∗ Corresponding author at: Medical University of Vienna, Department of Clinical Pharmacology, Waehringer Guertel 18-20, 1090 Vienna, Austria. E-mail address: [email protected] (B. Jilma). http://dx.doi.org/10.1016/j.phrs.2017.09.006 1043-6618/© 2017 Published by Elsevier Ltd.

transcription factor residing in the cytosol [5]. Upon activation, the GR has activating and inhibitory effects on gene-transcription [6]. The anti-inflammatory action is mediated through the inhibition of pro-inflammatory transcription factors whereas most of the unfavourable effects are associated with induction of gene-transcription. Advances in the understanding of molecular mechanisms have led to the discovery of compounds that target selective gene expression [7]. Ideally, such compounds have similar anti-inflammatory activity as conventional glucocorticoids, but should come with a reduced side effect profile [8]. In recent years, several promising molecules have been developed [9–12]. After passing through in vitro pharmacologic studies, a new antiinflammatory compound is tested in animal models for in vivo activity [13]. We have recently demonstrated that the infusion of low dose endotoxin (lipopolysaccharide; LPS) induces a release

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Fig. 1. Schematic of the experimental design. On the first day 80 healthy Beagles received escalating doses of oral GRM1 or GRM2 or prednisolone or placebo (8 per group). This was followed by iv. endotoxin infusion 2 h after drug administration. On the next day a safety and pharmacokinetic evaluation phase (Part II) of multiple doses was initiated and dogs received allocated treatment for 14 days (once daily, without endotoxin).

of the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-␣) in Beagle dogs [14] as seen in human endotoxemia [15]. Moreover, investigation of the standard glucocorticoid prednisolone showed similar pharmacokinetics and pharmacodynamics compared to human studies [14]. Thus, infusion of low dose endotoxin (0.1 ␮g LPS/kg) provides a tool for testing new anti-inflammatory compounds in a canine model of systemic inflammation. In a recently published series two novel selective glucocorticoid receptor modulators (GRM1: BI 607812 BS and GRM2: BI 653048 BS*H3PO4) were identified who maintained anti-inflammatory activity in vivo in a collagen induced arthritis mouse model with the potential for reduced side effects compared to the widely used synthetic glucocorticoid prednisolone [10–12]. The present study compared the anti-inflammatory activity of these selective glucocorticoid receptor modulators with prednisolone and placebo in a canine model of endotoxemia. This was followed by a 14-day evaluation study of safety and pharmacokinetics. 2. Materials and methods The study was performed based on Good Laboratory Practice Regulations of the European Commission and the OECD Principles of Good Laboratory Practice and consisted of two parts: Part I was a single dose lipopolysaccharide (LPS) cytokine evaluation study, followed by, part II, a 14-day evaluation of the safety and pharmacokinetics of GMR1, GMR2, prednisolone and placebo (Fig. 1). The study was conducted in eighty male Beagle dogs (age ranging from 10 to 29 months, weighing 6.9–13.0 kg; from an identical genetic pool). The study was approved by the competent ethics committee, and reported in accordance with the ARRIVE guidelines [16,17]. Dogs were allocated to 10 different study groups (n = 8 per group) employing a pseudo-random body weight stratification procedure that yielded groups with approximately equal mean body weight. On study day 1 all animals received 0.1 ␮g kg−1 body weight (bw) lipopolysaccharide (LPS, Escherichia coli 0111.B4; Sigma-Aldrich Chemie GmbH; Taufkirchen, FRG) diluted in 0.9% NaCl solution 0.1 mL kg−1 and injected intravenously as a bolus. This dose was chosen based on previous experiments [14]. LPS was a lyophilised powder and the test item solution was freshly prepared on the administration day adjusted to each animal’s current body weight in a total volume of 0.1 mL kg−1 bw 0.9% NaCl (B. Braun Melsungen AG, Melsungen, FRG). On days 1–15 the animals of the groups 2–5 received 1, 2.5, 5 and 10 mg/kg GRM1, respectively, the animals of groups 6–8 received 0.1, 0.25 and 1 mg/kg GRM2, respectively and the dogs of groups 9 and 10 received 0.25 and 0.5 mg/kg prednisolone, respectively. Chemical structure of GRM1 (MW 395 g/mole) and GRM2 (MW 515 g/mole) have been published (GRM1: compound 21 [11] and GRM2: compound (R)-39 [12]) and were provided by Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA. The doses of the test items given were based on biologic data (human in vitro, animal PK and in vivo pharmacology)

[11,12]. On day 1, test and reference drugs were administered orally 2 h prior to intravenous LPS infusion. The test item formulations were freshly prepared once or twice per week adjusted to each animal’s current body weight once weekly in vehicle [PEG400/water (75/25% m/m)]. An equal volume of vehicle was given as placebo in group 1. Drinking water was offered ad libidum and food (40 g/kg bw ssniff Hd-H V3234, Spezialdiäten GmbH, Soest, Germany) was served once daily for two hours (up to 8 h in case of poor appetite). The dogs were kept singly or by twos in kennels (including an inside yard and outside yard with a total floor space of total 9 m2 ) maintained at a temperature of 22 ◦ C ± 3 ◦ C (maximum range). The adverse event profile was assessed by body weight, thorax circumference, urine production, laboratory tests (hematology, blood chemistry, and coagulation), appearance of clinical sings of systemic toxicity and cortisol suppression, C-Peptide serum levels, osteocalcin levels, served as surrogate safety parameters. At the end of the study, no further examinations were performed and the animals were returned to the stock as a source of plasma or serum samples, e.g. for method development purposes only. 2.1. Blood sampling and laboratory measurements Blood samples were taken in order to obtain venous blood from the saphenous or cephalic vein of the hind or forelimb for analysis. Concentrations of TNF-␣, IL-6, C-reactive protein (CRP), cortisol, and insulin, C-peptide, osteocalcin were measured by using specific enzyme-immunoassays following the instructions ® of the manufacturer (TNF-␣, Quantikine Canine TNF-␣ [R&D ® Systems, Minneapolis, USA]; IL-6, Quantikine Canine IL-6 [R&D Systems, Minneapolis, USA]; CRP, Canine C-Reactive Protein ELISA Kit [BD Bioscience, San Diego, USA]; osteocalcin, CANINE Osteocalcin ELISA Kit [Cusabio Biotech, Wuhan, Hubei, China]). Plasma leves of GRM1, GRM2, prednisolone and cortisol were measured with liquid chromatography-tandem mass spectrometry by the Department of Bioanalytics of the Nuvisan GmbH (Neu-Ulm, Germany). Sampling times for cytokine determination were on day 1 (-2 [predose], 0 [LPS], 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 22 h relative to LPS infusion) and for biomarker determination on day 2 (0 [pre-dose] and 2 h), day 9 (0 h [pre-dose]), day 15 (0 h [pre-dose] and 2 h relative to dosing). Sampling times for pharmacokinetics, cortisol and prednisolone: on day 1 (0 [pre-dose], 0.25, 0.5, 1, 1.5, 2 [LPS], 4, 6, 8 and 12 h relative to dosing), and after the 14-day treatment period (part II, day 15 [0,0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 36 and 48 h] relative to dosing). 2.2. Pharmacokinetics (PK) Pharmacokinetic data of GRM1, GRM2, and prednisolone were determined from plasma concentrations and included maximum concentration (Cmax ), half-life (t½ ), time of the last quantifiable analyte concentration tlast (h), time to reach maximum concentration

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Fig. 2. Effects of prednisolone, GRM1 and GRM2 on the endotoxin (LPS) induced cytokine release. Abscissa represents time (h) relative to LPS infusion. Data (n = 8 per dose group) are expressed as means ± SEM.

Fig. 3. Increase in body temperature (◦ C) 5 h after administration of GRM1, GRM2 or placebo (3 h after LPS infusion). Data (n = 8 per dose group) are expressed as means (95% confidence interval). *P ≤ 0.05.

(tmax ), area under the concentration-time curve (AUC) from time of administration until tlast . 2.3. Statistical analysis Data are given as mean +/− SD or median and IQR unless otherwise stated. The repeated measures analysis of variance (ANOVA) was used for analysis of treatment and period effects (treatment = independent factor, period = independent factor, outcome variable = dependent factor). When significant, post hoc comparisons were performed with nonparametric tests, i.e. Mann–Whitney U test for comparison between groups, and the Wilcoxon matched pairs test for time dependent changes in outcome variables within groups. The Chi squared test was used for binary outcomes. The main outcome variable was TNF-␣. Statistical calculations were performed with commercially available statistical software (Statistica Version 6.1; Stat Soft, Tulsa, OK, USA). 3. Results 3.1. Adverse event profile Part I: After LPS infusion approximately half of the dogs developed reddened gingiva and 30% developed reddened ears in all treatment groups regardless of dose. In the placebo group only 1 dog had reddened gingiva. There was no difference in the rate of dogs having at least a single episode of vomiting in the GRM1 group (40%) compared with the placebo group (50%). Only 1 dog in the

prednisolone group (out of 16, p < 0.05 vs. placebo) and 2 dogs in the GRM2 group (out of 24, p < 0.01 vs. placebo) had a vomiting episode. Part II: During the 14-day treatment period, reddened gingiva occurred in all treatment groups and reddened ears in ∼40% of the treated dogs, whereas 5 dogs had reddened gingiva and no dogs had reddened ears in the placebo group. Vomiting developed only in 4 dogs (1 in each group). There were no differences in thorax circumference, body weight and normalized urine production between the groups after the 14-day treatment period (Table S1). Laboratory blood test showed that the highest dose of GRM2 reduced creatinine by 13% and increased alanine aminotransferase by a factor of 1.88 compared to placebo after the 14-day treatment period (Table S1). 3.2. Anti-inflammatory effects on endotoxin induced responses In the placebo group, a single bolus infusion of 0.1 ␮g LPS/kg increased the body temperature by 1.7 ◦ C (median) (p = 0.014; at 3 h). TNF-␣ concentrations increased approximately 2000-fold 1 h after LPS infusion and IL-6 concentrations increased 100-fold 3 h after LPS infusion (peak change, both p = 0.008 vs baseline; Fig. 2). Treatment with GRM1 significantly suppressed TNF-␣ release compared with placebo by 63% (2.5 mg/kg), 73% (5 mg/kg) and 76% (10 mg/kg) at 1 h (all p < 0.001). Similarly, GRM1 suppressed peak concentrations of IL-6 by 71% (p = 0.02; 2.5 mg/kg), 50% (p = 0.04; 5 mg/kg) and 71% (p = 0.005; 10 mg/kg) at 3 h. The lowest dose (1 mg/kg) had no significant effect on TNF-␣ or IL-6 peak con-

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Fig. 4. Serum C-reactive protein concentrations 24 h after LPS challenge. Data (n = 8 per dose group) are expressed as means (95% confidence interval).

Table 1 Pharmacokinetic parameters. Prednisolone

0.25 mg/kg (n = 8)

0.5 mg/kg (n = 8)

Part I (single dose prior to LPS) Cmax (ng/mL) tmax (h) tlast (h) AUC0-last (ngxh/mL) t1/2 (h)

109 ± 23 1.0 (0.25–1.0) 8.0 (4.0–12.0) 248 ± 63 2.05 ± 1.01

0.25 mg/kg (n = 8)

0.5 mg/kg (n = 8)

Part II (after 14 day treatment period) 208 ± 59 0.75 (0.25–1.5) 12.0 (8.0–12.0) 437 ± 55 2.73 ± 0.88

91 ± 16 0.5 (0.25–1.5) 10.0 (8.0–12.0) 230 ± 37 4.55 ± 4.96

133 ± 40 1.0 (0.5–4.0) 12.0 (8.0–12.0) 327 ± 80 1.99 ± 0.76

GRM 1

1 mg/kg (n = 8)

2.5 mg/kg (n = 8)

5 mg/kg (n = 8)

10 mg/kg (n = 8)

Part I (single dose prior to LPS) Cmax (ng/mL) tmax (h) AUC0-last (ngxh/mL) t1/2 (h)

49 ± 36 0.50 (0.25–1.0) 65 ± 49 2.91 ± 6.04

310 ± 130 0.75 (0.5–1.0) 478 ± 217 0.87 ± 0.16

1048 ± 142 1.0 (0.25–1.5) 1839 ± 465 1.09 ± 0.76

2468 ± 1161 0.75 (0.25–1.83) 5161 ± 2448 2.67 ± 4.30

393 ± 134 0.5 (0.25–1.0) 6.0 (6.0–8.0) 525 ± 196 0.81 ± 0.09

951 ± 448 0.75 (0.17–1.5) 8.0 (6.0–12.0) 1574 ± 750 1.03 ± 0.84

2502 ± 958 1.0 (0.5–1.5) 12.0 (8.0–12.0) 5075 ± 1819 1.07 ± 0.29

Part II (after 14 day treatment period without LPS) Cmax (ng/mL) 118 ± 182 tmax (h) 0.5 (0.25–1.0) tlast (h) 4.0 (4.0–36.0) AUC0-last (ngxh/mL) 147 ± 217 t1/2 (h) 0.91 ± 0.12 GRM 2

0.1 mg/kg (n = 8)

0.25 mg/kg (n = 8)

1 mg/kg (n = 8)

Part I (single dose prior to LPS) Cmax (ng/mL) tmax (h) AUC0-last (ngxh/mL) t1/2 (h)

133 ± 79 1.0 (0.25–1.0) 741 ± 977 2.84 ± 1.98

226 ± 54 0.5 (0.25–1.5) 651 ± 213 1.87 ± 0.24

1019 ± 469 1.0 (0.5–1.5) 7524 ± 8483 3.36 ± 1.81

Part II (after 14 day treatment period without LPS) 111 ± 60 Cmax (ng/mL) 1.0 (0.5–1.0) tmax (h) 7.0 (6.0–12.0) tlast (h) 512 ± 575 AUC0-last (ngxh/mL) 2.58 ± 1.21 t1/2 (h)

207 ± 57 0.5 (0.5–1.5) 8.0 (8.0–12.0) 689 ± 331 1.82 ± 0.28

1025 ± 658 1.0 (0.5–1.83) 12.0 (12.0–48.0) 8974 ± 10573 3.24 ± 1.29

Notes: mean +/− SD or median and IQR; maximum concentration (Cmax ), half-life (t½ ), time of the last quantifiable analyte concentration tlast (h), time to reach maximum concentration (tmax ), area under the concentration-time curve (AUC).

centrations. The highest dose of GRM1 reduced the mean body temperature increase by 33% (p = 0.015). Treatment with GRM2 significantly suppressed the TNF-␣ release by 51% (p = 0.04; 0.25 mg/kg) and by 76% (p < 0.001; 1 mg/kg) compared with placebo at 1 h. Similarly, peak concentrations of IL-6 were suppressed by 50% (p < 0.05; 0.25 mg/kg) and 75% (p = 0.005; 1 mg/kg) at 3 h. The lowest dose (0.1 mg/kg) had no significant effect on TNF-␣ or IL-6 peak concentrations. The highest dose of GRM2 reduced the mean body temperature increase by 27% (p = 0.05) (Fig. 3). Prednisolone reduced TNF-␣ release by 71% in the 0.25 mg/kg and by 82% in the 0.5 mg/kg dose group at 1 h and suppressed IL-6 production by 56% (p < 0.05; 0.25 mg/kg) and by 71% (p = 0.003; 0.5 mg/kg). The mean body temperature increase was 14% (0.5 mg/kg) and 19% (0.25 mg/kg) lower at 3 h after LPS infu-

sion, but the differences were not significant (p = 0.18 and p = 0.29, respectively) (Fig. 3). Treatment with GRM1, GRM2 or prednisolone did not significantly reduce serum C-reactive protein concentrations compared with placebo 24 h after LPS challenge (Fig. 4). 3.3. Hypophyseal-pituitary-adrenal axis (HPA-axis) Infusion of LPS increased plasma cortisol 10-fold (median) at 2 h in the placebo group (p = 0.008). Treatment with 5 mg/kg and 10 mg/kg GRM1, and 1 mg/kg GRM2 completely suppressed the peak plasma cortisol concentrations at 2 h (all p < 0.001) similar to prednisolone (Fig. 5). The lowest doses of both GRMs had no significant effects on plasma cortisol, whereas 0.25 mg/kg of GRM2 reduced plasma cortisol by 18% (p = 0.001). Treatment with the low-

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Fig. 6. Pharmacokinetics: Concentration time curve. Mean plasma concentrations of prednisolone, GRM1 and GRM2 (n = 8 per dose group) during low dose endotoxemia (day 1; LPS was given 2 h after drug administration) and after a 14-day treatment period (day 15). Fig. 5. Hypophyseal-pituitary-adrenal axis (mean ± SEM): Cortisol plasma concentrations in response to LPS. Prednisolone, GRM1 and GRM2 (n = 8 per dose group) was given orally 2 h before LPS infusion.

tions and the AUC were slightly higher on day 1 (LPS) as compared with day 15.

3.4. Pharmacokinetics (Fig. 6 & Table 1)

3.4.2. GRM 1 (BI 607812 BS) The AUC was 5161 ng h mL−1 (median tlast = 12 h) on day 1 in the group receiving 10 mg/kg. The corresponding mean Cmax was 2468 ng mL−1 (median tmax = 0.75 h). Mean Cmax and AUC values increased more than dose-proportionally over the entire dose range. Mean Cmax and AUC values were comparable between day 1 and day 15.

3.4.1. Prednisolone Prednisolone peak concentrations (Cmax ) increased with increasing dose from 109 ng mL−1 to 208 ng mL−1 on the first treatment day (part I, day 1) and from 91 ng mL−1 to 133 ng mL−1 after the 14-day treatment period (part II, day 15). The AUC increased from 248 ng h mL−1 to 437 ng h mL−1 on day 1 and from 230 ng h mL−1 to 327 ng h mL−1 on day 15. Peak plasma concentra-

3.4.3. GRM 2 (BI 653048 BS*H3PO4) The AUC was 8974 ng h mL−1 (median tlast = 12 h) on day 15 in the group receiving 1 mg/kg/day. The corresponding mean Cmax was 1025 ng/mL (median tmax = 1.0 h). Mean Cmax and AUC values increased dose-proportionally between the 0.1 and 1 mg/kg dose on day 1 and day 15, while the increase was less dose proportional between the 0.1 and 0.25 mg/kg dose groups. Mean Cmax and AUC

est dose of GRM1 (1 mg/kg) was associated with higher plasma cortisol concentrations at 4 h (uncorrected p-value for multiple comparisons: p = 0.0499).

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Fig. 7. Insulin (mean ± SEM) (n = 8 per dose group).

values were comparable between day 1 and day 15. Plasma concentrations were ∼30% lower (P < 0.01) on day 1 (4 h and 6 h after administration) compared with day 15 in the highest dose group, however Cmax and AUC did not differ significantly. 3.5. Biomarkers of metabolism and bone metabolism Daily oral administration of GRM1, GRM2 and prednisolone for 14 days had no significant effect on fasting serum glucose or serum insulin (Supplement Table S2 & Fig. 7). Serum C-peptide concentrations were similar in all treatment groups as compared to placebo during and after the 14-day treatment period with 95% confidence intervals overlapping (Fig. 8). The serum concentrations of osteo-

Fig. 8. C-peptide (mean ± SEM) (n = 8 per dose group).

calcin, a biomarker of bone formation [18], were also not different between groups (Fig. 9). 4. Discussion Infusion of low dose endotoxin (0.1 ␮g LPS/kg) induced a selflimiting systemic inflammatory response in all 80 Beagle dogs. Endotoxin infusion and treatment with GRM1, GRM2 and prednisolone, was well tolerated. Vomiting was most prominent in the placebo (4/8) and in all GRM1 groups (13/32), whereas far less frequent in all other treatment groups (∼1/8), indicating an antiemetic effect of GRM2 and prednisolone, which is a known effect of glucocorticoids [19]. During the 14-day treatment period, all dogs of the treatment groups developed reddened gingiva and approximately

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Fig. 9. Osteocalcin (mean ± SEM) (n = 8 per dose group).

half of them reddened ears, which disappeared after a 7-day treatment free period. In contrast, these symptoms were less frequent in the placebo group. However, this may be because of the small sample size in the placebo group. After the 14-day treatment period, there were no significant differences in thorax circumference, body weight and urine production. Laboratory blood test showed that the highest dose of GRM2 reduced serum creatinine by 13%, which was accompanied by a non-significant 22% increase in urine production. This indicates that high doses of GRM2 may increase glomerular filtration, which is a known effect of glucocorticoid compounds [20]. The highest dose of GRM2 also increased alanine aminotransferase by a factor of 1.88. This small increase might suggest an early signal for steroid hepatopathy, a common and species-specific condition in dogs secondary to glucocorticoid treatment [21,22]. Although alanine aminotransferase and creatinine had a high biological variation compared to other biochemical analytes in a prospective cohort of clinically healthy dogs [23], the observed alterations in these parameters may warrant close monitoring in further investigation. Inflammation markers TNF-␣, IL-6 increased several hundredfold and the body temperature rose by 1.7 ◦ C in the placebo group, which is in line with our previous published data [14]. The highest doses of GRM1 and GRM2 tested suppressed TNF-␣ and IL-6 peak levels by ∼70%, similar to 0.5 mg/kg prednisolone. This indicates that the highest doses of both tested GRMs have comparable inhibitory activity on LPS induced inflammatory response compared to prednisolone. Prednisolone reduced serum levels of TNF-␣ and IL-6 by 82% and 71%, respectively. This is in line with a trial in humans, in which a single dose of 30 mg prednisolone (equivalent to a dose of ∼0.4 mg/kg) inhibited LPS induced peak IL-6 by 87% and TNF-␣ by 61% after infusion of 4 ng/kg LPS [24]. Endotoxin infusion increased CRP concentrations 7-fold after 24 h (considering physiologic CRP concentrations values of ∼0.5 mg/dL in normal canine sera, [25]), which is comparable to the ∼10-fold increase during human endotoxemia [26]. Interestingly, none of the tested drugs significantly lowered CRP levels in our study. This may be different in humans in which ∼0.4 mg/kg prednisolone reduced LPS induced rise in CRP by 40% [24]. The main stimulus for CRP release and synthesis is IL-6 in the human endotoxin model [27]. However, the IL-6 concentration at which the liver induces a detectable systemic release of CRP is unknown. Although prednisolone and the tested GRMs reduced peak IL-6 by almost 75%, the IL-6 concentrations were still 30-fold higher as compared to baseline (1268 vs. 38 pg/mL). Thus, the reduction in IL-6 may have been insufficient to suppress the release of CRP in the current model. A limitation was that, while the CRP concentration was trendwise lower in the prednisolone treated group as compared to placebo, our trial had insufficient power to detect a significant effect on CRP levels. Both GRMs suppressed the LPS induced increase in body temperature in the highest dose groups. A 3 day course of 0.5 mg/kg/day prednisolone had also a limited effect in our canine model and inhibited the LPS induced rise in body temperature by ∼30% [27] (mean temp reduction 0.6 ◦ C vs. placebo). Similarly, prednisolone

had only a modest effect (mean temperature reduction of 1.1 ◦ C vs. placebo) on the febrile response in experimental human endotoxemia [24]. The limited antipyretic effect of glucocorticoids may be the consequence of their lack to block the conversion of arachidonic acid to prostaglandin E2 [28], which is the main mediator of fever [29]. In contrast, cyclooxygenase (COX) inhibitors like acetaminophen and ibuprofen act by inhibiting the COX system [30,31] and therefore may have a stronger antipyretic effect. Since multiple mechanisms mediate antipyretic action of glucocorticoids [28], it is possible that the tested GRMs may have an alternative target or a different potential to inhibit the thermoregulatory response to endotoxin. The LPS induced release of inflammatory cytokines was accompanied by a rise in plasma cortisol in the placebo group. An increase in plasma cortisol reflects the physiologic adaption of the HPAaxis in response to stress conditions like bacterial infection [32] and has been described in experimental human and canine endotoxemia [33,34]. Glucocorticoid therapy suppresses endogenous plasma cortisol levels in the absence or presence of inflammation [35,36]. Repeated measurements of plasma cortisol are a sensitive diagnostic tool to evaluate the pharmacodynamic activity of exogenous glucocorticoids [37]. As expected, GRM1 and GRM2 inhibited the increase in plasma cortisol concentrations in a dose-dependent manner. Comparison with prednisolone revealed that particularly the highest doses of GRM1 and GRM2 suppressed plasma cortisol similar to doses of 0.25 mg/kg and 0.5 mg/kg prednisolone. Treatment with the lowest dose of GRM1 (1 mg/kg) was associated with higher plasma cortisol concentrations at 4 h, though we cannot exclude that this was due to statistical chance. A 14-day treatment with GRM1, GRM2 and prednisolone did not alter biomarkers for glucose metabolism (glucose, insulin and C-peptide) [38] and bone metabolism (osteocalcin) [39]. This is in contrast to human experiments in which even a single dose of 10 mg prednisone had significant effects on glucose tolerance and bone formation markers within hours of treatment in young healthy adults [40]. Impaired glucose metabolism is a common side effect during glucocorticoid therapy. However, since a 4-week treatment period with 1.1 mg/kg prednisone did not alter insulin sensitivity or glucose tolerance in clinically normal dogs [41], it is likely that our dosing and treatment duration was not sufficient to induce changes in glucose metabolism. Glucocorticoid increase bone resorption [39] and patients taking glucocorticoids are at risk for osteoporosis and fractures [42]. Although loss of bone mass in dogs receiving 2 mg/kg of prednisone for 30 days occurs rapidly [43], we could not detect any change in osteocalcin. This is in contrast to a 50% reduction of serum osteocalcin 1 day after an infusion of 10 mg cortisol in the porcine species [44] and a 68% reduction 24 h after 60 mg prednisone in healthy men [45]. However, there is also a large heterogeneity among the canine species, considering that 33% of beagles treated with 1.3 mg kg−1 day−1 for 29 weeks were totally resistant to prednisolone-induced bone loss [46]. Pharmacokinetic data showed that mean Cmax and AUC of GRM1 increased more than dose-proportional and mean Cmax and AUC of GRM2 increased dose-proportionally. Synthetic glu-

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cocorticoids, such as prednisolone, usually have a sufficient oral bioavailability (70–80%) and reach peak concentrations in plasma 1–2 h after an oral dose [47]. Strengths of the current investigation are the placebo and active controlled study design. Over 100 dogs (including our previous published study) have undergone the low dose endotoxin protocol without any serious adverse events. Limiting factors are the small number of dogs investigated per group and the short duration of the inflammatory response induced by endotoxin. A considerable proportion of patients with rheumatoid arthritis receive glucocorticoid therapy [48]. This may be the reason why many emerging GRMs are initially tested in collagen/adjuvant -induced arthritis models [49–53]. However, glucocorticoids are also used in other inflammatory diseases, such as chronic obstructive pulmonary disease, systemic lupus erythematosus, polymyalgia rheumatic, giant cell arthritis [54–56], and are equally effective for the reduction of systemic inflammation [57]. As such, canine endotoxemia may provide a reliable model of systemic inflammation for testing the anti-inflammatory effect of GRMs and may contribute to translation of new drug targets into human clinical trials, which are the objective of ongoing investigations (NCT02224105, NCT02217631). Overall, our experiment shows that both GRMs have comparable anti-inflammatory effects to standard glucocorticoid treatment. Our data provides further information on the strengths and limitations of prednisolone in a safe and non-fatal canine model of systemic inflammation. Disclosure summary The authors have nothing to disclose. Acknowledgment This work was supported by Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phrs.2017.09. 006. References [1] L. Fardet, I. Nazareth, I. Petersen, Synthetic glucocorticoids and early variations of blood pressure: a population-based cohort study, J. Clin. Endocrinol. Metab. 100 (7) (2015) 2777–2783. [2] K. John, J.S. Marino, E.R. Sanchez, T.D. Hinds Jr., The glucocorticoid receptor: cause of or cure for obesity? Am. J. Physiol. Endocrinol. Metab. 310 (4) (2016) E249–E257. [3] Y. Wang, C. Yan, L. Liu, W. Wang, H. Du, W. Fan, K. Lutfy, M. Jiang, T.C. Friedman, Y. Liu, 11beta-Hydroxysteroid dehydrogenase type 1 shRNA ameliorates glucocorticoid-induced insulin resistance and lipolysis in mouse abdominal adipose tissue, Am. J. Physiol. Endocrinol. Metab. 308 (1) (2015) E84–E95. [4] S. Sutter, K.K. Nishiyama, A. Kepley, B. Zhou, J. Wang, D.J. McMahon, X.E. Guo, E.M. Stein, Abnormalities in cortical bone, trabecular plates, and stiffness in postmenopausal women treated with glucocorticoids, J. Clin. Endocrinol. Metab. 99 (11) (2014) 4231–4240. [5] E. Charmandari, T. Kino, T. Ichijo, K. Zachman, A. Alatsatianos, G.P. Chrousos, Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRalphaR477H and hGRalphaG679S associated with generalized glucocorticoid resistance, J. Clin. Endocrinol. Metab. 91 (4) (2006) 1535–1543. [6] P.J. Barnes, Glucocorticosteroids: current and future directions, Br. J. Pharmacol. 163 (1) (2011) 29–43. [7] T. Joshi, M. Johnson, R. Newton, M. Giembycz, An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics, Br. J. Pharmacol. 172 (5) (2015) 1360–1378.

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