Glucocorticoid receptor

The collagenase promoter activity in HeLa cells, LPS-induced IL-12p40 in human PBMC, and phytohemagglutinin aqueous solution of IFN-γ in human PBMC are all inhibited by methylprednisolone acetonate; the IC50 values for these reactions are 9.3, 16.8, and 15.2 nM, respectively [3]. The MMTV promoter and TAT activity are activated by methylprednisolone acetate, with EC50 values of 21.8 and 20.5 nM, respectively [3].

Methylprednisolone propyl acetate exhibits negligible systemic side effects and anti-inflammatory properties with an IC50 of 0.0015% when applied topically in a 50 μL croton oil-induced Evan's blue edema model [1]. When administered topically, mmethylprednisolone aceponate (0.0001%–0.1%) reduces mouse edema and irritating contact dermatitis, with an ED50 of 0.002% [3].

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The local antiinflammatory potency of methylprednisolone aceponate was equal to the very strong glucocorticoid clobetasol 17-propionate but higher than the potency of hydrocortisone 17-butyrate after topical application in 2 animal models of inflammation. Methylprednisolone aceponate is activated enzymatically in the skin. This activation proceeds faster in inflamed tissue. In contrast to clobetasol 17-propionate, methylprednisolone aceponate was devoid of systemic effects after topical application for 3 days. Finally, whereas clobetasol 17-propionate induced marked skin atrophy, methylprednisolone aceponate induced only slight atrophogenic changes after long-term application (up to 43 days) on rat skin, comparable to the effects of hydrocortisone 17-butyrate. Conclusions: Methylprednisolone aceponate combines high local antiinflammatory potency with very low systemic side effects and only minor local atrophogenic activity. The reason for the dissociation between local antiinflammatory and atrophogenic effects is not known so far. It may be speculated that one of the reasons for the very strong local antiinflammatory activity may reside in the faster enzymatic activation in inflamed tissue. Methylprednisolone aceponate represents a new corticosteroid with which it is possible to improve the dissociation between desired antiinflammatory activity and undesired side effects of topical glucocorticoids. [2]

Binding and receptor selectivity [3]
Receptor binding assays [3]
Extracts from Sf9 cells, infected with recombinant baculovirus coding for the human GR, progesterone receptor (PR), androgen receptor (AR) or mineralocorticoid receptor (MR) were used for the receptor binding assays (Schäcke et al., 2004). All receptor nomenclature follows the ‘Guide to receptors and channels’ (Alexander et al., 2008). For the binding assays for GR, PR, AR and MR [1,2,4,6,7-3H]dexamethasone (approximately 3.18 GBq·mmol−1, NEN) (∼20 nmol·L−1), [1,2,6,7-3H(N)]progesterone (approximately 3.7 GBq·mmol−1, NEN), [17a-methyl-3H]methyltrienolone (approximately 3.18 GBq·mmol−1, NEN) or D[1,2,6,7-3H(N)]aldosterone (approximately 2.81 GBq·mmol−1, NEN) respectively, SF9 cytosol (100–500 µg protein), test compounds and binding buffer (10 mmol·L−1 Tris/HCL pH 7.4, 1.5 mmol·L−1 EDTA, 10% glycerol) were mixed in a total volume of 50 µL and incubated for 1 h at room temperature. Specific binding was defined as the difference between binding of [1,2,4,6,7-3H]dexamethasone, [1,2,6,7-3H(N)]progesterone, [17a-methyl-3H]methyltrienolone and D[1,2,6,7-3H(N)]aldosterone in the absence and presence of 10 µmol·L−1 unlabelled dexamethasone, progesterone, metribolone or aldosterone respectively. After incubation, 50 µL of cold charcoal suspension was added for 5 min and the mixtures were transferred to microtiter filtration plates. The mixtures were filtered into Picoplates and mixed with 200 µL Microszint-40. The bound radioactivity was determined with a Packard Top Count plate reader. The concentration of test compound giving 50% inhibition of specific binding (IC50) was determined from Hill analysis of the binding curves.

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Selectivity assays [3]
The potential of the ZK 245186 SEGRA and competitor compounds to exhibit agonistic or antagonistic activity in oestrogen receptor α- (ERα-), PR-, AR- and MR-mediated transactivation assays was determined. Increasing concentrations of test compounds were added: (i) to MCF-7 cells stably transfected with a vit-tk luciferase reporter gene; (ii) to SK-N-MC cells stably co-transfected with the human PR and a mouse mammary tumour virus (MMTV)-luciferase reporter gene; (iii) to CV-1 cells transfected with the rat AR and an MMTV-luciferase reporter gene; and (iv) to COS-1 cells co-transfected with the human MR and a MMTV-luciferase reporter gene. Dose-dependent induction of reporter gene activity by test compounds via the respective nuclear receptors were determined and compared with the potency and efficacy of the reference estradiol (ERα), promegestone (PR), metribolone (AR) and aldosterone (MR). To test for antagonistic activity, the effects of ZK 245186 and competitor compounds on reporter gene activity stimulated by estradiol (ERα), promegestone (PR), metribolone (AR) or aldosterone (MR) were determined. Antagonistic potency and efficacy of test compounds in the respective transactivation experiments were compared with reference compounds: fulvestrant (ERα), mifepristone (PR), cyproterone acetate (AR) and the MR antagonist ZK 91587.

Anti-inflammatory/immunomodulatory activity in vitro (transrepression) [3]
Inhibition of collagenase promoter activity [3]
HeLa cells stably transfected with a luciferase reporter gene linked to the collagenase promoter were cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 3% charcoal absorbed foetal calf serum (FCS), 50 units·mL−1 penicillin and 50 µg·mL−1 streptomycin, 4 mmol·L−1 L-glutamine and 300 µg·mL−1 geneticin. Cells were then seeded onto 96-well dishes (1 × 104 cells per well). After 24 h, cells were incubated with inflammatory stimulus [10 ng·mL−1 12-o-tetradecanoylphorbol 13-acetate (TPA)] with or without increasing concentrations (1 pmol·L−1 to 1 µmol·L−1) of reference or test compounds. As negative control (unstimulated cells) cells were incubated with 0.1% dimethylsulphoxide (DMSO) and as positive control cells (stimulated cells) were incubated with 10 µg·mL−1 TPA plus 0.1% DMSO. After 18 h luciferase assay was carried out.

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Inhibition of cytokine secretion in stimulated human primary cells [3]
All blood cells were used with written consent of the donors in accordance with institutional ethical guidelines. Effects of compounds on monocytic secretion of IL-12p40 was determined after stimulation of peripheral blood mononuclear cells (PBMCs) from healthy donors with 10 ng·mL−1 lipopolysaccharide (Escherichia coli serotype 0127:B8). Effects on interferon (IFN)-γ secretion were determined after PBMC stimulation with 10 µg·mL−1 of the mitogenic lectin, phytohemagglutinin. After 24 h incubation (37°C, 5% CO2), cytokine concentrations in supernatants of treated cells were determined using specific ELISA kits: IFN-γ and IL-12p40 ELISA.

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Inhibition of lymphocyte proliferation in mixed lymphocyte reaction [3]
Human PBMCs obtained from healthy donors were isolated by centrifugation of heparinized blood on Histopaque-1077 and cultured in RPMI 1640 medium supplemented with FCS (10% v/v). For mixed leukocyte reaction (MLR), PBMC from one donor were incubated with 50 µg·mL−1 Mitomycin C for 30 min at 37°C followed by repeated washings with PBS and used as stimulator cells. PBMCs from an unrelated donor were used as responder cells and seeded (5 × 10~4 cells per well) together with Mitomycin C-treated stimulator PBMCs (1 × 105 cells per well) in 96-well round-bottom microtest plates. The cultures were set up in triplicate, compounds were added in concentrations as indicated in Figure 2 and plates were incubated at 37°C. On day 5 PBMCs were pulse-labelled with [methyl-3H]-thymidine (7.4 kBq per well) for 6 h and harvested on glass filters. [3H]-thymidine incorporation of the triplicate cultures was measured by liquid scintillation counts quantified by beta-plate scintigraphy (Zügel et al., 2002).

Animal/Disease Models: Irritant contact dermatitis in mice and rats [3]: 0.0001%-0.1%
Route of Administration: Topical application, 10 µL for mice and 20 µL for rats
Experimental Results: Significant suppression of ear inflammation.

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Anti-inflammatory activity in vivo [3]
Compounds were co-applied with croton oil or with dinitrofluorobenzene (DNFB) in ethanol containing 5% isopropylmyristate or acetone/olive oil 4/1 respectively.

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Irritant contact dermatitis in mice and rats [3]
Both ears of NMRI mice (10 per group) and Wistar rats (10 per group), respectively, were topically treated with compounds or vehicle that were dissolved in croton oil solution (10 µL of 1% for mice and 20 µL of 6.5% for rats). After 24 h, animals were killed and oedema was determined by measuring ear weight in mice or 10 mm diameter ear punch biopsy weight in rats as described earlier (Schäcke et al., 2004). As parameters for neutrophil infiltration, elastase activities were analysed in ear homogenate. The effect of ZK 245186 was compared with methylprednisolone aceponate (MPA) and MF. Each experiment has been repeated twice.

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Allergic contact dermatitis in mice and rats [3]
NMRI mice (10 per treatment group) were sensitized in the skin of the flank with 25 µL of 0.5% DNFB at days 0 and 1. On day 5, mice were challenged by topical application of 20 µL of 0.3% DNFB as described earlier (Zügel et al., 2002). Wistar rats were sensitized with 75 µL of 0.5% DNFB at day 0. On day 5, rats were challenged by topical application of 40 µL 0.4% DNFB. Test or reference compounds (ZK 245186 and methylprednisolone aceponate (MPA) respectively) were topically co-applied in up to four different concentrations (0.001%, 0.01%, 0.1% and 1%) with the hapten challenge. After 24 h, animals were killed to determine ear weight and elastase activity from ear homogenates as parameters for oedema and neutrophil infiltration. Each experiment has been repeated twice.

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In vivo dissociation and side effect parameters [3]
Determination of dissociated in vivo activity in mice as measured by its anti-inflammatory versus pro-diabetogenic effects Determination of the induction of enzymes such as TAT may serve as a suitable surrogate marker for measuring the potential of a compound to act via transactivation in vivo. Two groups of mice (n = 10) were treated on the same day under identical conditions. All compounds were applied s.c., ZK 245186 and methylprednisolone aceponate (MPA) in doses of 1, 3, 10, 30 mg·kg−1 body weight and MF in doses of 0.1, 0.3, 1 mg·kg−1 body weight. One group of mice was used in the croton oil model (main outcome parameter was inhibition of oedema formation) and in the second group of mice, TAT activity was determined from liver homogenates as described earlier (Schäcke et al., 2004). Induction of TAT activity was calculated in relation to vehicle-treated animals as baseline (-fold TAT induction). To determine the therapeutic window or the effect to side effect profile of the compounds, the per cent inhibition of oedema formation on the x-axis was plotted against TAT induction on the y-axis.

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Glucose tolerance test following topical application of ZK 245186 onto rat skin [3]
Hairless rats (hr/hr, n = 10) were treated topically with vehicle, ZK 245186 (0.1%), methylprednisolone aceponate (MPA) (0.1%) or clobetasol propionate (0.1%). Prednisolone (3 mg·kg−1 body weight) p.o. served as a positive control to demonstrate GC-induced changes on the glucose metabolism. Treatment was performed once daily over four consecutive days. On day 5 after an overnight fasting period, 1 g glucose per kg body weight in 10 mL·kg−1 body weight was given orally and blood glucose concentrations were determined immediately before (baseline = 0 min) and shortly afterwards (30, 60, 120, 180 min) using glucose test strips and a blood sugar meter. In house experience with marketed topical GCs showed that compounds that only minimally increase blood glucose concentrations of topically treated hairless rats display low to no risk of altering glucose metabolism in humans after topical use, when applied according to the manufacturer's instructions (H. Wendt et al. unpubl. data).

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Topical and systemic side effects following prolonged topical application [3]
The variables measured were skin fold thickness, skin breaking strength (topical side effects) and total body weight as well as weights of thymus, spleen and the adrenal glands (systemic side effects after topical administration) as described earlier (Schäcke et al., 2004). The treatment regimen was daily application for 3 weeks. Active compounds (ZK 245186, methylprednisolone aceponate (MPA) and MF) or vehicle (EtOH containing 5% isopropylmyristate) were topically applied onto a skin area of 3 cm × 3 cm of juvenile, hr/hr rats (n = 10 per group) in equivalent concentrations for 19 days in a volume of 75 µL. Two different concentrations of the active compounds have been chosen: 3× ED50 determined in the croton oil rat model (for ZK 245186, 0.042%; for methylprednisolone aceponate (MPA), 0.035%; for MF, 0.0027%) and a concentration that results in ca. 80% inhibition of oedema formation in this model (for ZK 245186 and methylprednisolone aceponate (MPA), 0.1% and for MF, 0.01%). Skin fold thickness and animal body weight were determined on days 1 (baseline), 5, 8, 12, 15 and 19. On day 20, animals were killed for determination of the weight of the following organs: thymus, spleen and adrenal glands. The correlation between the skin thinning effects in rodents and humans is well established (Kirby and Munro, 1976; van den Hoven et al., 1991; Woodbury and Kligman, 1992). In this paper, skin atrophy is defined as decrease of skin fold thickness. Body weight, spleen and thymus weight as well as the weight of the adrenal glands are used as measures of undesired systemic effects of topically applied GCs or similar compounds.

rat LD50 oral >2 gm/kg GASTROINTESTINAL: HYPERMOTILITY, DIARRHEA; KIDNEY, URETER, AND BLADDER: INCONTINENCE Yakuri to Chiryo. Pharmacology and Therapeutics., 19(3015), 1991
rat LD50 subcutaneous >3 gm/kg ENDOCRINE: OTHER CHANGES Yakuri to Chiryo. Pharmacology and Therapeutics., 19(3015), 1991

[1]. Ruzicka T. Methylprednisolone aceponate in eczema and other inflammatory skin disorders -- a clinical update. Int J Clin Pract. 2006 Jan;60(1):85-92.

[2]. Preclinical evaluation of a new topical corticosteroid methylprednisolone aceponate. 1994. 3 (s1), S32-S38.

[3]. Characterization of ZK 245186, a novel, selective glucocorticoid receptor agonist for the topical treatment of inflammatory skin diseases. Br J Pharmacol. 2009 Oct;158(4):1088-103.

Methylprednisolone aceponate is a corticosteroid hormone.

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Methylprednisolone aceponate (MPA) has been shown to provide rapid, reliable and highly effective treatment of eczematous disorders, with an efficacy comparable to that of most reference topical corticosteroids. It also has excellent local and systemic tolerability. MPA is effective in the treatment of facial and scalp eczema and sunburn and has shown promising results in the treatment of psoriasis. Its rapid efficacy and lack of undesirable local and/or systemic side effects make MPA particularly suitable for use in children and infants. The wide range of formulations (0.1%) of MPA, including cream, ointment, fatty ointment, milk and solution, enable treatment to be tailored to the individual patient. In addition, MPA has the advantage of once-daily application compared with twice-daily treatment for other topical corticosteroids, thereby improving patient safety and promoting patient compliance but without compromising efficacy. [1]

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Background and purpose: Glucocorticoids are highly effective in the therapy of inflammatory diseases. Their value, however, is limited by side effects. The discovery of the molecular mechanisms of the glucocorticoid receptor and the recognition that activation and repression of gene expression could be addressed separately opened the possibility of achieving improved safety profiles by the identification of ligands that predominantly induce repression. Here we report on ZK 245186, a novel, non-steroidal, low-molecular-weight, glucocorticoid receptor-selective agonist for the topical treatment of inflammatory dermatoses. Experimental approach: Pharmacological properties of ZK 245186 and reference compounds were studied in terms of their potential anti-inflammatory and side effects in functional bioassays in vitro and in rodent models in vivo. Key results: Anti-inflammatory activity of ZK 245186 was demonstrated in in vitro assays for inhibition of cytokine secretion and T cell proliferation. In vivo, using irritant contact dermatitis and T cell-mediated contact allergy models in mice and rats, ZK 245186 showed anti-inflammatory efficacy after topical application similar to the classical glucocorticoids, mometasone furoate and methylprednisolone aceponate. ZK 245186, however, exhibits a better safety profile with regard to growth inhibition and induction of skin atrophy after long-term topical application, thymocyte apoptosis, hyperglycaemia and hepatic tyrosine aminotransferase activity. Conclusions and implications: ZK 245186 is a potent anti-inflammatory compound with a lower potential for side effects, compared with classical glucocorticoids. It represents a promising drug candidate and is currently in clinical trials. [3]

C27H36O7

472.57

472.246

C, 68.62; H, 7.68; O, 23.70

86401-95-8

63019

White to off-white solid powder

1.2±0.1 g/cm3

595.8±50.0 °C at 760 mmHg

193.1±23.6 °C

0.0±3.8 mmHg at 25°C

1.560

4.01

1

7

7

34

980

8

CCC(=O)O[C@@]1(CC[C@@H]2[C@@]1(C[C@@H]([C@H]3[C@H]2C[C@@H](C4=CC(=O)C=C[C@]34C)C)O)C)C(=O)COC(=O)C

DALKLAYLIPSCQL-YPYQNWSCSA-N

InChI=1S/C27H36O7/c1-6-23(32)34-27(22(31)14-33-16(3)28)10-8-19-18-11-15(2)20-12-17(29)7-9-25(20,4)24(18)21(30)13-26(19,27)5/h7,9,12,15,18-19,21,24,30H,6,8,10-11,13-14H2,1-5H3/t15-,18-,19-,21-,24+,25-,26-,27-/m0/s1

[(6S,8S,9S,10R,11S,13S,14S,17R)-17-(2-acetyloxyacetyl)-11-hydroxy-6,10,13-trimethyl-3-oxo-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-yl] propanoate

Advantan; MPA; Methylprednisolone aceponate; 86401-95-8; Advantan; Adventan; Methylprednisolone aceponate [INN]; SH-440; Methylprednisoloni aceponas; Methylprednisoloni aceponas [Latin]; Methylprednisolone Aceponate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)