Glucocorticoid receptor

This study evaluated the effect of in vivo administration of prednisolone on Escherichia coli lipopolysaccharide endotoxin (LPS)-induced increases in plasma RNI and neutrophil mRNA for NOS II and production of RNI in the rat. We show that LPS rapidly induces mRNA for NOS II and production of RNI (NO2- and NO3- anion) in rat neutrophils within 2 hr after in vivo administration of a sublethal dose of 0.5 mg/kg, i.v. A pharmacologic dose of prednisolone (50 micrograms/kg, im) given 15 min before LPS-attenuated production of NO2- and NO3- by neutrophils and suppressed LPS-stimulated mRNA for NOS II. 3-Amino, 1,2,4-triazine inhibited NO2- and NO3- production without affecting gene expression for NOS II. These data demonstrate that LPS rapidly induces functional gene expression for NOS II and prednisolone prevents induction of NOS II activity by inhibiting transcription of its mRNA.[1]

Diaphragm atrophy and weakness occur after administration of massive doses of corticosteroids for short periods. In the present study the effects of prolonged administration of moderate doses of fluorinated and nonfluorinated steroids were investigated on contractile properties and histopathology of rat diaphragm. 60 rats received saline, 1.0 mg/kg triamcinolone, or 1.25 or 5 mg/kg i.m. prednisolone daily for 4 wk. Respiratory and peripheral muscle mass increased similarly in control and both prednisolone groups, whereas triamcinolone caused severe muscle wasting. Maximal tetanic tension averaged 2.23 +/- 0.54 kg/cm2 (SD) in the control group. An increased number of diaphragmatic bundles in the 5-mg/kg prednisolone group generated maximal tetanic tensions < 2.0 kg/cm2 (P < 0.05). In addition, fatigability during the force-frequency protocol was most pronounced in this group (P < 0.05). In contrast, triamcinolone caused a prolonged half-relaxation time and a leftward shift of the force-frequency curve (P < 0.05). Histological examination of the diaphragm showed a normal pattern in the control and 1.25-mg/kg prednisolone group. Myogenic changes, however, were found in the 5-mg/kg prednisolone group and, more pronounced, in the triamcinolone group. Selective type IIb fiber atrophy was found in the latter group, but not in the prednisolone groups. In conclusion, triamcinolone induced type IIb fiber atrophy, resulting in reduced respiratory muscle strength and a leftward shift of the force-frequency curve. In contrast, 5 mg/kg prednisolone caused alterations in diaphragmatic contractile properties and histological changes without fiber atrophy.[2]

Prednisolone treatment [3]
Concentrations of Prednisolone were selected on the basis of the average in vivo dosage administrated intravenously to children at ages between 1 month and 12 years old (details in supplementary data, file: CCRFCEM Cytotoxixity Assay.xls). Also, bioactivity in cortisol equivalents is estimated to be in the range of 40–200 nM. To ensure that the study covers these ranges, prednisolone was diluted to the following 12 concentrations: control, 10 nM, 100 nM, 1 μM, 5.5 μM, 11 μM, 22 μM, 44 μM, 88 μM, 175 μM, 350 μM, and 700 μM.

---

Cell proliferation assay [3]
Cell population counts were determined with the use of a NIHON KOHDEN CellTaq-α hematology analyzer. Cells were counted at the −24 h time point as well as 0 h, 4 h, 24 h, 48 h, and 72 h after initiation of exposure to prednisolone. For this purpose, 200 μl of cell suspensions were obtained from each flask and counted directly with the analyzer.

---

Protein extraction and Western blotting [3]
Cells were harvested after 1 h and 4 h exposure to different concentrations of Prednisolone. Protein extraction and Western blotting were performed as previously described. Total protein content was determined by the Bradford method using bovine serum albumin as a standard. Proteins were separated by SDS-PAGE and Western blotting was carried out, with anti-p65 antibodies

---

Microarray analysis [3]
cDNA microarray chips (1200 genes) were obtained from TAKARA (Human Cancer Chip v.40). Hybridization was performed with the CyScribe Post-Labeling kit as described by the manufacturer, utilizing the Cy3 and Cy5 fluorescent dyes. Slides were scanned with a microarray scanner. Images were generated with ScanArray microarray acquisition software. cDNAs from three experimental setups were used, each one consisting of three independent experiments. The experimental setups consisted of the three following pairs: control vs. 10 nM Prednisolone (designated as 0vs1), 10 nM prednisolone vs. 700 μM prednisolone (designated as 1vs3), control vs. 700 μM prednisolone (designated as 0vs3). This is a ‘simple loop’ experimental design, taking into account all possible combinations between samples, as previously described. Raw microarray data are available as supplementary data.

---

Real-time reverse transcription PCR (qRT-PCR) [3]
The GRIM19 (NDUFA13) gene was tested from three samples control, 10 nM and 700 μM Prednisolone at 4 h and 48 h treatment, using the one-step Plexor™ qRT-PCR kit. The set of primers was designed using the on-line tool Plexor™ Primer Design System v1.2 by Promega

Study design, animals, and treatment [2] 60 adult male Wistar rats, aged 14 wk, weighing 350-400 g, were randomized in quadruplets, into one of four treatment groups: control (C), saline, 0.05 ml/d i.m.; low dose Prednisolone (LD), 1.25 mg/kg per d i.m.; high dose Prednisolone (HD), 5 mg/kg per d i.m.; or triamcinolone-diacetate (TR), 1 mg/kg per d i.m. Dilution of medication was performed such that with each injection all animals received a similar volume (0.05 ml). During 4 wk the animals were injected daily in the left hindlimb. They were fed ad libitum and weighed twice weekly. After the treatment period, contractile properties, histological, and histochemical characteristics of the diaphragm were examined.[3]

Absorption, Distribution and Excretion
The Cmax of prednisolone acetate oral suspension equivalent to 15 mg prednisolone is 321.1 ng/hr, Tmax is 1–2 hours, and AUC is 1999.4 nghr/mL. The absorption pharmacokinetics of prednisolone acetate are not significantly different from those of prednisolone at equivalent doses. Prednisolone acetate is primarily excreted in the urine. The volume of distribution of the active metabolite prednisolone is 0.22/0.7 L/kg. Data on the clearance of prednisolone acetate are currently unavailable. Metabolism/Metabolites Prednisolone acetate undergoes ester hydrolysis to form prednisolone. After this step, the drug undergoes normal prednisolone metabolism. Prednisolone is reversibly metabolized to prednisone, which is then metabolized to 17α,21-dihydroxy-pregnane-1,4,6-trien-3,11,30-trione (M-XVII), 20α-dihydroprednisolone (MV), 6β-hydroxyprednisolone (M-XII), 6α-hydroxyprednisolone (M-XIII), or 20β-dihydroprednisolone (M-IV). 20β-dihydroprednisolone is metabolized to 17α,20ξ,21-trihydroxy-5ξ-pregnane-1-ene-3,11-dione (M-XVIII). Prednisolone is metabolized to Δ6-prednisolone (M-XI), 20α-dihydroprednisolone (M-III), 20β-dihydroprednisolone (M-II), 6α-hydroxyprednisolone (M-VII), or 6β-hydroxyprednisolone (M-VI). 6α-Hydroxyprednisolone is metabolized to 6α,11β,17α,20β,21-pentahydroxypregnane-1,4-dien-3-one (MX). 6β-Hydroxyprednisolone is metabolized to 6β,11β,17α,20β,21-pentahydroxypregnane-1,4-dien-3-one (M-VIII), 6β,11β,17α,20α,21-pentahydroxypregnane-1,4-dien-3-one (M-IX), and 6β,11β,17α,21-tetrahydroxy-5ξ-pregnane-1-en-3,20-dione (M-XIV). MVIII is metabolized to 6β,11β,17α,20β,21-pentahydroxy-5ξ-pregn-1-en-3-one (M-XV), and then to MXIV; while MIX is metabolized to 6β,11β,17α,20α,21-pentahydroxy-5ξ-pregn-1-en-3-one (M-XVI), and then to MXIV. These metabolites and their glucuronide conjugates are primarily excreted in the urine.
Biological Half-Life
The plasma half-life of orally administered prednisolone acetate is 2–3 hours.

Protein Binding
The active metabolite prednisolone has a protein binding rate of 70-90% in plasma. 5834 Rat subcutaneous LD50 >240 mg/kg, Japanese Pharmacopoeia, 6(713), 1982. 5834 Mouse subcutaneous LD50 3500 mg/kg, Japanese Pharmacopoeia, 6(713), 1982.

[1]. Proc Soc Exp Biol Med.1994 Mar;205(3):220-9;
[2]. J Clin Invest.1993 Sep;92(3):1534-42.
[3]. Leuk Res, 2009. 33(12): p. 1684-95.

Prednisolone acetate is a corticosteroid. Prednisolone acetate is formed by linking the prednisolone molecule to the acetate functional group via an ester bond. Prednisolone acetate was approved by the U.S. Food and Drug Administration (FDA) in 1955. There are reports that Rehmannia glutinosa contains prednisolone acetate, and relevant data are available for reference. Prednisolone acetate is the acetate form of prednisolone, a synthetic glucocorticoid with anti-inflammatory and immunomodulatory effects. As a glucocorticoid receptor agonist, prednisolone acetate binds to specific intracellular glucocorticoid receptors and promotes the translocation of the ligand-receptor complex to the cell nucleus, thereby initiating the transcription of glucocorticoid-responsive genes (such as various cytokines and lipocortin). Lipocorticoids inhibit phospholipase A2, thereby blocking the release of arachidonic acid from membrane phospholipids and inhibiting the synthesis of prostaglandins and leukotrienes, both of which are potent inflammatory mediators. This drug can also reduce the number of circulating lymphocytes, induce cell differentiation, and stimulate apoptosis in sensitive tumor cell populations. See also: Prednisolone (containing the active ingredient); Prednisolone acetate; Sodium sulfacetamide (one of the ingredients); Neomycin sulfate; Prednisolone acetate (one of the ingredients)... See more...

---

Drug Indications
Prednisolone acetate is indicated for the treatment of allergic diseases, skin diseases, gastrointestinal diseases, hematological diseases, ophthalmic diseases, nervous system diseases, kidney diseases, respiratory diseases, rheumatic diseases, or infectious diseases. It can be used as an anti-inflammatory drug or immunosuppressant. Prednisolone acetate is also indicated for organ transplant patients and endocrine or tumor patients.

---

Mechanism of Action
The short-term effects of corticosteroids are to reduce vasodilation and capillary permeability, and to reduce the migration of leukocytes to sites of inflammation. Corticosteroids bind to glucocorticoid receptors, mediating alterations in gene expression, thereby producing a variety of downstream effects over hours to days. Glucocorticoids inhibit neutrophil apoptosis and marginalization; inhibit phospholipase A2, thereby reducing the production of arachidonic acid derivatives; inhibit NF-κB and other inflammatory transcription factors; and promote the expression of anti-inflammatory genes such as interleukin-10. Low-dose corticosteroids have anti-inflammatory effects, while high-dose corticosteroids have immunosuppressive effects. Long-term use of high-dose glucocorticoids can bind to mineralocorticoid receptors, leading to increased sodium and decreased potassium levels.

---

Pharmacodynamics
Corticosteroids bind to glucocorticoid receptors, inhibiting pro-inflammatory signaling and promoting anti-inflammatory signaling. Prednisolone acetate has a short duration of action, with a half-life of 2-3 hours. Corticosteroids have a wide therapeutic window, and patients may need to take doses far exceeding the body's natural production. Patients taking corticosteroids should be informed of the risks of hypothalamic-pituitary-adrenal axis suppression and increased susceptibility to infection.

C23H30O6

402.48

402.204

C, 68.64; H, 7.51; O, 23.85

52-21-1

Prednisolone;50-24-8;Prednisolone disodium phosphate;125-02-0;Prednisolone hemisuccinate;2920-86-7;Prednisolone acetate-d8; Prednisolone acetate;52-21-1; 630-67-1 (sodium metazoate); 72064-79-0 (valerate acetate)

5834

White to off-white solid powder

1.3±0.1 g/cm3

579.8±50.0 °C at 760 mmHg

240-244 °C

198.4±23.6 °C

0.0±3.7 mmHg at 25°C

1.587

2.58

2

6

4

29

827

7

CC(=O)OCC(=O)[C@]1(CC[C@@H]2[C@@]1(C[C@@H]([C@H]3[C@H]2CCC4=CC(=O)C=C[C@]34C)O)C)O

LRJOMUJRLNCICJ-JZYPGELDSA-N

InChI=1S/C23H30O6/c1-13(24)29-12-19(27)23(28)9-7-17-16-5-4-14-10-15(25)6-8-21(14,2)20(16)18(26)11-22(17,23)3/h6,8,10,16-18,20,26,28H,4-5,7,9,11-12H2,1-3H3/t16-,17-,18-,20+,21-,22-,23-/m0/s1

[2-[(8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxoethyl] acetate

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)

Solubility in Formulation 1: ≥ 3 mg/mL (7.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 3 mg/mL (7.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 3 mg/mL (7.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 20 mg/mL (49.69 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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