Glucocorticoid receptor

Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis in childhood acute lymphoblastic leukemia. Prednisolone exerted a delayed biphasic effect on the resistant CCRF-CEM leukemic cell line, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone's mitogenic and cell death effects were counterbalanced. Early gene microarray analysis revealed notable differences in 40 genes. The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance [1].

Nitric oxide is believed to participate in nonspecific cellular immunity. Gram negative bacterial endotoxins increase the production of reactive nitrogen intermediates (RNI) in phagocytic cells by inducing the enzyme nitric oxide synthase II (NOS II). Anti-inflammatory glucocorticoids attenuate endotoxin-induced increases in RNI. 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 [2].

---

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 [3].

Prednisolone treatment [1]
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 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 [1]
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 [1]
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) [1]
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 [3]
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]

[1]. Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression. Leuk Res, 2009. 33(12): p. 1684-95.

[2]. Rapid induction of messenger RNA for nitric oxide synthase II in rat neutrophils in vivo by endotoxin and its suppression by prednisolone. Proc Soc Exp Biol Med. 1994 Mar;205(3):220-9.

[3]. Triamcinolone and prednisolone affect contractile properties and histopathology of rat diaphragm differently. J Clin Invest. 1993 Sep;92(3):1534-42.

ATL-2502(Colal-Pred) is an old drug (prednisolone metasulphobenzoate, a steroid) with a new delivery system that releases the medication only in the colon. Releasing the drug directly into the colon reduces the potential for significant side effects often experienced with steroid drugs. Colal-Pred is being studied for use in ulcerative colitis.
Prednisolone Sodium Metasulfobenzoate is a poorly adsorbed analog of prednisolone, a synthetic glucocorticoid with anti-inflammatory and immunomodulating properties. After cell surface receptor attachment and cell entry, prednisolone enters the nucleus where it binds to and activates specific nuclear receptors, resulting in an altered gene expression and inhibition of proinflammatory cytokine production. This agent also decreases the number of circulating lymphocytes, induces cell differentiation, and stimulates apoptosis in sensitive tumor cells populations. (NCI)

---

Drug Indication
Investigated for use/treatment in inflammatory bowel disease and ulcerative colitis.

---

Mechanism of Action
ATL-2502 is the combination of Alizyme's proprietary colonic drug delivery system, colal, and prednisolone metasulfobenzoate sodium ("PMSBS"), an approved steroid in Europe. colal-PRED has a starch coating that is only broken down in the gut by bacteria occurring in the colon. This leads to topical delivery of PMSBS to the colon rather than systemic delivery with the objective of providing efficacy, but without the side effects of steroids; however, the efficacy and safety of colal-PRED must be shown in well-controlled, prospective clinical trials and approved by the FDA.

---

In conclusion, this study shows: (a) prednisolone exerts a delayed biphasic effect on CCRF-CEM cells, necrotic at low doses and apoptotic at higher doses, (b) at low doses, prednisolone exerts a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone's mitogenic and cell death effects are counterbalanced. These mitogenic effects are of clinical importance in the case of resistant leukemic cells. It is of crucial importance if a certain administered dose of GCs to an ALL patient possesses proliferative rather than suppressive actions, (c) NF-κB is constitutively localized in the nucleus and its inhibition emerges as a possible candidate for the treatment of resistant leukemia, (d) prednisolone activates genes related to at least four different pathways upon 4 h treatment: apoptosis and tumor suppression, cell cycle progression, metabolism and intra- and extra-cellular signaling. Several of these genes manifested a biphasic differential expression profile. This approach might lead to the identification of genes candidates for future molecular drug targets in combination therapy with GCs. These drugs may affect the potential of GCs to inhibit growth of resistant leukemic cells. Also, this type of approach could identify potential early markers for GCs resistance. Early detection of resistance could facilitate the efficiency of ALL therapies.[1]

---

In conclusion, the present study shows that triamcinolone and prednisolone have different effects on morphology and contractile properties of the rat diaphragm. Fluorinated steroids such as triamcinolone caused severe muscle wasting due to selective type Ilb fiber atrophy, resulting in markedly reduced respiratory muscle strength. Prednisolone in four times higher doses caused a tendency towards lower tetanic tensions and increased fatigability of diaphragmatic muscle bundles, with distinct alterations in muscle histology.[3]

C28H31O9S-.NA+

566.59514

566.158

C, 59.36; H, 5.52; Na, 4.06; O, 25.41; S, 5.66

630-67-1

52-21-1 (acetate);72064-79-0 (valerate acetate);630-67-1 (sodium metazoate);125-02-0 (Na+ phosphate); 50-24-8 (free);

23675004

Typically exists as solid at room temperature

2

9

6

39

1210

7

C[C@]12C[C@@H]([C@H]3[C@H]([C@@H]1CC[C@@]2(C(=O)COC(=O)C4=CC(=CC=C4)S(=O)(=O)[O-])O)CCC5=CC(=O)C=C[C@]35C)O.[Na+]

RWFZSORKWFPGNE-VDYYWZOJSA-M

InChI=1S/C28H32O9S.Na/c1-26-10-8-18(29)13-17(26)6-7-20-21-9-11-28(33,27(21,2)14-22(30)24(20)26)23(31)15-37-25(32)16-4-3-5-19(12-16)38(34,35)36;/h3-5,8,10,12-13,20-22,24,30,33H,6-7,9,11,14-15H2,1-2H3,(H,34,35,36);/q;+1/p-1/t20-,21-,22-,24+,26-,27-,28-;/m0./s1

sodium;3-[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-oxoethoxy]carbonylbenzenesulfonate

PREDNISOLONE SODIUM METAZOATE; 630-67-1; Cortico-Sol; Prednisolone sodium metasulfobenzoate; Prednisolone methylsulfobenzoate; UNII-D345THM53T; Prednisolone sodium metazoate [USAN]; D345THM53T;

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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