Glucocorticoid receptor (GR)
Hydrocortisone phosphate is a prodrug that is hydrolyzed in vivo to release active hydrocortisone, which then binds to the cytosolic glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. Upon binding, the newly formed receptor-ligand complex translocates to the cell nucleus, where it binds to glucocorticoid response elements (GREs) in the promoter regions of target genes, leading to increased transcription of specific anti-inflammatory proteins. The primary anti-inflammatory mechanism involves the induction of lipocortin-1 (annexin-1), a protein that inhibits cytosolic phospholipase A2 (cPLA2). This inhibition prevents the release of arachidonic acid from cell membrane phospholipids, thereby blocking the biosynthesis of prostaglandins and leukotrienes—both potent inflammatory mediators. Additionally, cyclooxygenase (both COX-1 and COX-2) expression is suppressed, further reducing eicosanoid production. Glucocorticoids also stimulate lipocortin-1 release into the extracellular space, where it binds to leukocyte membrane receptors and inhibits various inflammatory events including epithelial adhesion, emigration, chemotaxis, phagocytosis, respiratory burst, and the release of inflammatory mediators from neutrophils, macrophages, and mast cells.

Hydrocortisone phosphate has no lethal effect on MH60 cells that are IL-6-independent, but it inhibits the biological activities of IL-6 and IL-3 with IC50s of 6.7 and 21.4 μM, respectively [3]. PHA responses in peripheral lymphocyte (PBL) and T lymphocyte cultures are inhibited by hydrocortisone phosphate (0.12-60 μM; 72 hours) [3].

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In vitro studies have characterized the stability and cellular effects of hydrocortisone phosphate. Research on the stability of hydrocortisone sodium phosphate (HCSP) during iontophoresis has demonstrated that drug stability is highly dependent on pH. Applied current induces pH shifts in the surrounding matrices due to ion migration and electrochemical reactions, which can lead to drug degradation. Addition of buffers to the matrix can protect HCSP from hydrolysis but introduces competing ions that reduce drug delivery efficiency. In HEp-2 cell cultures (human laryngeal carcinoma cells), exposure to hydrocortisone (the active form of the prodrug) for 24 or 48 hours resulted in a significant increase in mitochondrial activity as measured by MTT assay, while crystal violet proliferation assays showed similar behavior to control groups. Three-dimensional (3D) cultures of HEp-2 cells treated with hydrocortisone showed dispersed cells within 24 hours with reduced FAK (focal adhesion kinase) labeling, though no changes were observed by 48 hours.

In mice, oral hydrocortisone phosphate (30 mg/kg) given twice a day for five days decreases weight loss and enhances food intake [2].

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In vivo studies have demonstrated the biological effects of hydrocortisone phosphate across multiple animal models. In a primate study involving cynomolgus monkeys, subcutaneous implantation of osmotic pumps releasing hydrocortisone phosphate at 5 mg/day for one menstrual cycle elevated serum cortisol levels 1.4-fold and decreased serum adrenal androgens to 0.6 of baseline. Higher dose (15 mg/day) over two menstrual cycles elevated serum cortisol 1.7-fold and suppressed adrenal androgens to 0.4 of baseline. The lower dose increased serum estradiol (E2) levels by 77% during the follicular phase (p < 0.001) but did not affect gonadotropin levels. The higher dose raised serum luteinizing hormone (LH) levels by 111% and 96% across two follicular phases (p < 0.001) while decreasing follicle-stimulating hormone (FSH) levels by 45-50% (p < 0.05), demonstrating dose-dependent and hormone-selective effects of chronic glucocorticoid exposure. Interestingly, after cessation of treatment, serum cortisol remained suppressed to 0.8 and 0.6 of baseline for low and high doses, respectively, indicating prolonged HPA axis suppression.

The primary non-cellular method for characterizing the receptor binding activity of hydrocortisone (after hydrolysis of hydrocortisone phosphate) is the glucocorticoid receptor (GR) competitive binding assay. In this cell-free procedure, cytosolic glucocorticoid receptors are isolated from target tissues such as rat liver or thymus through homogenization and ultracentrifugation. The receptor preparation is incubated with a radiolabeled high-affinity glucocorticoid ligand, typically [³H]dexamethasone or [³H]hydrocortisone, in the presence of varying concentrations of unlabeled hydrocortisone. Incubation is carried out at 4°C for 12-24 hours to reach equilibrium while minimizing ligand degradation. Separation of bound radioligand from free ligand can be achieved using validated methods including hydroxylapatite adsorption, dextran-coated charcoal (DCC) adsorption, or rapid vacuum filtration through glass fiber filters (e.g., GF/B filters). The bound radioactivity retained is quantified by liquid scintillation counting. For the phosphate ester itself, stability studies are conducted using chromatographic methods (HPLC or LC-MS/MS) to assess hydrolysis rates under various pH conditions, which is critical for understanding prodrug activation.

A validated in vitro cell-based assay for evaluating hydrocortisone activity uses the HEp-2 cell culture model (human laryngeal carcinoma cells). In this protocol, HEp-2 cells are seeded into 96-well plates at a density of approximately 2 × 10⁴ cells per well and cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO₂. After reaching appropriate confluence, cells are exposed to varying concentrations of hydrocortisone (the active form of hydrocortisone phosphate) for 24 or 48 hours. Cell viability and mitochondrial activity are assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay: MTT solution is added to each well and incubated for 3-4 hours, followed by solubilization with DMSO or isopropanol, and absorbance measured at 570 nm. Cell proliferation is evaluated using the crystal violet staining method. For three-dimensional (3D) culture studies, HEp-2 cells are cultured in ultra-low attachment plates or Matrigel to form spheroids, then treated with hydrocortisone. Immunofluorescent labeling for fibronectin and focal adhesion kinase (FAK) is performed to assess cell adhesion and migration characteristics.

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats (200-220 g, 10-11 weeks) induced colitis [2]
Doses: 30 mg/kg
Route of Administration: Po twice (two times) daily for 5 days
Experimental Results: Significant reduction in disease Activity index (DAI) scores and myeloperoxidase (MPO) activity compared with the 2,4,6-trinitrobenzene sulfonic acid (TNBS) group. Weight gain.

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A representative in vivo protocol for studying hydrocortisone phosphate uses the cynomolgus monkey (Macaca fascicularis) model with subcutaneously implanted osmotic pumps. In this procedure, adult female cynomolgus monkeys with regular menstrual cycles are used. Alzet osmotic pumps are filled with hydrocortisone phosphate solution (prepared in saline) to deliver doses of 5 mg/day or 15 mg/day. Under anesthesia, the pumps are implanted subcutaneously in the interscapular region. For the lower dose (5 mg/day), pumps remain implanted for one menstrual cycle; for the higher dose (15 mg/day), pumps are implanted for two consecutive menstrual cycles. Control cycles (saline-filled pumps) are performed before and after each treatment period, separated by at least one menstrual cycle. Blood samples are collected at regular intervals throughout the study for hormone analysis. Serum cortisol, adrenal androgens (dehydroepiandrosterone sulfate), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2) are measured using validated radioimmunoassays (RIAs). Endpoints include changes in hormone levels, menstrual cycle length, and assessment of HPA axis suppression post-treatment.
For pharmacokinetic studies in rats, microdialysis sampling combined with radioimmunoassay (RIA) is used. Awake, free-roaming rats receive intravenous administration of hydrocortisone phosphate, and dialysate samples are collected every 2 minutes through a microdialysis probe inserted into the extracellular space. Unbound hydrocortisone levels are quantified by RIA, with pharmacokinetic parameters including half-life (t₁/₂ = 17-29 minutes) calculated from the data.

Absorption, Distribution and Excretion
Topical corticosteroids are absorbed through normal, intact skin. Skin inflammation and/or other conditions can increase percutaneous absorption. Corticosteroids are primarily metabolized in the liver and then excreted via the kidneys. Some topical corticosteroids and their metabolites are also excreted via bile. Metabolism/Metabolites Primarily metabolized in the liver via CYP3A4. Biological Half-Life 6-8 hours

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The pharmacokinetic properties of hydrocortisone phosphate are characterized by its role as a water-soluble prodrug of hydrocortisone. Key PK parameters include:
Absorption: Following intravenous administration, hydrocortisone phosphate is rapidly hydrolyzed to free hydrocortisone by plasma phosphatases. After topical ocular administration of 0.33% hydrocortisone sodium phosphate eye drops, peak hydrocortisone concentration in the aqueous humor reaches approximately 35.2 ± 20.2 ng/mL, with peak penetration observed 60-120 minutes post-instillation. The steroid persists at relatively stable concentrations until around 150 minutes, followed by a slow decline.
Hydrolysis and Activation: As a phosphate ester prodrug, hydrocortisone phosphate requires enzymatic hydrolysis to release the active parent drug. Stability studies have shown that the compound is susceptible to pH-dependent hydrolysis and may degrade during iontophoretic drug delivery if not properly buffered.
Plasma Protein Binding: After conversion to hydrocortisone, the drug is highly protein-bound (>90%), primarily to corticosteroid-binding globulin (CBG) and albumin.
Distribution: Free (unbound) hydrocortisone distributes into the extracellular space and can be sampled by microdialysis. The unbound fraction fluctuates rapidly in vivo, with changes observed between 2-minute sampling intervals.
Elimination Half-life: After intravenous administration in rats, unbound hydrocortisone decreases to predose endogenous concentrations in a first-order fashion with a half-life of 17-29 minutes.
Special Considerations: In primates, chronic administration of hydrocortisone phosphate (5-15 mg/day for 1-2 menstrual cycles) results in sustained serum cortisol elevation followed by prolonged HPA axis suppression after therapy cessation, with cortisol levels remaining below baseline for at least one subsequent menstrual cycle.

Protein Binding
95%

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The toxicological profile of hydrocortisone phosphate has been evaluated through clinical use and case reports:
Hypersensitivity Reactions: A documented case report describes a 62-year-old male patient who experienced a severe asthmatic attack approximately 5 minutes after intravenous administration of 200 mg Hydrocortone Phosphate Injection. Provocation challenge testing with 100 mg of the drug produced dramatic confirmation of the reaction. Interestingly, the patient showed no adverse reactions to other corticosteroid products including Solu-Cortef (hydrocortisone hemisuccinate), dexamethasone phosphate, prednisolone phosphate, prednisolone hemisuccinate, oral hydrocortisone, or oral methylprednisolone. Skin tests to crude hydrocortisone phosphate were negative, but skin tests to diluents were idiosyncratically positive. Passive cutaneous anaphylaxis testing in Japanese monkey skin showed positive results for hydrocortisone phosphate but negative for the diluent, suggesting that the reaction may be specific to the phosphate ester or a unique hypersensitivity mechanism.
Endocrine Toxicity: Subchronic administration of hydrocortisone phosphate in primates (5-15 mg/day for 1-2 menstrual cycles) results in sustained suppression of adrenal androgens to 0.4-0.6 of baseline levels, with prolonged HPA axis suppression persisting after treatment cessation. Serum cortisol remained suppressed to 0.6-0.8 of baseline in the cycle following treatment.
HPA Axis Suppression: As with all systemic glucocorticoids, prolonged or high-dose use of hydrocortisone phosphate can cause hypothalamic-pituitary-adrenal (HPA) axis suppression, leading to adrenal insufficiency upon drug withdrawal.
Ocular Side Effects: Topical ophthalmic use of hydrocortisone requires consideration of the lowest clinically effective dose to minimize steroid exposure in the anterior chamber and avoid side effects including intraocular pressure increase and cataract development.
Drug Stability and Degradation: Hydrocortisone phosphate is susceptible to pH-dependent hydrolysis during administration (e.g., during iontophoresis), and degradation products may have unknown toxicity profiles.

[1]. Inhibitory effects of anti-inflammatory drugs on interleukin-6 bioactivity. Biol Pharm Bull. 2001 Jun;24(6):701-3.

[2]. In vitro and in vivo application of pH-sensitive colon-targeting polysaccharide hydrogel used for ulcerative colitis therapy. Carbohydr Polym. 2015 Oct 5;130:243-53.

[3]. The immunosuppressive potency in vitro of physiological and synthetic steroids on lymphocyte cultures. Int J Immunopharmacol. 1987;9(4):469-73.

Cortisol phosphate is a steroidal phosphate, a 21-O-phosphate derivative of cortisol. It is a cortisol ester, steroidal phosphate, 11β-hydroxysteroid, 3-oxo-Δ⁴steroid, 17α-hydroxysteroid, and tertiary α-hydroxy ketone. It is the conjugate acid of cortisol phosphate (2-). Pharmacological Indications: Used to relieve inflammatory and pruritus symptoms of corticosteroid-sensitive skin diseases. Also used to treat endocrine (hormonal) disorders (adrenal insufficiency, Addison's disease). It is also used to treat various immune and allergic diseases, such as arthritis, lupus, severe psoriasis, severe asthma, ulcerative colitis, and Crohn's disease. Mechanism of Action: Hydrocortisone binds to cytoplasmic glucocorticoid receptors. After binding to the receptor, the newly formed receptor-ligand complex translocates to the nucleus and binds to multiple glucocorticoid response elements (GREs) in the promoter region of target genes. DNA-binding receptors then interact with basic transcription factors, leading to increased expression of specific target genes. The anti-inflammatory effects of corticosteroids are thought to be related to lipocortin, a phospholipase A2 inhibitor that controls the biosynthesis of prostaglandins and leukotrienes by inhibiting arachidonic acid. Specifically, glucocorticoids induce the synthesis of lipocortin-1 (annexin-1), which binds to the cell membrane, preventing phospholipase A2 from contacting its substrate, arachidonic acid. This results in reduced arachidonic acid production. The expression of cyclooxygenases (COX-1 and COX-2) is also inhibited, thereby enhancing the above effects. In other words, both major products of inflammation—prostaglandins and leukotrienes—are inhibited by glucocorticoids. Glucocorticoids can also stimulate lipocortin-1 to escape into the extracellular space. Lipocortictin-1 binds to leukocyte membrane receptors, inhibiting various inflammatory responses, including epithelial cell adhesion, migration, chemotaxis, phagocytosis, respiratory burst, and the release of various inflammatory mediators (lysosomal enzymes, cytokines, tissue plasminogen activator, chemokines, etc.) from neutrophils, macrophages, and mast cells. Furthermore, glucocorticoids suppress the immune system through mechanisms including decreased lymphatic system function, reduced immunoglobulin and complement concentrations, lymphopenia, and interference with antigen-antibody binding.

C21H31O8P

442.43984

442.176

3863-59-0

Hydrocortisone 17-butyrate;13609-67-1;Hydrocortisone acetate;50-03-3;Hydrocortisone 17-valerate;57524-89-7;Hydrocortisone hemisuccinate;2203-97-6;Hydrocortisone;50-23-7; 6000-74-4 (Hydrocortisone phosphate sodium)

441407

Typically exists as solid at room temperature

1.42g/cm3

669.9ºC at 760mmHg

358.9ºC

1.596

1.898

4

8

4

30

848

7

C[C@@]12[C@](C(COP(O)(O)=O)=O)(O)CC[C@@]1([H])[C@]3([H])CCC4=CC(CC[C@]4(C)[C@@]3([H])[C@@H](O)C2)=O

BGSOJVFOEQLVMH-VWUMJDOOSA-N

InChI=1S/C21H31O8P/c1-19-7-5-13(22)9-12(19)3-4-14-15-6-8-21(25,17(24)11-29-30(26,27)28)20(15,2)10-16(23)18(14)19/h9,14-16,18,23,25H,3-8,10-11H2,1-2H3,(H2,26,27,28)/t14-,15-,16-,18+,19-,20-,21-/m0/s1

[2-[(8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-17-yl]-2-oxoethyl] dihydrogen phosphate

Hydrocortisone phosphate; 3863-59-0; Cortisol 21-phosphate; Hydrocortisone 21-phosphate; 2Y87E22X71; DTXSID7048160; NSC-529660; PREGN-4-ENE-3,20-DIONE, 11,17-DIHYDROXY-21-(PHOSPHONOOXY)-;

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.)