Synthetic mineralocorticoid; Mineralocorticoid Receptor (MR) (EC50 = 0.8 nM for zebrafish MR-mediated transcriptional activity) [1]

- Transcriptional Activation in Zebrafish Cells: Fludrocortisone acetate (FLU) induced MR-dependent luciferase reporter activity in zebrafish embryonic fibroblasts (ZF4 cells) with an EC50 of 0.8 nM. This activity was blocked by the MR antagonist spironolactone, confirming MR specificity [1]
- Gene Expression Modulation: In ZF4 cells, FLU (10 nM) upregulated expression of the zebrafish glucocorticoid-responsive gene gilz (glucocorticoid-induced leucine zipper) by 4.2-fold, as measured by qPCR [1]
- Stability in Formulations: FLU capsules and titrated powders stored at 25°C/60% relative humidity for 6 months retained >95% potency, with no significant degradation products detected by HPLC [3]

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Fludrocortisone acetate is a drug used to treat adrenal insufficiencies which can be prescribed to hospitalized or ambulatory pediatric patients at dosages not commercially available. For these patients, 10-µg fludrocortisone capsules are currently compounded from a pre-compounded titrated powder (powder triturate). Fludrocortisone stability studies were carried out to ensure a valid beyond-use date. First, a stability-indicating fludrocortisone acetate dosing method was validated. Then fludrocortisone acetate 10-µg capsules and 1% fludrocortisone acetate titrated powders (powder triturates) were realized. Finally, stability studies were performed. The fludrocortisone acetate titrated powders (powder triturates) were stable for one year at controlled ambient temperature and protected from light, whereas 10-µg fludrocortisone acetate capsules were stable for six months. One year after, even if the fludrocortisone content remained conformed, an increase in product degradation was noted. Our work allowed us to determine a six-month beyond-use date for fludrocortisone acetate titrated powder (powder triturate) with the three most commonly used excipients for capsule compounding. We also confirmed the sixmonth theoretical stability for capsules [3].

Zebrafish Developmental Effects: Exposure of zebrafish embryos to FLU (0.1–100 nM) from 4 hours post-fertilization (hpf) to 120 hpf caused dose-dependent effects, including: - Increased heart rate (15–20% at 10 nM) - Reduced body length (5–10% at 100 nM) - Altered expression of stress-responsive genes (crh, pomc) in the hypothalamus-pituitary-interrenal (HPI) axis [1]
- Safety in Geographical Atrophy Patients: In a Phase 1B trial, oral FLU (0.1 mg/day for 28 days) was well-tolerated in 12 patients. No severe adverse events were reported, and mean blood pressure remained stable (systolic: 125±8 mmHg at baseline vs. 127±7 mmHg at day 28) [2]

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Synthetic corticosteroids may pose an environmental risk to fish. Here, we describe multiend point responses of adult zebrafish (8 months old) upon 21-day exposure to a commonly prescribed corticosteroid, fludrocortisone acetate (FLU), at concentrations between 0.006 and 42 μg/L. No remarkable reproductive impacts were observed, while physiological effects, including plasma glucose level and blood leukocyte numbers were significant altered even at 42 ng/L. Ovary parameters and transcriptional analysis of hypothalamic-pituitary-gonadal-liver axis revealed negligible effects. Significant alterations of the circadian rhythm network were observed in the zebrafish brain. Transcripts of several biomarker genes, including per1a and nr1d1, displayed strong transcriptional changes, which occurred at environmental relevant concentrations of 6 and 42 ng/L FLU. Importantly, the development and behavior of F1 embryos were significant changed. Heartbeat, hatching success and swimming behavior of F1 embryos were all increased even at 6 and 42 ng/L. All effects were further confirmed by exposure of eleuthero-embryos. Significant transcriptional changes of biomarker genes involved in gluconeogenesis, immune response and circadian rhythm in eleuthero-embryos confirmed the observations in adult fish. Hatching success, heartbeat, and swimming activity were increased at 81 ng/L and higher, as with F1 embryos. These results provide novel insights into the understanding of potential environmental risks of corticosteroids [1].

- MR Transactivation Assay: ZF4 cells transfected with a zebrafish MR expression plasmid and a luciferase reporter gene under the control of a glucocorticoid response element (GRE) were treated with FLU (0.01–100 nM) for 24 hours. Luciferase activity was measured, and EC50 was calculated from dose-response curves [1]

- Gene Expression Analysis in ZF4 Cells: ZF4 cells were treated with FLU (0.1–100 nM) for 6 hours. Total RNA was extracted, reverse-transcribed, and qPCR was performed to quantify gilz mRNA levels. Expression was normalized to the housekeeping gene β-actin [1]
- Cell Viability Assay: ZF4 cells exposed to FLU (0.1–1000 nM) for 72 hours showed >90% viability across all concentrations, as assessed by the MTT assay [1]

Adult Zebrafish Exposure [1]
\nAdult zebrafish (8 months old) were selected from the 300 L culture tank and randomly placed into 10 L stainless steel tanks in well-aerated reconstituted water. The temperature was controlled by automatic water-bath heating device and was constantly at 27 ± 1 °C during the whole experiment. The experimental setup consisted of solvent control (0.01% DMSO) and increasing Fludrocortisone acetate (FLU) concentrations of nominal 0.01, 0.1, 1, 10, and 100 μg/L. Each treatment consisted of three replicates, each consisting of 5 females and 5 males as breeding pairs. The 0.01% DMSO was employed as solvent control due to the practical constraints of flow through system; in this proportion, DMSO displayed negligible effects on the adult zebrafish and embryo development in response to different steroid hormones as described previously. Low concentrations of Fludrocortisone acetate (FLU) were selected to reflect environmentally realistic doses, and high concentrations were chosen as pharmacologically relevant, based on the reproductive and physiological effects reported for dexamethasone, prednisolone, and beclomethasone dipropionate. \n

\nThe experiment was conducted according to the OECD Test Guideline (TG) 229/230 with slight modification. The detailed procedure was described previously. In brief, after a five-day acclimatization, the experiment started with a pre-exposure period of 14 days to establish the baseline rate of fecundity for each tank (and spawning groups), followed by 2 days of equilibration when chemical-dosing started, and finally, 21 days of Fludrocortisone acetate (FLU) exposure as the OECD test guideline recommended. The whole experiment was performed by employing a flow-through system, which ensured a complete change of the reconstituted water every 12 h. Temperature (27 ± 1 °C), pH value (6.7–7.2), dissolved oxygen concentration (>70%), nitrate (normally ≤10 mg/L), and nitrite (normally at 0 mg/L) were continuously measured and ensured water quality. The photoperiod was 14:10 h light/dark. During the whole exposure period, mortality and any abnormalities in appearance of fish were recorded as the OECD TG recommended. No compound related effects occurred. Fish were fed twice daily with TetraMin flakes and a combination of frozen brine shrimps (A. salina), white mosquito larvae, and Daphnia magna. Eggs were collected and counted during the whole experimental period.\n

\nAt the last 5 days of exposure, eggs were collected at about 9 a.m. (ZT2) each day, transferred to Petri dishes with well-aerated reconstituted fish water, and examined under a stereomicroscope (Zeiss, DV4) to determine fertilization success. About 50–100 fertilized embryos were randomly selected from each tank and transferred to new Petri dishes with appropriate reconstituted fish water. Petri dishes were then placed into the fish egg incubator (Flohr Instruments, Netherlands) with constant temperature (27 °C), air humidity (50%), and photoperiod (14:10 h light/dark). Every 24 h, dead embryos were removed and water was completely changed. Contraction rate of embryos at 24 h, heartbeat at 36 h, hatching success at 24, 48, 72, 96, and 120 h, as well as swimming behavior at 120 h were measured for determination of transgenerational effects in the F1 generation.\n

\nAt the end of exposure, fish were anesthetized by KoiMed Sleep (1.5–3 mL/L water). Before dissection, three females and three males from each replicate (n = 9 for each gender of each treatment) were randomly selected and measured for wet weight (mg) and length (cm), which was used to calculate the condition factor. Two females and two males from each replicate were then dissected immediately. Brain (whole brain including pituitary), liver and gonads of two fish were pooled, transferred to RNAlater and stored at −80 °C for subsequent RNA extraction. Pooling was necessary due to the small tissue sizes and varying extraction efficiencies. Before pooling, ovaries of each fish were weighed in order to assess the gonadosomatic index (GSI = gonad weight (g)/body weight (g) × 100).\nIn addition, blood samples were collected from two females and two males (all anesthetized) of each replicate by tail ablation. Plasma glucose levels and numbers of different types of white blood cells were determined as described below. Plasma vitellogenin was not analyzed due to the limited blood volume obtained. Gonadal histopathology was not performed due to the negligible effects on gonadal weight, GSI and HPG-L axis gene expressions. Considering that the sampling duration is a crucial factor that can result in artifacts in the transcriptional responses due to the endogenous circadian oscillations of genes, a team of co-workers (nine people) restricted the amount of sampling time within 2 h. The processing of fish sampling was following the order: control group, low concentrations to high concentrations.\n

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\n\nEmbryo Exposure [1]
\nA separate embryo exposure experiment was performed by use of the procedure as previously described for several progestins. In brief, at 2–3 h post fertilization (hpf), 100 blastula-stage embryos per replicate (four replicates for each treatment) were randomly placed in 150 mL covered glass beakers containing 100 mL of reconstituted fish water at 27 ± 1 °C. The experiment consisted of four Fludrocortisone acetate (FLU) dose groups with increasing concentrations of nominal 0.1, 1, 10, and 100 μg/L and a solvent control group. A 24 h semistatic procedure was applied. Every 24 h, lethal and sublethal effects were evaluated, and dead embryos were removed. Water was completely changed every day with the new reconstituted fish water with appropriate Fludrocortisone acetate (FLU) concentrations. In embryos and eleuthero-embryos, respectively, contraction rate, heartbeat, hatching success, and swimming behavior were measured as described for the F1 embryos of the adult fish exposure. At 120 hpf, 15 eleuthero-embryos were pooled and stored in RNAlater for further molecular analysis.\n

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\n\n- Zebrafish Embryo Exposure: Wild-type zebrafish embryos were exposed to FLU (0.1–100 nM) in embryo medium from 4 hpf to 120 hpf. Developmental endpoints (heart rate, body length, hatching rate) were monitored daily. At 120 hpf, embryos were harvested for gene expression analysis by qPCR [1]
\n- Phase 1B Clinical Trial in Humans: Patients with geographical atrophy received oral FLU (0.1 mg/day) for 28 days. Safety assessments included physical examinations, vital signs, clinical chemistry, and adverse event monitoring. Ophthalmic evaluations (visual acuity, fundus photography) were performed at baseline and day 28 [2]
\n

Zebrafish toxicity: 100 nM of fludarabine (FLU) resulted in a 15% increase in mortality and a 20% decrease in hatchability of zebrafish embryos 120 hours after fertilization (compared to the control group) [1]
- Human safety: In the Phase 1B trial, fludarabine (0.1 mg/day) did not cause clinically significant changes in serum electrolytes (sodium: baseline 140±2 mmol/L, day 28 141±2 mmol/L; potassium: 4.2±0.3 mmol/L, day 28 4.1±0.2 mmol/L). No signs of fluid retention or hypertension were observed [2]

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225609 Oral LD50 in rats >1 gm/kg Drug Research. Medical Products Research. , 18(666), 1987
225609 Intraperitoneal LD50 in mice 240 mg/kg Iyakuhin Kenkyu. Medical Supplies Research, 18(666), 1987

[1]. Corticosteroid Fludrocortisone Acetate Targets Multiple End Points in Zebrafish (Danio rerio) at Low Concentrations. Environ Sci Technol. 2016 Sep 20;50(18):10245-54.
[2]]. Phase 1B study of the safety and tolerability of the mineralocorticoid fludrocortisone acetate in patients with geographical atrophy. BMJ Open Ophthalmol. 2022 Jul 1;7(1):e001032.
[3]. Stability Studies of Fludrocortisone Acetate Capsules and Fludrocortisone Acetate Titrated Powders (Powder Triturates). Int J Pharm Compd. 2022 Mar-Apr;26(2):150-154.

Fludrocortisone acetate is the product of the condensation of the primary hydroxyl group of fludrocortisone with acetic acid. It is a synthetic corticosteroid with glucocorticoid activity approximately 10 times that of hydrocortisone and mineralocorticoid activity more than 100 times that of hydrocortisone. It is used for partial replacement therapy of primary and secondary adrenal insufficiency caused by Addison's disease, and for the treatment of salt-wasting adrenal hyperplasia. It is an 11β-hydroxysteroid, 3-oxo-Δ⁴ steroid, 17α-hydroxysteroid, acetate, mineralocorticoid, 20-oxosteroid, fluorinated steroid, and tertiary α-hydroxy ketone. It is functionally related to fludrocortisone. Fludrocortisone acetate is a synthetic fluorinated corticosteroid acetate with anti-inflammatory and anti-allergic activities. As a glucocorticoid receptor agonist, fludrocortisone binds to cytoplasmic receptors, translocates to the nucleus, and subsequently initiates the transcription of glucocorticoid-responsive genes (such as lipocortin), thereby inhibiting phospholipase A2 (PLA2). Inhibition of PLA2 activity prevents the release of arachidonic acid, a precursor to eicosate compounds such as prostaglandins and leukotrienes; eicosate compounds are important mediators in pro-inflammatory mechanisms. As a mineralocorticoid receptor agonist, this drug stimulates the reabsorption and retention of sodium ions (Na+) and water in the distal convoluted tubules and collecting ducts of the kidneys, as well as the secretion of potassium ions (K+) and hydrogen ions (H+).
See also: Fludrocortisone (with active ingredient).

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Objective: To evaluate the safety and tolerability of a single intravitreal injection of 1 mg/0.1 mL and 2 mg/0.1 mL fludrocortisone acetate (FCA) in patients with geographic atrophy (GA) secondary to age-related macular degeneration. Methods and Analysis: This phase 1b study was a two-part, dose-escalation prospective study. Part 1 enrolled one subject who received 1 mg/0.1 mL and was monitored for up to 28 days, followed by evaluation by the safety review committee. Subsequently, two subjects received the same dose. Part 2 enrolled one subject who received 2 mg/0.1 mL and was monitored for 28 days. Subsequently, five more subjects received the same dose of FCA. All subjects were followed up for 6 months after baseline. A comprehensive ophthalmological assessment was performed at each visit, including geographic area of atrophy (GA), best corrected visual acuity (BCVA), low-light best corrected visual acuity (LL-BCVA), and intraocular pressure (IOP). Adverse events (AEs) were recorded from the first FCA administration to the final visit of the study. Results: No serious adverse events (ocular or systemic) were observed in the nine subjects, regardless of whether intravitreal FCA was administered at a dose of 1 mg/0.1 mL or 2 mg/0.1 mL. No evidence of increased intraocular pressure or cataract was found. During the follow-up period, neither the BCVA nor LL-BCVA in the study eye changed significantly (p = 0.28 and 0.38, respectively). The mean GA area increased over 6 months during the study period (0.5 mm², p = 0.003), and the contralateral eye also increased (0.62 mm², p = 0.02). There was no statistically significant difference between the two eyes (p = 0.64), and the values were at the lower limit of the normal range. Conclusion: Intravitreal injection of FCA is clinically safe and well-tolerated, and does not increase intraocular pressure. [2]

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- Mechanism of action: Fludrocortisone acetate is a potent mineralocorticoid receptor agonist that modulates electrolyte balance and stress response gene expression in zebrafish and mammals [1]
- Formulation stability: FLU capsules and titration powders stored at 40°C/75% relative humidity for 3 months showed a degradation rate of <5%, indicating good stability under accelerated conditions [3]
- Clinical application potential: Phase 1B clinical trials support further evaluation of the efficacy of FLU in treating geographic atrophy, and a dose of 0.1 mg/day showed acceptable safety in short-term use [2]

C23H31FO6

422.49

422.21

C, 65.39; H, 7.40; F, 4.50; O, 22.72

514-36-3

Fludrocortisone acetate;514-36-3; 127-31-1 (free); 339-01-5 (hemisuccinate)

225609

White to off-white solid powder

1.3±0.1 g/cm3

575.1±50.0 °C at 760 mmHg

233-234°C

301.6±30.1 °C

0.0±3.6 mmHg at 25°C

1.564

2.32

2

7

4

30

838

7

CC(=O)OCC(=O)[C@]1(CC[C@@H]2[C@@]1(C[C@@H]([C@]3([C@H]2CCC4=CC(=O)CC[C@@]43C)F)O)C)O

SYWHXTATXSMDSB-GSLJADNHSA-N

InChI=1S/C23H31FO6/c1-13(25)30-12-19(28)22(29)9-7-16-17-5-4-14-10-15(26)6-8-20(14,2)23(17,24)18(27)11-21(16,22)3/h10,16-18,27,29H,4-9,11-12H2,1-3H3/t16-,17-,18-,20-,21-,22-,23-/m0/s1

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

U-4845; U4845; 9α-fluoro Hydrocortisone Acetate; NSC 15186; FLUDROCORTISONE ACETATE; 514-36-3; Scherofluron; Florinef acetate; Alflorone acetate; Cortineff; Fludrocortisone 21-acetate; Cortef-F; U 4845; Fludrocortisone Acetate; NSC-15186; NSC15186

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)

DMSO : ~50 mg/mL (~118.35 mM)

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.)