Synthetic mineralocorticoid

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].

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].

Adult Zebrafish Exposure [1]
Adult 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.

The 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.

At 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.

At 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). In 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.

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Embryo Exposure [1]
A 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.

225609 rat LD50 oral >1 gm/kg Iyakuhin Kenkyu. Study of Medical Supplies., 18(666), 1987
225609 mouse LD50 intraperitoneal 240 mg/kg Iyakuhin Kenkyu. Study of Medical Supplies., 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 an acetate ester resulting from the formal condensation of the primary hydroxy group of fludrocortisone with acetic acid. A synthetic corticosteroid, it has glucocorticoid actions about 10 times as potent as hydrocortisone, while its mineralocorticoid actions are over 100 times as potent. It is used in partial replacement therapy for primary and secondary adrenocortical insufficiency in Addison's disease and for the treatment of salt-losing adrenal hyperplasia. It is an 11beta-hydroxy steroid, a 3-oxo-Delta(4) steroid, a 17alpha-hydroxy steroid, an acetate ester, a mineralocorticoid, a 20-oxo steroid, a fluorinated steroid and a tertiary alpha-hydroxy ketone. It is functionally related to a fludrocortisone.
Fludrocortisone Acetate is the acetate salt of a synthetic fluorinated corticosteroid with antiinflammatory and antiallergic 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 lipocortins to inhibit phospholipase A2 (PLA2). Inhibition of PLA2 activity prevents the release of arachidonic acid, a precursor of eicosanoids such as prostaglandins and leukotrienes; eicosanoids are important mediators in the pro-inflammatory response mechanism. As a mineralocorticoid-receptor agonist, this agent stimulates Na+ reabsorption and water retention and K+ and H+ secretion in the distal tubules and collecting ducts of the kidney.
See also: Fludrocortisone (has active moiety).

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Objective To evaluate the safety and tolerability of a mineralocorticoid, in a single-dose intravitreal (IVT) injection of 1 mg/0.1 mL and 2 mg/0.1 mL fludrocortisone acetate (FCA) in subjects with geographical 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 involved a single participant treated with 1 mg/0.1 mL and monitored up to 28 days before being reviewed by a safety review committee. Two subsequent participants were then dosed with the same dose. Part 2 involved a single participant dosed with 2 mg/0.1 mL and monitored up to 28 days when a further five participants were dosed. All participants were followed up for 6 months after baseline. A full ophthalmic assessment was performed at study visits which included GA area, best-corrected visual acuity (BCVA), low-luminance BCVA (LL-BCVA) and intraocular pressure (IOP). Adverse events (AEs) were reported from the first dose of FCA until the end-of-study visit. Results There were no serious AEs (ocular or systemic) observed with IVT FCA at either 1 mg/0.1 mL or 2 mg/0.1 mL among nine participants. There was no evidence of increased IOP or cataract development. Neither BCVA or LL-BCVA changed significantly in the study-eye over the follow-up period (p=0.28 and 0.38, respectively). Mean GA area increased in the study (0.5 mm2, p=0.003) and fellow-eyes (0.62 mm2, p=0.02) over 6 months. Differences between eyes were not significant (p=0.64), and at the lower end of population norms. Conclusion IVT FCA is clinically safe and well tolerated and did not increase IOP.[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.)