Synthetic mineralocorticoid

醋酸氟屈可的松是一种用于治疗肾上腺功能不全的药物，可以以商业上无法获得的剂量开给住院或门诊的儿科患者。对于这些患者，目前10µg氟屈可的松胶囊是由预先配制的滴定粉末（研磨粉）配制而成的。进行了氟屈可的松稳定性研究，以确保有效期超过使用日期。首先，验证了一种稳定性指示醋酸氟屈可的松给药方法。然后制成10µg醋酸氟氢可的松胶囊和1%醋酸氟氢可的松滴定粉末（粉末研制物）。最后，进行了稳定性研究。醋酸氟屈可的松滴定粉末（粉末研制物）在受控环境温度下避光稳定一年，而10µg醋酸氟屈可的松胶囊稳定六个月。一年后，即使氟屈可的松含量保持一致，也注意到产品降解的增加。我们的工作使我们能够确定醋酸氟屈可的松滴定粉（研磨粉）与三种最常用的胶囊配制赋形剂的使用日期后六个月。我们还证实了胶囊的六个月理论稳定性[3]。

合成皮质类固醇可能对鱼类构成环境风险。在这里，我们描述了成年斑马鱼（8个月大）在暴露于浓度在0.006至42μg/L之间的常用皮质类固醇醋酸氟氢可的松（FLU）21天后的多终点反应。没有观察到显著的生殖影响，而生理效应，包括血浆葡萄糖水平和血白细胞数量，即使在42 ng/L的浓度下也发生了显著变化。卵巢参数和下丘脑-垂体-性腺-肝轴的转录分析显示，影响可以忽略不计。在斑马鱼的大脑中观察到昼夜节律网络的显著变化。包括per1a和nr1d1在内的几个生物标志物基因的转录物显示出强烈的转录变化，这些变化发生在6和42 ng/L FLU的环境相关浓度下。重要的是，F1胚胎的发育和行为发生了显著变化。即使在6和42 ng/L的浓度下，F1胚胎的心跳、孵化成功率和游泳行为都有所增加。所有这些影响都通过暴露于刺五加胚胎得到了进一步证实。在刺五加胚胎中，参与糖异生、免疫反应和昼夜节律的生物标志物基因的显著转录变化证实了成年鱼的观察结果。孵化成功率、心跳和游泳活动在81 ng/L及以上时增加，与F1胚胎一样。这些结果为理解皮质类固醇的潜在环境风险提供了新的见解[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.

---

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).

---

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

100.9

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。