Androgen receptor; metabolite of testosterone; oxidized testosterone

In a few teleosts, 11-Ketotestosterone (11-KT) is the most powerful androgen and is non-aromatizable. Testosterone and 17β-estradiol concentrations increased in the culture medium of hepatic explants and ovarian explants after incubation with 11-Ketotestosterone (10 and 100 μM) for five days in vitro; the concentration of vitellogenin also increased in the culture medium of hepatic explants[1].

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HSD11B2 is induced by SF-1 and involved in the 11-KT production [1]
Human ovarian granulosa cell tumor-derived KGN cells have low steroidogenic capacity under basal conditions. However, they can be transformed to produce a range of steroid hormones by infection with an adenovirus expressing SF-1 and its coactivator. To examine changes in gene expression in this transformation, we used a DNA microarray with GFP- or SF-1-introduced KGN cells. SF-1 introduction induced a number of genes, including steroidogenic enzymes such as CYP11A1, HSD3B2, CYP17A1, and CYP19A1 (Supplemental Table 2), which are known SF-1 targets. In addition to these genes, HSD11B2 was a strong SF-1-inducible candidate gene (Supplemental Table 2) and was almost undetectable in GFP-introduced cells (Figure 1, A and B). Consistent with DNA microarray data, Q-PCR and immunoblotting analyses revealed that introduction of SF-1 strongly induced HSD11B2 mRNA and proteins (Figure 1, A and B). In previous studies, it was demonstrated that HSD11B2 is involved in 11-KT production from testosterone with CYP11B1 (Supplemental Figure 1) in murine gonads or from adrenal androgens by in vitro analysis. The DNA microarray analyses suggest that CYP11B1 is an SF-1-inducible gene, as well as other steroidogenic genes that are involved in testosterone and E2 synthesis (Figure 1C and Supplemental Table 2, and Supplemental Figure 1). In fact, the introduction of SF-1 induced not only the production of testosterone and E2 but also the CYP11B1 expression and 11-KT production (Figure 1, C and D).
To verify the role of HSD11B2 during 11-KT production in human steroidogenic cells, it was ectopically expressed in human adrenocortical H295R cells. H295R cells expressed CYP11B1 and other steroidogenic enzymes for testosterone synthesis (Supplemental Figure 2), whereas endogenous HSD11B2 was undetectable (Figure 2A). Even though H295R cells produce testosterone at relatively high levels, its conversion to 11-KT was marginal (Figure 2, C and D). Transient transfection of the HSD11B2 expression vector (Figure 2B) markedly increased 11-KT production compared with the control group (Figure 2D), whereas testosterone concentrations were similar in both groups (Figure 2C). These results indicate that the expression of human HSD11B2 could be an important factor for the production of 11-KT in steroidogenic cells.

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Effects of 11-KT Treatment in vitro [2]
Histological Observation of Ovary [2]
The ovarian development of samples in vitro was confirmed by histological observation and results showed all samples developed into stage II (Figure 6).

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Gene Expression Profiles of era, erb, ar, lpl, cyp19a1, Vtg in Hepatic Explant [2]
From the reference primers screened, b-actin was best suited (SD = 0.22, in BestKeeper; SD = 0.57 in Delta CT; SD= 0.052 in Normfinder; P < 0.05, Table 1). For cyp19a1, expression in the 100 μm group was 6.4 times higher than that in the control group (P = 0.001) and 3 times higher than that in the 10 μM group (P = 0.002) (Figure 7A). For era, expression in the 100 μm group was 2.2 times higher than that in the control group (P = 0.033) and 1.3 times higher than that in the 10 μM group (P = 0.108) (Figure 7B). Expression for erb increased slightly with increasing 11-KT concentrations but there were no differences showed among groups (P = 0.838, Figure 7C). Expression for ar and lpl in the experimental groups were higher than those in the corresponding control group (P = 0.017; P ≤ 0.001, respectively) but no significant differences were detected between the two experimental groups (P = 0.784; P = 0.999, respectively) (Figures 7D,E). Ar expression was 1.93 times higher in the 10 μM group (P = 0.017) and 2.18 times higher in the 100 μM group (P = 0.019) than that in the control group (Figure 7D). The expression of lpl was 1.3 times higher in the 10 μM group (P = 0.001), and 1.5 times higher in the 100 μM group (P ≤ 0.001) than that in the control group (Figure 7E). Notably, 11-KT had a marked effect on vtg expression. In the 100 μM group, it was 30 times higher than that in the 10 μM group (P = 0.002), and 80 times higher than that in the control group (P = 0.01) (Figure 7F).

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hanges in T, E2, and Vtg Concentrations in the Haptic Explants Culture Medium [2]
Concentrations of T and E2 in the following 5 days of liver explant culture increased significantly for culture medium supplemented with 100 μM 11-KT. T concentrations were higher in the 100 μM group (151.30 ± 73.50 ng/mL) than both those in 10 μM groups (14.14 ± 10.90 ng/mL) (P = 0.058) and the control group (0.01 ± 0.00 ng/mL) (P = 0.026) (Figure 8A). Concentrations of E2 in the 100 μM group (96.88 ± 42.63 pg/mL) was 6 times more than that in the 10 μM group (16.61 + 6.40 pg/mL) (P = 0.002), and 60 times more than that in the control group (1.59 + 0.69 pg/mL) (P = 0.001) (Figure 8B). Vtg showed an upward trend with increasing 11-KT concentrations (P = 0.035). The concentration in the 100 μM (0.18 ± 0.00 ng/mL) was 1.8 times as high as that of the 10 μM (0.10 ± 0.02 ng/mL) (P = 0.026), and 6 times as high as that in the control group (0.03 ± 0.01 ng/mL) (Figure 8C).

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Gene Expression Profiles for foxl2, cyp19a1, era, erb, ar, Vtgr in Ovarian Explants [2]
After incubation of ovarian explants in a culture medium with or without 11-KT for 5 days, changes in expression levels were only observed for era in both experimental groups as compared to the control group (P = 0.005, Figure 9C). The expression of other genes foxl2, cyp19a1, and erb displayed a rising trend with increasing 11-KT concentration, but there was no significant difference between groups (P = 0.095, Figure 9A; P = 0.214, Figure 9B; P = 0.838, Figure 9D, respectively).

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Changes in T and E2 Concentrations in the Ovarian Explants Culture Medium [2]
Both T and E2 concentrations increased in the ovarian tissue culture medium with 11-KT supplementation. Concentrations of T were higher in the experimental groups than in the control group (0.02 ± 0.01 ng/mL, P = 0.001), with no significant differences between the 10 μM group (140.75 ± 28.87 ng/mL) and the 100 μM group (40.27 ± 40.54 ng/mL) (P = 0.058, Figure 10A). E2 concentrations were 4.8 and 33 times higher in the 100 μM group (66.36 ± 22.79 pg/mL) than in the 10 μM group (13.77 ± 5.63 pg/mL; P = 0.024) and the control group (2.17 ± 1.63 pg/mL; P = 0.002) (Figure 10B).

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Changes in T and E2 Concentrations in the Ovarian Explants Culture Medium [2]
Both T and E2 concentrations increased in the ovarian tissue culture medium with 11-KT supplementation. Concentrations of T were higher in the experimental groups than in the control group (0.02 ± 0.01 ng/mL, P = 0.001), with no significant differences between the 10 μM group (140.75 ± 28.87 ng/mL) and the 100 μM group (40.27 ± 40.54 ng/mL) (P = 0.058, Figure 10A). E2 concentrations were 4.8 and 33 times higher in the 100 μM group (66.36 ± 22.79 pg/mL) than in the 10 μM group (13.77 ± 5.63 pg/mL; P = 0.024) and the control group (2.17 ± 1.63 pg/mL; P = 0.002) (Figure 10B).

30 days are spent implanting silastic strips with 11-Ketotestosterone (5 or 25 mg/kg) in vivo in previtellogenic cultured sterlets (Acipenser ruthenus). There is no evidence of sex reversal or ovarian masculinization. 11-Diffusion-dependent sterlet ovarian development is promoted by ketotestosterone[1].

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11-ketotestosterone (11-KT) is a non-aromatizable and the most potent androgen in a few teleost. It has been reported that 11-KT in serum had a high concentration and increased sharply before the period of yolk deposition in females of few fishes. The aim of this study was to analyze the role of 11-KT both in vivo and in vitro on ovarian development, related gene expression levels, Vitellogenin (Vtg) synthesis, and serum sex steroid concentrations in previtellogenic cultured sterlet (Acipenser ruthenus). Silastic strips embedded with 11-KT (5 or 25 mg/kg) were implanted in vivo for 30 days. Ovarian masculinization or sex reversal was not observed. Histological analysis showed that 11-KT promoted sterlet ovarian development in a dose-dependent manner. Vtg and testosterone (T) increased significantly, while 17β-estradiol (E2) decreased with no significant difference among groups. The expression of genes androgen receptor (ar), vtg and lipoprotein lipase (lpl) were significantly increased in liver. However, 11-KT had no effect on the expression of foxl2 and cyp19a1 in ovary. In vitro, after incubation with 11-KT (10 and 100 μM) for 5 days, both T and E2 concentrations increased in both hepatic explants and ovarian explants culture medium; the concentration of Vtg also increased in hepatic explants culture medium. The expression of ar, era, vtg, and lpl increased significantly in hepatic explants. However, only the expression of era significantly increased in cultured ovarian explants. Altogether, these results suggest that 11-KT induced ovarian development, as well as Vtg and lipid synthesis, and could be an important factor facilitating the initiation of Vtg synthesis in the liver of the previtellogenic sterlet. [2]

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11-KT is produced in gonads and is one of the major androgens in human [1]
To elucidate the 11-KT synthesis pathway, we investigated the expression of CYP11B1 and HSD11B2 in human gonads (Figure 3, A and B). Q-PCR and Western blot analyses showed that both genes were detectable in the testis and ovary at the mRNA and protein levels (Figure 3, A and B). Immunohistochemical analysis showed that both proteins are localized on testicular Leydig cells and ovarian theca cells, even though HSD11B2 is also detectable in some populations of ovarian granulosa cells (Figure 3C). These results strongly suggest that 11-KT is produced in testicular Leydig cells and ovarian theca cells. To confirm this hypothesis, we investigated the production of 11-KT in human Leydig cells. In support of the immnohistochemical analyses, Leydig cells expressed CYP11B1 and HSD11B2 genes (Supplemental Figure 3A). They can produce progesterone, testosterone, and 11-KT under basal conditions (Figure 3D and Supplemental Figure 3B). cAMP treatment moderately increased the production of these steroid hormones.

Then, we measured plasma concentrations of 11-KT, testosterone, and E2 in both sexes (Figure 3, E–G). Testosterone levels in men were about 22-fold higher than those in women, whereas 11-KT levels were similar between the sexes (Figure 3, E and F). It is noteworthy in women that 11-KT concentrations were similar to testosterone concentrations and about 5-fold higher than E2 concentrations (Figure 3H). Human AR-mediated transactivation was significantly increased by 11-KT at concentrations more than 10−9M in KGN cells (Figure 4). This level is lower than that for the induction by other androgens, although transactivation was increased to similar levels by DHT and testosterone at 10−8M and 10−7M, respectively. These results suggest that 11-KT may play some roles in human as one of the major androgens.

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11-KT is not convertible to estrogenic hormones [1]
In female individuals, testosterone acts as both an androgen and a precursor for estrogen. To determine whether 11-KT is a precursor for estrogen, we performed a luciferase assay using a reporter plasmid containing the ERE in human breast cancer-derived MCF-7 cells, which endogenously express aromatase and ERα. In contrast to E2, androgens had no effect on luciferase activity at lower concentrations (10−11M and 10−10M) (Figure 5A). However, testosterone activated ER-dependent transcription at high concentrations and at 10−7M testosterone was effective as E2. This activity was completely suppressed by an aromatase inhibitor, fadrozole (Figure 5B). DHT weakly activated ER-mediated transactivation in an aromatase-independent manner at 10−7M. In contrast, 11-KT had no effect on ER-mediated transactivation, even at 10−6M (Figure 5A; data not shown). These results indicate that 11-KT is a nonaromatizable androgen, and does not convert to a compound that activates ER-mediated transactivation.

Measurements by enzyme immunoassays (EIAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [1]
Concentrations of testosterone, 11-KT, and E2 in culture media of KGN, H295R, and human Leydig cells were determined by competitive EIA . Each sample was diluted with EIA buffer and analyzed using progesterone, testosterone, 11-KT, and E2 EIA kit following the manufacturer’s instructions in a microplate reader. On the other hand, concentrations of steroid hormones in human plasma were measured by LC-MS/MS for optimal quantification of the clinical samples. Processing of human plasma samples and quantification of testosterone, 11-KT, and E2 by LC-MS/MS are based on methods as described previously.

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Measurement of Sex Steroid Hormone and Vtg [2]
About 0.8 mL of plasma was used for sex steroid hormone and Vtg concentration determination. According to manufacturer's instructions, Vtg was measured using the Vitellogenin Elisa Kit. T was measured by the Testosterone Radioimmunoassay Kit. The minimum detectable limit of the method was 0.02 ng/ML for T. all antibodies had <0.1% cross-reactivity with closely-related steroids, such as dihydrotestosterone, 1.1 × 10−2%; 17β-estradiol, 2.1 × 10−2%; estriol, 6.2 × 10−15%; progesterone, 3.2 × 10−2%; and 11-KT, 1.2 × 10−2%. E2 was measured by chemiluminescent immunoassay using the Quantitative Determination Kit 17β-estradiol. The minimum detectable limit of the method was <4.0 pg/ml for E2. Cross-reactivity of the antibody, as provided by the manufacturer, was as follows: estriol, <0.5%; progesterone <1.5%; and testosterone <0.01%. The standard curve was built with linear model logit (%B/B0) vs. log concentrations. All samples were analyzed in duplicate. Intra-assay variance averaged 8.38, 5.9, and 6.4% for Vtg, T, and E2, respectively, which fell within acceptable levels.

Proliferation assay [1]
MCF-7 cells or AR-introduced cells were seeded with DMEM/F-12 supplemented with 10% or 2% charcoal/dextran-stripped-FBS at 1 × 103 cells/well in 96-well plates. At 24 hours after seeding, the cells were treated with the media containing various concentrations of DHT, testosterone, 11-KT, or E2. Six days (parental cells) or 9 days (AR-introduced cells) after incubation, cell proliferation was evaluated using a CellTiter 96 Aqueous One Solution kit following the manufacturer’s instructions. To evaluate the effect of an aromatase inhibitor, fadrozole or an ER antagonist, fulvestrant, on the growth of MCF-7 cells, a proliferation assay was performed with or without these agents.

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Experiment 2: in vitro Effects of v on Target Gene Expression, Sex Steroid Concentrations, and Vtg Synthesis in Hepatic and Ovarian Explants [2]
A stock solution of 1,000 μM (3,000 ng/mL) 11-KT (MW = 302.408) was prepared by dissolving in 40 μL ethanol (0.16% of final incubation volume), and then added to 5 mL with DMEM/F12.
Three 28-month old female sterlets were selected through endoscopic determination at the previtellogenic stage. After being anesthetized with 400 ppm of MS222, the sterlets were briefly submerged in 75% ethanol prior to the removal of ovaries and liver under sterile conditions. A portion consistently resected from the central part of ovaries was fixed in Bouin's solution for histological analysis. Following adipose tissue removal, the remaining ovarian tissue and livers were washed separately by cold PBS (1X, PH = 7.4) and cut into 1 cm3 fragments in culture medium (DMEM/F12, 1:1, 1X, phenol-red free). Using 6-well Costar culture plates, fragments were incubated in 2.5 mL culture medium for 5 days at 25°C. Three replicate incubations were performed for each treatment and for each individual. The culture medium consisted of DMEM/F12 (1:1, 1X, phenol-red free) supplemented with 1% penicillin-streptomycin solution, 20% fetal bovine serum and 0, 10, 100 μM 11-KT (0, 30, or 300 ng/mL). At the end of incubation, explants were flash frozen in liquid nitrogen and stored at −80°C until analysis. Culture medium from each dish was also collected to determine T,E2, and Vtg concentrations.

Animals and Synthetic 11-KT Powder [2]
For these experiments, 11-KT powder was synthetized in Academy of Military Medical Sciences, through reducing C17 ketone group of adrenosterone to hydroxyl with the catalytic reaction of sodium borohydride. Using standard 11-KT as control, the identification and purity of synthetized 11-KT was 99.9% checked by HPLC (High Performance Liquid Chromatography), and then stored at 4°C.

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Experiment 1: in vivo Effects of 11-KT on Ovarian Development, Target Gene Expression, Sex Steroid Concentrations and Vtg Synthesis [2]
Manufacture of Slow-Release 11-KT Silastic Strips [2]
The dry 11-KT was mixed and thoroughly homogenized with unpolymerized medical elastomer base and coagulator silastic MDX4-4210. After uniform mixing, the paste was dried and processed into silastic strips (1.5 mm in diameter and 30 mm in length). Each strip carried 25 mg 11-KT. All strips were kept at 4°C in aluminum foil until use.

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Animals and 11-KT Implantation [2]
Twenty-eight-month old sterlets were randomly collected on Aug. 2015. After endoscopic detection under anesthesia, eighteen previtellogenic females were selected for implantation and divided into three balanced groups: one control group (355.30 ± 27.93 g, n = 6), two treatment groups consisting of a lower dose group (5 mg/kg, 375.12 ± 50.37 g, n = 6), and a high dose group (25 mg/kg, 405.83 ± 49.84 g, n = 6). No significant difference existed between groups (P = 0.142). Fish were fed with commercial standard diets daily. After being anesthetized with 400 ppm of MS222, a small ventral midline incision was performed on all sterlets. In the treatment groups, the appropriate length of 11-KT silastic strips were cut and implanted to achieve the corresponding dose (5 or 25 mg/kg, respectively). In the control group, silastic strips devoid of 11-KT were implanted in an identical manner as in the treated groups. Following surgery, the incisions were daubed erythromycin ointment to prevent wound infection. Then, sterlets were transferred to indoor cylinder tanks (1 m3) and reared in flowing water for 30 days. Water temperature in the tanks ranged from 16.8 to 21.4°C.

[1]. Effects of 11-Ketotestosterone on Development of the Previtellogenic Ovary in the Sterlet, Acipenser ruthenus. Front Endocrinol (Lausanne). 2020 Mar 25;11:115.

[2]. 11-Ketotestosterone Is a Major Androgen Produced in Human Gonads. J Clin Endocrinol Metab. 2016 Oct;101(10):3582-3591.

11-oxotestosterone is a 3-oxo Delta(4)-steroid that is testosterone carrying an additional oxo substituent at position 11. It has a role as an androgen, a marine xenobiotic metabolite and a human metabolite. It is a 3-oxo-Delta(4) steroid, an 11-oxo steroid, an androstanoid and a 17beta-hydroxy steroid. It is functionally related to a testosterone. It derives from a hydride of an androstane.

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Context: 11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. Objective: The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. Results: Cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. Conclusions: The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen. [1]

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In summary, we demonstrated that 11-KT is a major androgen and produced in gonads. Because androgens are essential for reproduction and physiology, their excess and deficiency often induce pathogenesis. Then, it is possible that 11-KT could be responsible for, and the novel target of therapies against, such diseases. In addition, it might provide novel insights for elucidating ambiguous AR-mediated phenomena.[1]

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In summary, we report that11-KT induced ovarian development without ovarian masculinization or sex reversal in vivo, as well as Vtg and lipid synthesis in vivo and in vitro. To our knowledge, this is the first report in sturgeon to describe the 11-KT effect on the development of the previtellogenic sterlet. Through detection of T, E2, and 11-KT concentrations in gonadal development stages of breeding Siberia sturgeon during a natural breeding season, Hamlin et al. reported 11-KT in females increased, beginning at the previtellogenic stage and peaking at the germinal vesicle stage, with a concomitant increase in E2 concentrations. Serum 11-KT concentrations were low in previtellogenic females of the Amur sturgeon (A. schrenckii), but increased at the beginning of vitellogenesis and peaked before E2 concentrations. Therefore, it appears that in sturgeon, 11-KT is an important factor initiating Vtg synthesis at previtellogenic stage, potentially through the activation of E2 secretion via Ar and Era signal pathways. However, detailed understanding of these pathways requires additional studies, such as RNA-sequencing or microRNA regulation, to decipher the molecular mechanisms involved. [2]

C19H26O3

302.41

302.188

564-35-2

5282365

White to off-white solid powder

1.2±0.1 g/cm3

476.8±45.0 °C at 760 mmHg

256.3±25.2 °C

0.0±2.7 mmHg at 25°C

1.569

1.67

1

3

0

22

577

6

C[C@]12CCC(=O)C=C1CC[C@@H]3[C@@H]2C(=O)C[C@]4([C@H]3CC[C@@H]4O)C

WTPMRQZHJLJSBO-XQALERBDSA-N

InChI=1S/C19H26O3/c1-18-8-7-12(20)9-11(18)3-4-13-14-5-6-16(22)19(14,2)10-15(21)17(13)18/h9,13-14,16-17,22H,3-8,10H2,1-2H3/t13-,14-,16-,17+,18-,19-/m0/s1

(8S,9S,10R,13S,14S,17S)-17-hydroxy-10,13-dimethyl-2,6,7,8,9,12,14,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-3,11-dione

11-Ketotestosterone; 11-Oxotestosterone; 11-Keto-testosterone; UNII-KF38W1A85U; KF38W1A85U; 17beta-Hydroxyandrost-4-ene-3,11-dione; Androst-4-ene-3,11-dione, 17-hydroxy-, (17beta)-; ...; 564-35-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (330.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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