Aromatase

In vitro activity: Aminoglutethimide displays aromatase inhibition in vitro assay with human placental aromatase, which is an enzyme involved in the conversion of androgens into estrogens, and an important target for the endocrine treatment of breast cancer. Aminoglutethimide inhibits ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. Aminoglutethimide significantly suppresses steroid secretion and the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 μM AG, 5±1%; 30 μM AG, 64±1%; 3 μM AG, 108±19% compared with control cells, 100±11%) by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability, in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. Aminoglutethimide inhibits aromatase in a dose-dependent fashion with IC50 of 13 μM in 6 breast tumor homogenates, placental aromatase with IC50 of 6 μM and hypothalamic aromatase with IC50 of 8 μM.

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Cell Assay: The NCI-h295 tumor cell line is maintained in RPMI 1640 medium supplemented with transferrin (0.1 mg/mL), insulin (5 μg/mL), selenium (5.2 μg/mL) and 2% FCS. The cells are incubated for 48 hours with Aminoglutethimide (3, 30, 300 μM). Then cells are examined by trypan blue staining for cell viability, counted with a coulter counter. For the assessment of ACTH-R mRNA, cells are harvested, and total RNA is extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe.

Aminoglutethimide accelerates its own metabolism from a basal value of 2.6±0.3 (S.E.) liters/24 hours to 5.3±1.4 liters/24 hours after 1 to 2 weeks of Aminoglutethimide administration, and markedly accelerates the metabolism of the synthetic glucocorticoid and dexamethasone, from basal values of 145±26.6 liters/24 hours to 568±127 liters/24 hours (p < 0.02) after 2 weeks of Aminoglutethimide administration. Aminoglutethimide (150 mg/kg) abolishes the induction of ornithine decarboxylase (ODC) and almost depletes the gonads and plasma of progesterone or testosterone elicited by human chorionic gonadotropin (hCG) in the ovary of adult female mice and the testis of immature male mice, which is related to an inhibition of cAMP-dependent protein kinase (IC50 287 μM) rather than blockade of the steroidogenic pathway.

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Use of steroid biosynthesis inhibitors to suppress estrogen production is a logical strategy in the treatment of women with hormone-dependent breast cancer. The clinical availability of aminoglutethimide as an inhibitor of cytochrome P-450-mediated steroid hydroxylations prompted study of the precise pharmacological and biochemical effects of this drug. Pharmacokinetic studies revealed that aminoglutethimide alters its own metabolic clearance rate as well as that of dexamethasone, a synthetic glucocorticoid. The metabolic clearance rates of other steroids such as hydrocortisone, medroxyprogesterone acetate, and androstenedione, and estrone are not altered by aminoglutethimide. These findings led to development of a practical regimen of escalating aminoglutethimide dosage in combination with hydrocortisone for treatment of patients with breast carcinoma. Further studies focused upon the biochemical mechanism of estrogen suppression with aminoglutethimide. In vivo, isotopic kinetic data demonstrated that aminoglutethimide inhibits peripheral aromatase by 95 to 98% in postmenopausal women. In vitro experiments indicated that aminoglutethimide can effectively block aromatase directly in human breast tumors as well. With respect to relative potency, aminoglutethimide is a 10-fold more potent aromatase inhibitor than is testololactone but is less potent than are 4-hydroxyandrostenedione and several brominated androstenedione derivatives. Taken together, these studies suggest that aminoglutethimide blocks estrogen production at three sites in women with breast carcinoma: the adrenal cortex, extraglandular peripheral tissues containing aromatase, and breast carcinoma tissue itself [1].

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Aminoglutethimide is a steroidogenesis inhibitor and inhibits a cholesterol side-chain cleavage enzyme (CYP11A1) that converts cholesterol to pregnenolone in mitochondria. We investigated histopathological changes induced by 5-day administration of AG in mice. Cytoplasmic vacuoles of various sizes and single cell necrosis were found in zona fasciculata cells in AG-treated mice. Some vacuoles were positive for adipophilin, whereas others were positive for lysosome-associated membrane protein-2 on immunohistochemical staining, indicating they were enlarged lipid droplets and lysosomes, respectively. Electron microscopy revealed enlarged lysosomes containing damaged mitochondria and lamellar bodies in zona fasciculata cells, and they were considered to reflect the intracellular protein degradation processes, mitophagy and lipophagy. From these results, we showed that AG induces excessive lipid accumulation and mitochondrial damage in zona fasciculata cells, which leads to an accelerated lysosomal degradation in mice [4].

Kinase Assay: The microsomal protein (30 μg), [1β-3H]androstenedione (6.6 × 105 dpm) and NADPH (270 μM) are used for the concentration–response experiment with an incubation time of 20 minutes. The Aminoglutethimide is initially tested at 10 μM and 100 μM concentrations, followed by a full concentration–response study with at least 8 concentrations ranging from 0.01 μM to 160 μM. For the initial velocity study the concentration of [1β-3H]androstenedione is varied from 7.5 to 100 nM and the incubation time is set to 5 minutes. The tritiated water formed during the conversion of the tritiated substrate, [1β-3H]androstenedione, to estrone is quantified by liquid scintillation counting. Each assay is performed three times in duplicate and the results are treated by nonlinear regression analysis allowing the determination of the half-maximal inhibitory concentration (IC50).

Dissociated cortical culture and cell death assay [3]
Primary cultures of dissociated cortical neurons were prepared from the cerebral cortex of fetal Wistar rats (17–19 days of gestation) derived from 16 mother rats in total, as described previously (Shirakawa et al., 2002). Single cells mechanically dissociated from the whole cerebral cortex were seeded onto 48-well plates coated with polyethylenimine (for cell death assay) or onto glass coverslips coated with polyethylenimine (for Ca2+ measurement) at a density of 4.5 × 105 cells cm−2. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere in Eagle's minimal essential medium supplemented with glutamine (2 mM), glucose (11 mM in total), NaHCO3 (24 mM), HEPES (10 mM), and 10% heat-inactivated fetal bovine serum (1–7 DIV) or 10% heat-inactivated horse serum (8–12 DIV). Proliferation of non-neuronal cells was arrested by addition of 10 μM cytosine arabinoside at 6 DIV.
At 11 DIV, glutamate was added to the medium for 24 h at a final concentration of 300 μM, and cell death was evaluated by LDH release assay as mentioned above for slice culture experiments, except that 15 μl of culture medium was mixed with 30 μl of the LDH substrate mixture and 60 μl of 10 mM phosphate-buffered saline. Cultures treated with 10 mM glutamate for 24 h were used to determine the degree of the standard injury in each set of experiments. Absorbance values were normalized with the absorbance in cultures that received standard injury as 100%.
We also evaluated cell viability by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cultured cells were incubated in Eagle's medium containing 0.5 mg ml−1 MTT for 2 h and then solubilized with isopropanol, and the absorbance at 595 nm was measured. Viability was expressed as % of control, by setting the value of control cultures as 100% and the value of cultures receiving the standard injury (10 mM glutamate for 24 h) as 0%.

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Measurement of intracellular Ca2+ concentration [3]
Glutamate-induced increases in intracellular Ca2+ concentration ([Ca2+]i) were estimated with a Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl ester, and a fluorescence imaging system. Dissociated cortical neurons at 11–12 DIV cultured on a polyethylenimine-coated glass coverslip were incubated in Krebs–Ringer buffer (137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 25 mM glucose, pH 7.4) containing 5 μM fura-2 acetoxymethyl ester and 0.01% cremophore EL for 30 min at 37°C. After postincubation in fura-2-free Krebs–Ringer buffer for at least 30 min, the coverslip was transferred to a recording chamber settled on the stage of an inverted fluorescence microscope. Fura-2 fluorescence obtained by excitation at wavelengths of 340 and 380 nm was recorded every 3 s at room temperature.

Animal experiments [4]
Male Crlj:CD1(ICR) mice were housed in a plastic cage with softwood chip bedding under controlled conditions (12 h light/dark cycle), and fed a standard diet and tap water ad libitum. Aminoglutethimide (AG) was orally administrated to 7-week-old mice at 0, 125, 250, 500 mg/kg/day in 0.5% methylcellulose solution once daily ...

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Body weight and adrenal weight [4]
In Aminoglutethimide (AG)-treated mice, body weight tended to decrease at 500 mg/kg/day, and adrenal weight appeared to increase at 125–500 mg/kg/day, though these changes were not statistically significant (Table 1). In addition, 3 of the 4 animals treated with 500 mg/kg/day were in a moribund state characterized by loss of locomotor activity, hypothermia, and bradypnea, and they were euthanized on Day 3 (data not shown)....

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Dissolved in DMSO and diluted in saline; 150 mg/kg; i.p. injection

Swiss CD1 male and female mice

Absorption, Distribution and Excretion
This product is rapidly and completely absorbed from the gastrointestinal tract. The bioavailability of the tablets is comparable to that of the equivalent dose solution. Following a single oral dose, 34%–54% of the drug is excreted unchanged in the urine within 48 hours, with a further portion excreted as an N-acetyl derivative. Cytadren is rapidly and completely absorbed after oral administration. In six healthy male volunteers, the mean peak plasma concentration of Cytadren was 5.9 μg/mL 1.5 hours after administration of a 250 mg tablet. The bioavailability of the tablets is comparable to that of the equivalent dose solution. Aminoglutethimide can cross the placenta… It is currently unknown whether aminoglutethimide is excreted into breast milk. Following a single oral dose, 34%–54% of the drug is excreted unchanged in the urine within the first 48 hours, with a further portion excreted as an N-acetyl derivative. For more complete data on the absorption, distribution, and excretion of aminoglutethimide (7 types), please visit the HSDB record page. Metabolism/Metabolites: Hepatic metabolism. 34-54% of the administered dose is excreted unchanged in the urine within the first 48 hours, with a further portion excreted as N-acetyl derivatives. Hepatic metabolism; the major metabolite is N-acetylamydimide; acetylation rates may vary genetically between individuals. Four aminoglutethimide metabolites have been identified in the urine of patients taking aminoglutethimide long-term. These metabolites are products of hydroxylation of 3-ethylpiperidine-2,6-dione residues, namely 3-(4-aminophenyl)-3-ethyl-5-hydroxypiperidine-2,6-dione and its acetamido analog 3-(4-aminophenyl)-3-(1-hydroxyethyl)piperidine-2,6-dione, and 3-(4-aminophenyl)-3-(2-carboxamidoethyl)tetrahydrofuran-2-one (a lactone formed by the rearrangement of 3-(4-aminophenyl)-3-(2-hydroxyethyl)piperidine-2,6-dione). These new metabolites are present in lower amounts compared to aminoglumite and the previously identified major metabolites 3-(4-acetamidophenyl)-3-ethylpiperidine-2,6-dione and 3-(4-hydroxyaminophenyl)-3-ethylpiperidine-2,6-dione. Significant species differences exist between rats and humans because almost all metabolites in rat urine are N-acetylated, while most metabolites are not. However, 5-hydroxylation of piperidine dione residues exhibits the same stereoselectivity in both species, yielding only the cis isomer. The synthesized cis-3-(4-aminophenyl)-3-ethyl-5-hydroxypiperidine-2,6-dione does not inhibit the activity of the target enzyme system dehydrogenases and aromatases in vitro; therefore, like other reported metabolites, it is an inactivating product of the drug. A novel aminoluminate metabolite—hydroxyaminoluminate (3-(4-aminophenyl)-3-ethyl-2,6-piperidine dione)—has been identified in the urine of patients who have been taking aminoluminate (3-(4-aminophenyl)-3-ethyl-2,6-piperidine dione) for a long period. This metabolite was separated by reversed-phase thin-layer chromatography and characterized by comparing its mass spectrometric and chromatographic properties with those of the synthesized compound. Hydroxyaminoluminate is unstable; it is readily oxidized to nitrosoluminate and undergoes a disproportionation reaction in mass spectrometry to generate this compound and aminoluminate. In the four patients studied, the metabolite was not detected in urine after the first dose. It was detected in one patient after the second dose, and in two other patients within seven to eight days, suggesting that its formation is drug-induced and may be a contributing factor to the shortened half-life of aminoluminate during long-term treatment. In one patient, metabolite profiles were analyzed using high-performance liquid chromatography (HPLC) after the first dose and six weeks after treatment, indicating that hydroxylamine formation occurred at the expense of the major metabolite, N-acetylglutamine. High-performance liquid chromatography (HPLC) was used to quantify hydroxyaminoglutamine [3-ethyl-3-(4-hydroxyaminophenyl)piperidin-2,6-dione] (HxAG), aminoglutamine [3-(4-aminophenyl)-3-ethylpiperidin-2,6-dione] (AG), and N-acetylaminoglutamine (N-AcAG) in 24-hour urine samples from a patient receiving long-term aminoglutamine therapy without steroid supplementation. Since the [HxAG]/[AG] ratio increased over time, HxAG is a product of the major aminoglutamine-induced metabolic pathway. Conversely, the [N-AcAG]/[AG] ratio decreased over time. A rapid and simple colorimetric method was used to quantify HxAG in the urine of male and female patients taking different doses of AG, demonstrating that induced metabolism is a common phenomenon even at low doses (125 mg twice daily).
Extensive metabolism occurs in all species except dogs and humans, with N-acetaminoglutamine being the major metabolite. In the latter two species, the unchanged drug is the major excretion product. A metabolite previously undetected in human urine, 3-(4-acetaminophenyl)-3-(2-carboxamidoethyl)tetrahydrofuran-2-one, was identified. Long-term administration of aminoglutamine to rats did not cause detectable changes in drug excretion or metabolic patterns. However, long-term administration of phenobarbital reduced the amount of (14)C excreted in urine over 72 hours. The residual (72-hour) (14)C levels in rat, guinea pig, and rabbit tissues were less than 1 microgram (14)C-aminoglutamine equivalent/gram of tissue. At this time, a considerable amount of (14)C remained in canine tissues.
Hepatic metabolism: 34%–54% of the administered dose is excreted unchanged in the urine within the first 48 hours, with a further portion excreted as an N-acetyl derivative. Elimination pathway: Following a single oral administration, 34%-54% is excreted unchanged in the urine within the first 48 hours, with a further portion excreted as an N-acetyl derivative. Half-life: 12.5 ± 1.6 hours. After long-term (2 to 32 weeks) treatment, the duration of treatment is shortened to 7 hours because aminoglutethimide induces liver enzymes and accelerates its own metabolism.

Toxicity Summary
Aminoglutamate reduces the production of D5-pregnenolone and blocks several other steps in steroid synthesis, including C-11, C-18, and C-21 hydroxylation, as well as the hydroxylation required for the aromatization of androgens into estrogens. These hydroxylations are mediated by the binding of aminoglutamate to the cytochrome P-450 complex. Specifically, the drug binds to and inhibits aromatase, which is crucial for the synthesis of estrogens from androstenedione and testosterone. The decrease in adrenocorticotropic hormone (ACTH) secretion from the pituitary gland increases after the adrenocortical corticotropin secretion decreases, thereby counteracting the blocking effect of aminoglutamate on adrenocortical steroid synthesis. Concomitant administration of hydrocortisone can inhibit this compensatory increase in ACTH secretion. Because aminoglutamate accelerates the metabolic rate of dexamethasone but does not affect the metabolic rate of hydrocortisone, the latter is more suitable as an adrenocortical glucocorticoid replacement therapy. Although aminoglutethimide inhibits thyroid hormone synthesis, the compensatory increase in thyroid-stimulating hormone (TSH) is usually sufficient to offset the inhibitory effect of aminoglutethimide on thyroid hormone synthesis. Despite the increase in TSH levels, aminoglutethimide does not cause an increase in prolactin secretion. Protein binding rate: 21-25% Toxicity data: Oral LD50 (mg/kg): Rat, 1800; Dog, >100. Intravenous LD50 (mg/kg): Rat, 156; Dog, >100. Interactions: Aminoglutethimide may inhibit the adrenal response to adrenocorticotropic hormone (ACTH); this may interfere with the therapeutic effect of ACTH. Concomitant use with central nervous system depressants may result in an additive effect of central nervous system depression. Concomitant use with diuretics may cause hyponatremia. Cytadren can accelerate the metabolism of dexamethasone…
For more complete data on interactions of aminoglutethimide (12 in total), please visit the HSDB record page.

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Non-human toxicity values
Intraperitoneal LD50 in mice: 625 mg/kg

[1]. Eur J Med Chem. 2009 Oct;44(10):4121-7;
[2]. Cancer Res. 1982 Aug;42(8 Suppl):3353s-3359s.
[3]. Br J Pharmacol. 2006 Feb 6;147(7):729–736
[4]. Exp Toxicol Pathol. 2017 Sep 5;69(7):424-429.

Therapeutic Uses

Adrenocortical Inhibitor; Antitumor Drug
Aminoglutamine is indicated for temporary adrenal suppression in some patients with Cushing's syndrome, including those with adrenal carcinoma, ectopic adrenocorticotropic hormone (ACTH)-secreting tumors, or adrenal hyperplasia. /Included on US Product Label/
Aminoglutamine is indicated for the treatment of postmenopausal metastatic breast cancer, particularly unresectable or recurrent breast cancer that is hormone-dependent and resistant to tamoxifen, to achieve the effect of "medical adrenalectomy". /Included on US Product Label/
Aminoglutamine is indicated for the treatment of prostate cancer that is unresponsive to hormone or surgical treatment. /Included on US Product Label/
For more complete data on the therapeutic uses of aminoglutamine (7 types), please visit the HSDB record page.
Drug Warnings

Cytadren may cause adrenocortical insufficiency, especially in stressful situations such as surgery, trauma, or acute illness. Patients should be closely monitored and given hydrocortisone and mineralocorticoid supplements as needed. Dexamethasone is contraindicated. Cytadren may also suppress aldosterone production by the adrenal cortex and may cause orthostatic hypotension or persistent hypotension. Blood pressure should be monitored regularly in all patients. Patients should be informed of the possible symptoms of hypotension, such as weakness and dizziness, and what to do if these symptoms occur. Cytadren may cause harm to the fetus in pregnant women. In early experience with approximately 5,000 patients using this drug, two cases of pseudohermaphroditism were reported, involving female infants born to mothers treated with Cytadren… If this drug must be used during pregnancy, or if a patient becomes pregnant while using the drug, the patient should be informed of the potential harm to the fetus. Patients should be warned of the possibility of drowsiness and therefore should not drive, operate potentially dangerous machinery, or engage in other activities that may become dangerous due to decreased alertness. For more complete data on drug warnings for aminoglutethimide (19 in total), please visit the HSDB record page.

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Pharmacodynamics
Aminoglutamate inhibits the enzymatic conversion of cholesterol to D5-pregnenolone, thereby reducing adrenal glucocorticoids, mineralocorticoids, estrogens, and androgens.Aminoglutamate is a clinically available drug that inhibits steroid biosynthesis by inhibiting enzymes such as cytochrome P450scc and aromatase. Since many neurosteroids regulate glutamate receptors, we investigated the effect of aminoglutamate on cell death induced by excessive activation of glutamate receptors in neurons of the central nervous system. Long-term pretreatment of organoid cerebral cortical slice cultures with aminoglutamate (100–1000 μM) for 6 days or longer showed that aminoglutamate concentration-dependent inhibition of NMDA-induced neuronal cell death was achieved. Aminoglutamate (1000 μM) also inhibited the neurotoxicity of AMPA and fucoidan, but had no inhibitory effect on the neurotoxicity of ionomycin or astrococcus. The protective effect of aminoglutethimide against NMDA receptor cytotoxicity cannot be mimicked by other steroid synthesis inhibitors (including tramostan and exemestane) and cannot be reversed by concurrent administration of steroids such as pregnenolone, estrone, 17β-estradiol, and estriol. In in vitro rat cortical cell cultures, long-term administration of aminoglutethimide (10–1000 μM) attenuated NMDA receptor-mediated glutamate cytotoxicity but had no significant effect on glutamate-induced intracellular Ca2+ elevation. Both short-term and long-term pretreatment with aminoglutethimide (30–1000 μM) prevented NMDA receptor-dependent ischemic neuronal injury. Organoid cortical slice cultures were associated with inhibition of glutamate release during ischemic injury. These results suggest that aminoglutethimide (independent of neurosteroid synthesis) can protect central nervous system neurons from excitotoxicity and ischemic injury. The development of aminoglutethimide analogs with neuroprotective properties may have therapeutic value. [3]

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Abstract: Aromatases are enzymes involved in the conversion of androgens to estrogens and are important targets for endocrine therapy of breast cancer. Aromatase inhibition is usually achieved using steroids associated with the catalytic substrate structure or azole nonsteroidal compounds. Substituted androstenedione derivatives with Δ(1), Δ(6), and Δ(1,6) unsaturated bonds and 6-alkyl/6-phenyl aliphatic substitutions are among the most potent steroid aromatase inhibitors known to date. In this paper, we combined the common pharmacophore and shape features of these molecules to construct a new pharmacophore model for virtual screening of large compound databases. We extracted a small subset of the best-fitting anti-aromatase candidate compounds from the NCI database and tested them in an in vitro human placental aromatase assay. We identified new and highly potent aromatase inhibitors, such as compounds 8 and 14. Given that the crystal structures of aromatases are currently unknown, this ligand-based approach is an effective tool for virtual screening of novel aromatase inhibitors. [2]

C13H16N2O2

232.28

232.121

C, 67.22; H, 6.94; N, 12.06; O, 13.78

125-84-8

125-84-8; 23734-88-5 (phosphate); 57344-88-4 [(R)-(+)-Aminoglutethimide L-Tartrate]; 57288-03-6 (S-isomer); 57288-04-7 [S-(-)-Aminoglutethimide D-tartrate]; 62268-19-3 (S-isomer tartrate); 55511-44-9 (R-isomer); 57344-88-4 (R-isomer tartrate);

2145

White to off-white solid powder

1.2±0.1 g/cm3

457.4±45.0 °C at 760 mmHg

152-154 °C(lit.)

230.4±28.7 °C

0.0±1.1 mmHg at 25°C

1.566

1.41

2

3

2

17

321

0

CCC1(C2=CC=C(N)C=C2)CCC(NC1=O)=O

ROBVIMPUHSLWNV-UHFFFAOYSA-N

InChI=1S/C13H16N2O2/c1-2-13(8-7-11(16)15-12(13)17)9-3-5-10(14)6-4-9/h3-6H,2,7-8,14H2,1H3,(H,15,16,17)

3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.76 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 (10.76 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 (10.76 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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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 8 mg/mL

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