Enzalutamide (formerly MDV3100; MDV-3100; trade name: Xtandi) is an orally bioavailable, 2nd-generation and non-steroidal androgen-receptor (AR) antagonist with potential antineoplastic activity. It inhibits AR with an IC50 of 36 nM in LNCaP cells. Enzalutamide belongs to the nonsteroidal antiandrogen (NSAA) class of medication and has been approve for treating prostate cancer. It has higher affinity to the AR compared to the first-generation AR inhibitors. It acts by blocking the binding of androgens to the AR, AR nuclear translocation, and the association of the AR with DNA. The AR is a 919-amino acid member of the steroid receptor transcription factor superfamily with different domains including an N-terminal regulation domain.

Physicochemical Properties

Molecular Formula	C21H16F4N4O2S
Molecular Weight	464.44
Exact Mass	464.093
Elemental Analysis	C, 54.31; H, 3.47; F, 16.36; N, 12.06; O, 6.89; S, 6.90
CAS #	915087-33-1
Related CAS #	N-desmethyl Enzalutamide;1242137-16-1;N-desmethyl Enzalutamide-d6;Enzalutamide carboxylic acid;1242137-15-0;Deutenzalutamide-d3;1443331-82-5;Enzalutamide-d6;1443331-94-9
PubChem CID	15951529
Appearance	White to off-white solid powder
Density	1.5±0.1 g/cm3
Index of Refraction	1.630
LogP	2.13
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	3
Heavy Atom Count	32
Complexity	839
Defined Atom Stereocenter Count	0
InChi Key	WXCXUHSOUPDCQV-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H16F4N4O2S/c1-20(2)18(31)28(12-5-4-11(10-26)15(8-12)21(23,24)25)19(32)29(20)13-6-7-14(16(22)9-13)17(30)27-3/h4-9H,1-3H3,(H,27,30)
Chemical Name	4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-sulfanylideneimidazolidin-1-yl]-2-fluoro-N-methylbenzamide
Synonyms	MDV-3100; MDV3100; MDV 3100; MDV3100; 4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide; Enzalutamide (MDV3100); XTANDI; trade name: Xtandi.
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	AR/androgen-receptor (IC50 = 36 nM in LNCaP cells)[1]
ln Vitro	Enzalutamide (MDV3100) in castration-resistant LNCaP/AR cells had a greater affinity for AR than ICI 176334 (AR-overexpressing) in a competitive assay using 16β-[18F]fluoro-5α-DHT (18-FDHT). On LNCaP/AR prostate cells, ezetimibe had no agonistic effects. Enzalutamide inhibits the activation of transmembrane serine protease 2 (TMPRSS2) and prostate-specific antigen (PSA) by binding to the synthetic androgen R1881, which is present in parental LNCaP cells. The transcriptional activity of the mutant AR protein (W741C, Trp741 altered to Cys) is inhibited by enzalutamide [1]. Additionally, enzalutamide inhibits the recruitment of coactivators and the nuclear translocation of ligand-receptor complexes [2].
ln Vivo	When administered at a dose of 10 mg/kg to castrated male mice harboring LNCaP/AR xenografts, enzalutamide (MDV3100) significantly reduces tumor growth [1]. The pharmacokinetics of enzalutamide show dose-independency when administered orally at dosages ranging from 0.5 to 5 mg/kg [4]. MDV3100/enzalutamide and RD162 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.[1] Enzalutamide Treatment Decreased Tumor Volume, Increased Body Weight, and Induced Apoptosis in a Mouse LNCaP-AR Xenograft Model[2] To study the effects of enzalutamide and bicalutamide in vivo, a mouse xenograft CRPC model was developed using castrated male animals implanted with human LNCaP-AR cells that over-express wild-type AR 25. Animals were administered enzalutamide (1–50 mg/kg/day) or bicalutamide (50 mg/kg/day), and tumor volume and mouse body weight were measured at 2- to 3-day intervals for 28 days. Bicalutamide (50 mg/kg/day) inhibited tumor growth through Day 16 when compared with the vehicle control group. After Day 16, however, tumors in these mice grew continuously up to 154% of baseline by Day 28 (Fig. 3A, Table I). In contrast, enzalutamide (10 mg/kg/day) inhibited tumor growth significantly during the first 6 days of treatment compared with vehicle- and bicalutamide-treated mice (mean ± SE percentage tumor growth relative to baseline: vehicle, 119 ± 5%; enzalutamide 10 mg/kg, 86 ± 6%, bicalutamide 50 mg/kg, 106 ± 8%). By Day 13, enzalutamide treatment resulted in a 19% decrease in tumor volume at doses of 10 mg/kg/day or greater compared with the initial tumor size (Fig. 3A). Some tumors in the enzalutamide-treated groups (1 and 50 mg/kg) decreased in size significantly so as to be beyond the measurement limits (Table I, non-measurable tumors/group). These tumors were not included in further analysis. Tumor volume continued to decrease through Day 24 for the 10 mg/kg/day-enzalutamide group and through the last measured time point at Day 28 for the 50-mg/kg/day group (Table I). Maximal effect on tumor regression relative to initial tumor volumes in each group occurred at Day 28 or beyond of enzalutamide treatment (Fig. 3A). The mean ± SE relative tumor volume decline after 27 days of enzalutamide treatment was 41 ± 7% at 10 mg/kg and 68 ± 13% at 50 mg/kg compared with baseline. In contrast, after 27 days of vehicle or 50 mg/kg bicalutamide treatments, tumor volume increased 54% when compared with the baseline (Table I). Pharmacokinetic analyses[4] The areas under the plasma concentration–time curve (AUC) and the first moment curve (AUMC) were calculated using the linear trapezoidal method, extrapolated to time = infinity. The terminal half-life (T½) was calculated as 0.693/λ, where λ is the slope of the log-linear portion of the concentration–time profile. The systemic clearance (CL), mean residence time (MRT), and the volume of the distribution at steady state (Vss) were calculated as dose/AUC, AUMC/AUC, and MRT·CL, respectively. The extent of absolute oral bioavailability (F) was estimated by dividing the AUC after oral administration by the AUC after intravenous administration of the respective dose. The peak concentration (Cmax) and the time to reach Cmax (Tmax) were read directly from individual plasma concentration–time profiles. The tissue-to-plasma partition coefficient (Kp) for enzalutamide was calculated by dividing the mean AUCtissue by the mean AUCplasma after administration. To obtain the pharmacokinetic parameters above, all plasma and tissue concentration–time profiles were analyzed using a non-compartmental method with non-linear least squares regression using the WinNonlin software
Enzyme Assay	Estimation of hepatic intrinsic clearance of enzalutamide in rat liver microsomes[4] Rat liver microsomes were used to estimate the intrinsic clearance of enzalutamide. A typical reaction mixture (500 µL) consisted of rat liver microsomal protein (final concentration = 0.5 mg protein/mL incubation mixture) and an NADPH regenerating system (final concentrations: 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride) in 100 mM potassium phosphate buffer (pH 7.4). The mixture was pre-incubated in a water bath at 37 °C for 5 min and an aliquot of enzalutamide solution added to a final concentration of 2 µM. Aliquots (50 µL) of the mixture were sampled at 0, 5, 15, and 30 min after initiation of the reaction. Immediately after collection, a stop solution (100 µL ice-chilled methanol) was added to the sample to terminate the reaction. After vigorous vortexing, and centrifuging at 10,000×g for 5 min, an aliquot (50 µL) of the supernatant was assayed. The concentration of enzalutamide remaining in the sample was plotted against the reaction time to determine the metabolic rate constant of the reaction. Estimation of the fraction of enzalutamide bound to plasma protein[4] We conducted a protein-binding study to determine the fraction of unbound enzalutamide in rat plasma. Binding of test material was assessed via equilibrium dialysis using RED® devices. All assessments were made in triplicate. After 200 µL samples of plasma containing 2 µg/mL enzalutamide were placed into a sample chamber, 350 µL of phosphate buffer (pH 7.4) was added to the buffer chamber. The device containing the samples was incubated at 37 °C for 4 h in a shaking water bath. After incubation, enzalutamide in was assayed in plasma and buffer.
Cell Assay	Nuclear Translocation Assay[3] The yellow fluorescent protein (YFP)-AR plasmid was donated by Marc I. Diamond and was stably transfected into HEK293 cells. Cells were seeded at 1.5 × 105 cells/cm2 in optical microplates in phenol red-free DMEM/F12 medium supplemented with 10% hormone-depleted FBS. After 2 days of cultivation, the cells were pre-treated with enzalutamide (1 µM) or bicalutamide (1 µM) for 2 hr and then co-treated with 1 nM DHT for 1 hr in the presence of enzalutamide or bicalutamide. Cells were then washed in phosphate-buffered saline, incubated with the nuclear fluorescent marker DAPI (1 µg/ml) for 30 min and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were visualized using a Qimaging digital camera coupled to an Olympus X71 fluorescence microscope using a YFP filter. Nuclear and total cellular AR-YFP fluorescence intensities (integrated density) were quantified using ImageJ software. Nuclear AR-YFP fluorescence was quantified in areas defined by image segmentation based on DAPI fluorescence, and the nuclear:total intensity ratio calculated. At least 14 cells were quantified per condition per independent experiment (n = 3). For live cell imaging experiments, cells were pre-treated with enzalutamide (1 or 10 µM) or bicalutamide (1 or 10 µM) for 2 hr and then co-treated with 1 nM DHT for 3 hr in the presence of enzalutamide or bicalutamide. Cells were imaged immediately before DHT addition (t′ = 0) and then at 60-min intervals over 3 hr.
Animal Protocol	Mouse Xenograft Model[3] Following a 5-day acclimation period, 5- to 9-week-old male CB17SCID mice were castrated and allowed to recover for an additional 5 days before inoculation with tumor cells. LNCaP cells co-expressing exogenous AR and the AR-dependent reporter construct ARR2-Pb-Luc were used to generate a xenograft model of human prostate cancer. Before implantation, LNCaP-AR-Lux cells were prepared by the addition of trypsin-EDTA, washed with complete medium, collected and resuspended at 20 × 106 cells/ml. Cell suspensions were diluted with Matrigel to 2 × 106 cells/0.2 ml and delivered subcutaneously in the suprascapular region. Tumor growth was monitored to the volume of 100 mm3 when treatment began (∼80 days). The observed rate of tumor take with LNCaP-AR-Lux cells is between 70% and 80%. Body weight and tumor volumes (width2 × length/2) were measured two to three times per week with a digital caliper, and the average tumor volumes were determined. Test drugs were diluted in Tween 80:PEG 400, and stored at 4°C until administration by oral gavage. Each group of mice (n = 7) was treated daily for 28 consecutive days with 1, 10, or 50 mg/kg enzalutamide, vehicle control, or 50 mg/kg bicalutamide. At the end of the treatment period or when tumor volume exceeded 1,000 mm3, animals were euthanized and blood and tissue samples were collected for analysis. Intravenous and oral administration of enzalutamide in rats[4] Enzalutamidewas dissolved in vehicle (10 % DMSO, 45 % polyethylene glycol 400, and 45 % saline) . As a single dose, the administration routes were an intravenous bolus via the tail vein (n = 3) and an oral gavage dose (n = 4). Dosing volume was 1 mL per kg (body weight) and the dosing range was 0.5, 2, and 5 mg/kg. The use of 2 mg/kg was reported in a previous study (Song et al. 2014). A blood sample (200 µL) was collected from the jugular vein using a heparinized syringe to ensure anticoagulation at 0.08 (intravenous only), 0.33, 1, 3, 6, 10, 24, 48, and 72 h after dosing. During blood sampling, rats were placed in a restrainer. To prepare plasma samples, all blood samples were centrifuged at 13,500×g for 5 min. The samples were stored at −20 °C until analyzed. Determination of urinary and fecal excretion[4] Male SD rats (n = 3) were administered enzalutamide through the tail vein (intravenous) and by oral gavage at 1 mg/kg and were kept in metabolic cages after dosing. Urine and feces samples were collected over the following time intervals after dosing: 0–2, 2–4, 4–6, 6–10, 10–24, 24–48, and 48–72 h. The metabolic cages were rinsed with distilled water, and residues were added to the urine samples at 72 h. To extract the enzalutamide present in the feces, samples were shaken vigorously for 12 h with 50 % methanol. Formulated in 1% carboxymethyl cellulose, 0.1% Tween-80, 5% DMSO; 10 mg/kg; Oral gavage Castration-resistant LNCaP/HR xenografts in male SCID mice
ADME/Pharmacokinetics	Absorption, Distribution and Excretion The median Tmax is 1 hour (0.5 to 3 hours) following a single 160 mg dose of capsules and 2 hours (0.5 to 6 hours) following a single 160 mg dose of tablets. Enzalutamide achieves steady-state by Day 28 and its AUC accumulates approximately 8.3-fold relative to a single dose. At steady-state, the mean (%CV) maximum concentration (Cmax) for enzalutamide and N-desmethyl enzalutamide is 16.6 µg/mL (23%) and 12.7 µg/mL (30%), respectively, and the mean (%CV) minimum concentrations (Cmin) are 11.4 µg/mL (26%) and 13.0 µg/mL (30%), respectively. Enzalutamide is primarily eliminated by hepatic metabolism. 71% of the dose is recovered in urine (including only trace amounts of enzalutamide and N-desmethyl enzalutamide), and 14% is recovered in feces (0.4% of the dose as unchanged enzalutamide and 1% as N-desmethyl enzalutamide). The mean (%CV) volume of distribution after a single oral dose is 110 L (29%). The mean apparent clearance (CL/F) of enzalutamide after a single dose is 0.56 L/h (0.33 to 1.02 L/h). Metabolism / Metabolites Enzalutamide is metabolized by CYP2C8 and CYP3A4. CYP2C8 is primarily responsible for the formation of the active metabolite (N-desmethyl enzalutamide). Carboxylesterase 1 metabolizes N-desmethyl enzalutamide and enzalutamide to the inactive carboxylic acid metabolite. Biological Half-Life The mean terminal half-life (t1/2) for enzalutamide in patients after a single oral dose is 5.8 days (range 2.8 to 10.2 days). Following a single 160 mg oral dose of enzalutamide in healthy volunteers, the mean terminal t1/2 for N-desmethyl enzalutamide is approximately 7.8 to 8.6 days. Researchers characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5-5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 80.4-86.3 mL/h/kg, 1020-1250 mL/kg, and 9.13-10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5-5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism. [4]
Toxicity/Toxicokinetics	Hepatotoxicity In preregistration controlled trials, serum aminotransferase elevations occurred in up to 10% patients treated with enzalutamide, but similar somewhat high rates occurred in patients receiving placebo (~9%). The liver test abnormalities were generally mild, transient and not associated with symptoms or jaundice. ALT elevations above 5 times the ULN were rare (0.2%) and also no more frequent than with placebo therapy. In addition, clinically apparent liver injury with jaundice was not reported in the preregistration trials of enzalutamide, and clinically apparent liver injury and hepatitis are not mentioned in the product label. Since the approval and more wide scale use of enzalutamide, there have been no publications or descriptions of the clinical features of hepatotoxicity with jaundice associated with its use. Thus, clinically apparent liver injury due to enzalutamide must be rare, if it occurs at all. Likelihood score: E (unlikely cause of clinically apparent liver injury). Protein Binding Enzalutamide is 97% to 98% bound to plasma proteins, primarily albumin. N-desmethyl enzalutamide is 95% bound to plasma proteins.
References	[1]. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science, 2009, 324 (5928), 787-790. [2]. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet, 2010, 375(9724), 1437-1446. [3]. Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer. Prostate. 2013 Sep;73(12):1291-305. [4]. Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats. Arch Pharm Res. 2015 Nov;38(11):2076-82.
Additional Infomation	Pharmacodynamics Enzalutamide is a second-generation antiandrogen that blocks the activity of androgen and androgen receptor (AR) in prostate cancer. AR activity and prostate cancer progression are closely related due to the normal physiology of prostate cells, providing the rationale for androgen deprivation therapy (ADT). However, resistance will eventually arise after the commencement of ADT in 2-3 years due to the accumulation of mutations, including constitutively active mutation, AR overexpression, and changes in AR splicing variants. Enzalutamide was therefore designed to exploit these mutations. In vitro experiments in human prostate cancer cell line VCaP showed that enzalutamide can suppress cell growth and induce apoptosis while other antiandrogens like bicalutamide did not. Clinical trials on prostate cancer patients indicated that enzalutamide can lead to a decrease in serum PSA for at least 12 weeks, although this response can be short-lived and thus resulting in enzalutamide resistance. Patients receiving enzalutamide also had a 37% decreased in the risk of death compared to placebo. Metastatic prostate cancer is treated with drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease called castration-resistant prostate cancer, driven by elevated expression of the androgen receptor. Here we characterize the diarylthiohydantoins RD162 and MDV3100, two compounds optimized from a screen for nonsteroidal antiandrogens that retain activity in the setting of increased androgen receptor expression. Both compounds bind to the androgen receptor with greater relative affinity than the clinically used antiandrogen bicalutamide, reduce the efficiency of its nuclear translocation, and impair both DNA binding to androgen response elements and recruitment of coactivators. RD162 and MDV3100 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.[1] Background: MDV3100 is an androgen-receptor antagonist that blocks androgens from binding to the androgen receptor and prevents nuclear translocation and co-activator recruitment of the ligand-receptor complex. It also induces tumour cell apoptosis, and has no agonist activity. Because growth of castration-resistant prostate cancer is dependent on continued androgen-receptor signalling, we assessed the antitumour activity and safety of MDV3100 in men with this disease. Methods: This phase 1-2 study was undertaken in five US centres in 140 patients. Patients with progressive, metastatic, castration-resistant prostate cancer were enrolled in dose-escalation cohorts of three to six patients and given an oral daily starting dose of MDV3100 30 mg. The final daily doses studied were 30 mg (n=3), 60 mg (27), 150 mg (28), 240 mg (29), 360 mg (28), 480 mg (22), and 600 mg (3). The primary objective was to identify the safety and tolerability profile of MDV3100 and to establish the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT00510718. Findings: We noted antitumour effects at all doses, including decreases in serum prostate-specific antigen of 50% or more in 78 (56%) patients, responses in soft tissue in 13 (22%) of 59 patients, stabilised bone disease in 61 (56%) of 109 patients, and conversion from unfavourable to favourable circulating tumour cell counts in 25 (49%) of the 51 patients. PET imaging of 22 patients to assess androgen-receptor blockade showed decreased (18)F-fluoro-5alpha-dihydrotestosterone binding at doses from 60 mg to 480 mg per day (range 20-100%). The median time to progression was 47 weeks (95% CI 34-not reached) for radiological progression. The maximum tolerated dose for sustained treatment (>28 days) was 240 mg. The most common grade 3-4 adverse event was dose-dependent fatigue (16 [11%] patients), which generally resolved after dose reduction. Interpretation: We recorded encouraging antitumour activity with MDV3100 in patients with castration-resistant prostate cancer. The results of this phase 1-2 trial validate in man preclinical studies implicating sustained androgen-receptor signalling as a driver in this disease. [2] Background: Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR-dependent gene expression and cell apoptosis. Methods: The expression of AR target gene prostate-specific antigen (PSA) was measured in LnCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of CRPC. Differential gene expression in LNCaP cells was measured using Affymetrix human genome microarray technology. Results: We found that unlike bicalutamide, enzalutamide lacked AR agonistic activity at effective doses and did not induce PSA expression or AR nuclear translocation. Additionally, it is more effective than bicalutamide at inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced the regression of tumor volume in a CRPC xenograft model and apoptosis in AR-over-expressing prostate cancer cells. Finally, gene expression profiling in LNCaP cells indicated that enzalutamide opposes agonist-induced changes in genes involved in processes such as cell adhesion, angiogenesis, and apoptosis. Conclusions: These results indicate that enzalutamide efficiently inhibits AR signaling, and we suggest that its lack of AR agonist activity may be important for these effects.[3]

Solubility Data

Solubility (In Vitro)

DMSO: 92 mg/mL (198.1 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.38 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.38 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. Solubility in Formulation 3: 2.5 mg/mL (5.38 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 4: 15% DMSO +85% PEG 300 : 10mg/mL Solubility in Formulation 5: 10 mg/mL (21.53 mM) in 1% Tween-80 in PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1531 mL	10.7657 mL	21.5313 mL
	5 mM	0.4306 mL	2.1531 mL	4.3063 mL
	10 mM	0.2153 mL	1.0766 mL	2.1531 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.