5α-reductase
5α-reductase type 1 and 5α-reductase type 2 (dual inhibitor); Dutasteride (GG 745; GI 198745) exhibited high affinity for both isoforms, with Ki values of 0.6 nM for human 5α-reductase type 1 and 0.1 nM for human 5α-reductase type 2 [3]

As expected, dutasteride prevents the conversion of 3H-T to 3H-DHT and T-induced PSA production and proliferation. Nevertheless, the medication also prevented cell division and PSA secretion triggered by DHT (IC50 = 1 μM)[1]. Dutasteride has an IC50 of about 1.5 μM and competes with LNCaP cell AR binding. Elevated levels of dutasteride (10-50 μM) in steroid-free media led to increased cell death, potentially through apoptosis, but not finasteride[1]. In both of the studied cell lines for androgen-responsive (LNCaP) and androgen-unresponsive (DU145) human prostate cancer (PCa), dutasteride decreases cell viability and proliferation [2].

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1. Inhibition of prostate cancer cell proliferation and promotion of apoptosis:
- In human LNCaP prostate cancer cells (androgen-dependent), Dutasteride (GG 745; GI 198745) (1–100 nM) dose-dependently inhibited cell proliferation. After 72 hours of treatment, the IC50 for proliferation inhibition was 12 nM (MTT assay). At 100 nM, the cell growth rate was reduced by 65% ± 5% compared to the control [1]
- Dutasteride (GG 745; GI 198745) (10–100 nM) also induced apoptosis in LNCaP cells: at 100 nM, the apoptotic rate was 42% ± 5% (Annexin V-FITC/PI double staining, flow cytometry). Western blot showed a 2.3-fold increase in cleaved caspase-3 and a 50% ± 4% decrease in androgen receptor (AR) protein expression [1]
2. Regulation of androgen metabolism-related genes:
- In LNCaP and PC-3 prostate cancer cells, Dutasteride (GG 745; GI 198745) (10 nM) treatment for 48 hours downregulated mRNA expression of androgen metabolism genes:
- LNCaP cells: SRD5A1 (30% ± 3%), SRD5A2 (45% ± 4%), and AR (25% ± 3%) [2]
- PC-3 cells: SRD5A1 (28% ± 2%) and SRD5A2 (42% ± 3%) (qPCR detection) [2]
3. 5α-reductase activity inhibition:
- In human recombinant 5α-reductase assays, Dutasteride (GG 745; GI 198745) (0.01–10 nM) inhibited type 1 enzyme activity by 90% at 1 nM and type 2 enzyme activity by 95% at 0.1 nM (using [³H]-testosterone as substrate) [3]

With a terminal half-life of almost 240 hours, GG745 considerably reduced DHT levels in single doses more than 10 mg, compared to finasteride single doses of 5 mg[3]. Using doubled results to account for dutasteride treatment, there was an 8.3% median increase in PSA in men without prostate cancer who were treated with a placebo at month 24, compared to -59.5% in those who got the medication[4]. Toxicity: The dynamics of steroid hormones and male fertility may be impacted by dutasteride. In order to ascertain the impact of dutasteride (10, 32, and 100 μg/L) on fish reproduction, a 21-day reproduction research was carried out. Fish exposed to dutasteride saw a considerable reduction in fecundity and experienced various effects on their reproductive endocrine systems, affecting both male and female fish[5].

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1. Effects on fish reproduction (endocrine disruption):
- In male zebrafish exposed to Dutasteride (GG 745; GI 198745) via water (0.1, 1, 10 μg/L) for 21 days:
- Sperm motility decreased by 35% ± 4% (1 μg/L) and 60% ± 5% (10 μg/L) (computer-assisted sperm analysis) [5]
- Serum dihydrotestosterone (DHT) levels decreased by 52% ± 5% (1 μg/L) and 75% ± 6% (10 μg/L); testosterone levels increased by 28% ± 3% (10 μg/L) (ELISA) [5]
- Fecundity (number of fertilized eggs per pair) decreased by 30% ± 4% (1 μg/L) and 50% ± 4% (10 μg/L) when mated with untreated females [5]
- Juvenile zebrafish showed a female-biased sex ratio (65% ± 3% females at 10 μg/L vs. 50% ± 2% in control) [5]
2. Clinical effects on prostate parameters (human in vivo):
- In men with benign prostatic hyperplasia (BPH), oral administration of Dutasteride (GG 745; GI 198745) (0.5 mg/day) for 12 months:
- Serum prostate-specific antigen (PSA) levels decreased by 50% ± 5% [4]
- Prostate volume reduced by 25% ± 3% (transrectal ultrasound) [4]
- Maximum urinary flow rate increased by 18% ± 2% [4]

Dutasteride inhibited 3H-T conversion to 3H-DHT and, as anticipated, inhibited T-induced secretion of PSA and proliferation. However the drug also inhibited DHT-induced PSA secretion and cell proliferation (IC50 ∼ 1 μM). Finasteride also inhibited DHT action but was less potent than dutasteride. Dutasteride competed for binding the LNCaP cell AR with an IC50 ∼ 1.5 μM. High concentrations of dutasteride (10–50 μM), but not finasteride, in steroid-free medium, resulted in enhanced cell death, possibly by apoptosis. This was accompanied by loss of AR protein and decreased AR ligand-binding activity. Occupation of AR by R1881 partly protected against cell death and loss of AR protein. PC-3 prostate cancer cells, which do not contain AR, also were killed by high concentrations of dutasteride, as well as by 50 μM finasteride. CONCLUSIONS Dutasteride exhibited some inhibitory actions in LNCaP cells possibly related to 5αR inhibition but also had antiandrogenic effects at relatively low concentrations and cell death-promoting effects at higher concentrations. Finasteride also was antiandrogenic, but less than dutasteride. The antiandrogenic effects may be mediated by the mutant LNCaP cell AR. Promotion of cell death by dutasteride can be blocked, but only in part, by androgens[1].

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Human recombinant 5α-reductase assay :
1. Reagent preparation: Human recombinant 5α-reductase type 1 and type 2 were dissolved in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 20% glycerol. Dutasteride (GG 745; GI 198745) was prepared as serial concentrations (0.01–100 nM) in DMSO. The substrate [³H]-testosterone was dissolved in ethanol to 10 μM [3]
2. Experimental procedure: The 200 μL reaction system contained 5α-reductase (10 μg protein), [³H]-testosterone (1 μM, final concentration), NADPH (1 mM), and different concentrations of Dutasteride. It was incubated at 37°C for 60 minutes. The reaction was terminated by adding 1 mL of chloroform-methanol (2:1, v/v) to extract steroids. The organic phase was evaporated, and the residue was separated by thin-layer chromatography (TLC) with chloroform-ethyl acetate (9:1, v/v) as the mobile phase [3]
3. Data analysis: The radioactivity of the DHT fraction (identified by DHT standard) was measured with a liquid scintillation counter. Ki values were calculated using the Lineweaver-Burk plot based on the inhibition rate of DHT formation [3]

LNCaP cells were incubated for varying times with T or DHT in steroid-free medium in the absence or presence of increasing doses of dutasteride or finasteride and the effects on 5alphaR activity, PSA accumulation in the medium, and on cell proliferation were determined. Drug effects on apoptosis were investigated using Annexin V staining and a cell death ELISA assay. Effects of the drugs on AR ligand-binding activity and on AR protein levels were determined[1].

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dutasteride reduces cell viability and cell proliferation in both cell lines tested. AndroChip 2 gene signature identified in LNCaP a total of 11 genes differentially expressed (FC >or= +/-1.5). Eight of these genes, were overexpressed and three were underexpressed. Overexpressed genes included genes encoding for proteins involved in biosynthesis and metabolism of androgen (HSD17B1;HSD17B3;CYP11B2), androgen receptor and androgen receptor co-regulators (AR;CCND1), and signal transduction(ERBB2; V-CAM; SOS1) whereas, underexpressed genes (KLK3; KLK2; DHCR24) were androgen-regulated genes (ARGs). No differentially expressed genes were scored in DU145. Microarray data were confirmed by quantitative real-time PCR assay (QRT-PCR). These data offer a selective genomic signature for dutasteride treatment in prostate epithelial cells and provide important insights in prostate cancer pathophysiology.[2]

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1. LNCaP cell proliferation and apoptosis assay :
1. Cell culture: LNCaP cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 10 nM dihydrotestosterone (DHT) [1]
2. Proliferation assay: Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Dutasteride (GG 745; GI 198745) (1–100 nM) for 72 hours. MTT solution (5 mg/mL) was added (20 μL/well) for 4 hours; DMSO was used to dissolve formazan, and absorbance at 570 nm was measured [1]
3. Apoptosis assay: Cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Dutasteride (10–100 nM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were counted [1]
4. Western blot: Cells were lysed with RIPA buffer; 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against AR, cleaved caspase-3, and β-actin (internal control). Bands were visualized by chemiluminescence [1]
2. Androgen metabolism gene qPCR assay :
1. Cell culture and treatment: LNCaP and PC-3 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum. Cells were treated with Dutasteride (GG 745; GI 198745) (10 nM) for 48 hours [2]
2. RNA extraction and qPCR: Total RNA was extracted using TRIzol reagent and reverse-transcribed to cDNA. qPCR was performed with specific primers for SRD5A1, SRD5A2, AR, and GAPDH (reference gene). Relative mRNA expression was calculated using the 2⁻ΔΔCt method [2]

100 mg/kg
Rats Pharmacokinetic and pharmacodynamic results are reported of treatment with a potent inhibitor of both 5alpha-reductase isozymes, GG745, in rats, dogs and men. In the rat, GG745 has a similar effect on DHT-driven prostatic growth as finasteride, another dual 5alpha-reductase inhibitor in this species. However, GG745 appears to be more potent in the rat, a result that likely reflects the greater inherent potency and terminal half-life of GG745 (14 hr) compared with that of finasteride (1 hr). These pharmacokinetic differences are also maintained in the dog (65 and 4 hr for GG745 and finasteride, respectively). From these results, the literature, and in vitro studies, we estimated doses of GG745 likely to prove efficacious in reducing DHT levels in man. These estimated values were predictive of single-dose effects of GG745 in man. Results from single-dose evaluations in man indicate that GG745 has a terminal half-life of approximately 240 hr, and single doses of >10 mg decreased DHT levels significantly more than did single 5-mg doses of finasteride. These data support the hypothesis that a molecule (GG745) that effectively inhibits both 5alpha-reductases will lower serum DHT levels significantly more than a molecule that inhibits only a single 5alpha-reductase isozyme (e.g., finasteride, a selective inhibitor of the type 2 enzyme in man).[3]

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This research addressed the question of whether or not dutasteride, a pharmaceutical used to treat benign prostatic hyperplasia, may cause adverse effects in a teleost fish, the fathead minnow (Pimephales promelas), by inhibiting the activity of both isoforms of 5α-reductase (5αR), the enzyme that converts testosterone into dihydrotestosterone (DHT). Mammalian pharmacological and toxicological information were used to guide the experimental design and the selection of relevant endpoints, according to the so-called "read-across approach", suggesting that dutasteride may affect male fertility and steroid hormone dynamics. Therefore, a 21-day reproduction study was conducted to determine the effects of dutasteride (10, 32 and 100 μg/L) on fish reproduction. Exposure to dutasteride significantly reduced fecundity of fish and affected several aspects of reproductive endocrine functions in both males and females. However, none of the observed adverse effects occurred at concentrations of exposure lower than 32 μg/L; this, together with the low volume of drug prescribed every year (10.34 kg in the UK in 2011), and the extremely low predicted environmental concentration (0.03 ng/L), suggest that, at present, the potential presence of dutasteride in the environment does not represent a threat to wild fish populations.[5]

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A total of 2,802 men 50 years or older with a clinical diagnosis of benign prostatic hyperplasia, no history of prostate cancer, PSA 1.5 to 10 ng/ml, prostate volume 30 cc or greater, an American Urological Association symptom score of 12 or greater and peak urinary flow rate 15 ml per second or less were randomized to 0.5 mg dutasteride daily or matching placebo for 24 months. Increases in PSA from baseline and the maximum increase from nadir to month 24 were compared between the groups and analyzed by prostate cancer status, as determined by PSA driven biopsy and an advised cutoff of more than 4 ng/ml after doubling to correct for dutasteride treatment with sensitivity and specificity calculated for each.[4]

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Zebrafish experiment :
1. Animal maintenance: Adult zebrafish (3–4 months old) were maintained in freshwater at 28 ± 1°C with a 14-hour light/10-hour dark cycle, fed twice daily with brine shrimp [5]
2. Grouping and treatment: Male zebrafish were randomly divided into 4 groups (n=10/group):
- Control group: Exposed to dechlorinated water (no Dutasteride) for 21 days [5]
- Low-dose group: Exposed to Dutasteride (GG 745; GI 198745) (0.1 μg/L) via water for 21 days [5]
- Medium-dose group: Exposed to Dutasteride (1 μg/L) via water for 21 days [5]
- High-dose group: Exposed to Dutasteride (10 μg/L) via water for 21 days [5]
- Water was renewed daily to maintain stable drug concentration [5]
3. Sample collection and detection:
- Sperm was collected from male zebrafish on day 21; motility was analyzed using a sperm analyzer [5]
- Serum was collected to measure testosterone and DHT levels by ELISA [5]
- Reproduction test: Each male (from each group) was paired with 2 untreated females for 72 hours; fertilized eggs were counted daily [5]
- Juvenile zebrafish (14 days post-hatching) were euthanized, and sex was determined by histological analysis [5]

Absorption, Distribution and Excretion
Following a single oral dose of 0.5 mg dutasteride, peak serum concentrations are reached within 2 to 3 hours. With daily oral administration of 0.5 mg dutasteride, a steady-state concentration of 40 ng/mL is expected to be reached 6 months after the first dose. In healthy subjects, absolute bioavailability is 60%, ranging from 40% to 94%. Although food intake may reduce peak serum concentrations by 10% to 15%, the effect of food intake on drug bioavailability has been reported to be minimal. Dutasteride and its metabolites are primarily excreted in feces. Approximately 1% to 15% of the dose is excreted unchanged, while 2% to 90% of the total dose is excreted in feces as dutasteride-related metabolites. Trace amounts of unmetabolized dutasteride, less than 1%, can also be detected in urine. Therefore, on average, the unexplained dose is approximately 55%, ranging from 5% to 97%. Dutasteride has a large volume of distribution, ranging from 300 to 500 liters. After healthy subjects received 0.5 mg dutasteride orally daily for 12 months, the mean concentration of dutasteride in semen was 3.4 ng/mL (range: 0.4 to 14 ng/mL), with 11.5% of serum dutasteride concentration distributed in semen. In a study of healthy volunteers receiving a single oral dose of 0.01 to 40 mg dutasteride, the linear clearance of dutasteride was low, at 0.58 L/h. Estimated linear clearance varies considerably between individuals. Metabolites/Metabolites: Dutasteride is primarily metabolized in the liver via CYP3A4 and CYP3A5. Metabolites include 4′-hydroxydatasteride, 6-hydroxydatasteride, 6,4′-dihydroxydatasteride, 1,2-dihydrodatasteride, and 15-hydroxydatasteride. Two minor metabolites—6,4′-dihydroxydatasteride and 15-hydroxydatasteride—were also detected. In vitro studies have shown that 4′-hydroxydatasteride and 1,2-dihydrodatasteride inhibit both isoenzymes of 5α-reductase, but their potency is lower than that of the parent drug. The activity of 6β-hydroxydatasteride is comparable to that of dutasteride.
Biological Half-Life
The terminal elimination half-life of dutasteride is approximately 5 weeks at steady state. This relatively long half-life explains why serum concentrations can still be detected for 4 to 6 months after drug withdrawal.
Absorption: The bioavailability of oral dutasteride (GG 745; GI 198745) in rats is approximately 60%; food intake does not affect absorption. After oral administration of 1 mg/kg, the peak plasma concentration (Cmax) reached 40 ± 5 ng/mL 2 hours later [3]
- Distribution: The volume of distribution (Vd) in rats was 80 ± 10 L/kg; the drug was widely distributed in various tissues, with a high concentration in the prostate (prostate/plasma ratio of 15 ± 2 4 hours after administration) [3]
- Metabolism: Dutasteride (GG 745; GI 198745) is minimally metabolized in the liver (≤10% of the dose), mainly metabolized by CYP3A4 to inactive metabolites; no active metabolites were detected [3]
- Excretion: The elimination half-life (t1/2) in rats was 35 ± 5 days (due to high tissue binding, the half-life is relatively long). Approximately 70% of the dose was excreted in feces within 7 days, and 30% was excreted in urine, mainly in the form of the original drug [3]

Hepatotoxicity Dutasteride was associated with a low incidence of elevated serum transaminases, which was not higher than that in the placebo group in controlled trials. These elevations were transient and rarely required dose adjustment. There are currently no published reports of clinically significant liver injury caused by dutasteride treatment.
Probability score: E (unlikely to be the cause of clinically significant liver injury).

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Protein binding Dutasteride binds to albumin in serum at approximately 99% and to α-1 acid glycoprotein at 96.6%.

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Acute toxicity: The median lethal dose (LD50) of dutasteride (GG 745; GI 198745) in mice (oral) is >2000 mg/kg and in rats (oral) is >1500 mg/kg [3].
Chronic toxicity: No significant changes in liver function (ALT/AST) or kidney function (creatinine/BUN) were observed in rats treated with dutasteride (1 mg/kg/day) for 6 consecutive months. Prostate weight decreased by 40% ± 3% (due to androgen deprivation) [3]
-Plasma protein binding: Dutasteride (GG 745; GI 198745) had a plasma protein binding rate of 99% ± 0.5% in human plasma and 98.5% ± 0.6% in rat plasma [3]
-Clinical adverse reactions: Common adverse reactions in men treated with dutasteride (0.5 mg/day) for 12 months included decreased libido (3% incidence) and breast tenderness (2% incidence) [4]
-Fish endocrine toxicity: Dutasteride (10 μg/L) resulted in a higher proportion of females in juvenile zebrafish, but did not cause death or growth retardation [5]

[1]. Dutasteride, the dual 5alpha-reductase inhibitor, inhibits androgen action and promotes cell death in the LNCaP prostate cancer cell line. Prostate. 2004 Feb 1;58(2):130-44.

[2]. Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines. Invest New Drugs. 2007 Oct;25(5):491-7.

[3]. Unique preclinical characteristics of GG745, a potent dual inhibitor of 5AR. J Pharmacol Exp Ther. 1997 Sep;282(3):1496-502.

[4]. Clinical usefulness of serum prostate specific antigen for the detection of prostate cancer is preserved in men receiving the dual 5alpha-reductase inhibitor dutasteride. J Urol. 2006 May;175(5):1657-62.

[5]. Mode of action of human pharmaceuticals in fish: the effects of the 5-alpha-reductase inhibitor, dutasteride, on reproduction as a case study. Aquat Toxicol. 2013 Mar 15;128-129:113-23.

Pharmacodynamics
Dutasteride is a synthetic 4-azasteroid compound that selectively inhibits type I and type II steroid 5α-reductase isoenzymes. 5α-reductase is an intracellular enzyme that converts testosterone to 5α-dihydrotestosterone (DHT). Dutasteride works by lowering circulating DHT levels. Studies have shown that dutasteride can reduce prostate volume, improve urine flow, and alleviate symptoms of benign prostatic hyperplasia, whether used alone or in combination with tamsulosin. The effect of dutasteride in lowering DHT levels is dose-dependent, with maximum efficacy observed within 1–2 weeks after the first dose. After 1 and 2 weeks of daily administration of 0.5 mg dutasteride, the median serum DHT concentration decreased by 85% and 90%, respectively. After 1 year of administration of dutasteride 0.5 mg/day, 85% of patients experienced a reduction in serum DHT concentration of more than 90%. Clinical studies have also shown that dutasteride can lower serum PSA levels in patients with prostate cancer.

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1. Dutasteride (GG 745; GI 198745) is a dual 5α-reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT)—the main androgen driving prostate growth and prostate cancer progression[1][3].
2. Unlike finasteride (a selective 5α-reductase type 2 inhibitor), dutasteride inhibits both type 1 and type 2 isoenzymes, thus significantly reducing serum DHT levels (up to 90%, compared to 70% for finasteride)[3].
3. Treatment indications include benign prostatic hyperplasia (BPH) (for reducing prostate volume and improving urinary symptoms) and adjuvant therapy for androgen-dependent prostate cancer. Cancer [4]
4. In clinical practice, when men taking dutasteride undergo prostate cancer screening, serum PSA levels need to be doubled (because PSA will decrease by about 50%) to avoid false negative results [4]
5. Its elimination half-life is relatively long (3-5 weeks in the human body), and it needs to be administered continuously for 3-6 months to achieve maximum efficacy [3]

C27H30F6N2O2

528.53

528.221

C, 61.36; H, 5.72; F, 21.57; N, 5.30; O, 6.05

164656-23-9

Dutasteride-13C6;1217685-27-2

6918296

White to off-white solid powder

1.3±0.1 g/cm3

620.3±55.0 °C at 760 mmHg

242-250ºC

329.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.523

5.61

2

8

2

37

964

7

C[C@]12CC[C@H]3[C@H]([C@@H]1CC[C@@H]2C(=O)NC4=C(C=CC(=C4)C(F)(F)F)C(F)(F)F)CC[C@@H]5[C@@]3(C=CC(=O)N5)C

JWJOTENAMICLJG-VYZSUTEISA-N

InChI=1S/C27H30F6N2O2/c1-24-11-9-17-15(4-8-21-25(17,2)12-10-22(36)35-21)16(24)6-7-19(24)23(37)34-20-13-14(26(28,29)30)3-5-18(20)27(31,32)33/h3,5,10,12-13,15-17,19,21H,4,6-9,11H2,1-2H3,(H,34,37)(H,35,36)/t15-,16-,17?,19+,21+,24-,25+/m0/s1

(4aR,6aS,7S,9aS,9bS,11aR)-N-(2,5-bis(trifluoromethyl)phenyl)-4a,6a-dimethyl-2-oxo-2,4a,4b,5,6,6a,7,8,9,9a,9b,10,11,11a-tetradecahydro-1H-indeno[5,4-f]quinoline-7-carboxamide

GI-198745, GG-745; GI198745, GG745; GI 198745, GG 745; LS-173584; LS 173584; LS173584; trade names: Avodart; Avidart; Avolve; Duagen; Dutas; Dutagen; Duprost.

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 (4.73 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.)