GLS1 (IC50 = 31 nM)

GLS-1, also known as kidney-type or KGA, and GLS-2, also known as liver-type or LGA, are the two known isoforms of glutaminase. GLS-2 expression seems to be mostly restricted to the liver, whereas GLS-1 is widely expressed. IPN60090 has no effect against GLS-2, with an IC50 of >50000 nM, and inhibits purified recombinant human GLS-1 (GAC isoform) with an IC50 of 31 nM in a dual-coupled enzyme assay[2]. IPN60090 has an IC50 of 26 nM and prevents A549 cell proliferation[2].

IPN60090 (3 mg/kg for i.v.; 10 mg/kg for p.o.) exhibits exceptional pharmacokinetic characteristics, including CL=4.1 mL/min/kg, t1/₂=1 hour, Cmax=19 μM, and F%=89%[2]. IPN-60090 (oral administration; 100 mg/kg; twice daily; 30 days) exhibits comparable effectiveness and engagement with the target as CB-839 when taken orally at a dose of 250 mg/kg twice a day. Furthermore, for the upcoming model study, the tolerated dose of IPN-60090 is 100 mg/kg BID[2]. IPN-60090 (oral administration; 100 mg/kg; twice daily; 30 days; monotherapy or in combination with TAK228 ) inhibits the growth of tumors. In a dose-dependent manner, IPN-60090 exhibits strong in vivo target engagement on its own. Day 4 and Day 28 show lower glutamate/glutamine ratios and free plasma concentrations of IPN-60090 at 4 hours post-dose[2]. Additionally, IPN-60090 alone results in a 28% in vivo tumor growth inhibition, whereas IPN-60090 in combination with TAK228 strongly causes an 85% tumor growth inhibition[2].

GLS-1 Enzyme Assay. [2]
The glutaminase dual coupled fluorescence assay was performed in a 384 well, black, Greiner, non-binding, plate (Greiner, Catalog #784900) with assay buffer consisting of 50 mM Hepes (pH 7.4), 250 μM EDTA (pH 8), and 0.12 mM Triton-X 100. All final concentrations refer to a 20 μL volume. Stock solutions of the test compounds were prepared in 100% DMSO and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:50 in assay buffer, and 5 μL/well were transferred to the assay plate. A 4x stock of glutaminase and potassium phosphate dibasic trihydrate diluted in assay buffer was preincubated at RT for 10 min. 5 μL/well of the glutaminase with potassium phosphate dibasic trihydrate were added to the microplate (final concentrations 2 nM and 50 mM, respectively) followed by a 10 min incubation at RT. The coupled reaction consisted of glutamate oxidase, Amplex UltraRED, glutamine), and horseradish peroxidase. 5 μL/well of glutamate oxidase and Amplex UltraRED diluted in assay buffer (final concentrations 100 mU/mL and 75 μM, respectively) and 5 μL/well of glutamine and horseradish peroxidase diluted in assay buffer (final concentrations 1 mM and 100 mU/mL, respectively) were added to the microplate under subdued light followed by a 20 min incubation at RT. The resorufin signal was measured using a PerkinElmer Envision plate reader: excitation-535 nm, emission-590 nm. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.[2]

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GLS-2 Enzyme Assay. [2]
The GLS2 dual coupled fluorescence assay was performed in a 384 well, black, Greiner, non-binding, plate with assay buffer consisting of 50 mM Hepes (pH 7.4), 250 μM EDTA (pH 8), and 0.12 mM Triton-X 100. All final concentrations refer to a 20 μL volume. Stock solutions of the test compounds were prepared in 100% DMSO and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:50 in assay buffer, and 5 μL/well were transferred to the assay plate. A 4x stock of GLS-2 and potassium phosphate dibasic trihydrate diluted in assay buffer was preincubated at RT for 10 min. 5 μL/well of the GLS-2 with potassium phosphate dibasic trihydrate were added to the microplate (final concentrations 33 nM and 50 mM, respectively) followed by a 10 min incubation at RT. The coupled reaction consisted of glutamate oxidase, Amplex UltraRED (Molecular Probes, Catalog #A36006), glutamine, and horseradish peroxidase. 5 μL/well of glutamate oxidase and Amplex UltraRED diluted in assay buffer (final concentrations 100 mU/mL and 75 μM, respectively) and 5 μL/well of glutamine and horseradish peroxidase diluted in assay buffer (final concentrations 3 mM and 100 mU/mL, respectively) were added to the microplate under subdued light. The resorufin signal was measured continuously for 20 min using a PerkinElmer Envision plate reader: excitation-535 nm, emission-590 nm. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.[2]

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Microsomal stability. [2]
Microsomal stability assays were conducted on a Beckmann Biomek FXp laboratory automation system. The liver microsomal incubation mixture consisted of liver microsomes (0.5 mg microsomal protein/mL), the test compound (1 uM), MgCl2 (3 mM), and EDTA (1 mM) in potassium phosphate buffer (100 mM, pH 7.4). Midazolam and Ketanserin were used as the assay control substrates. The reaction was initiated with the addition of an NADPH regeneration solution (1.3 mM NADPH) and maintained at 37 °C with shaking. At five time points ranging from 0 to 45 min, aliquots (50 uL) were removed and quenched with acetonitrile (100 μL) containing an internal standard (imipramine). After vortex and centrifugation, samples were analyzed by LC-MS/MS. Calculation of the in vitro half-lives and clearance followed literature guidelines.[2]

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Plasma protein binding. [2]
Plasma protein binding (PPB) assays were conducted using the Rapid Equilibrium Dialysis (RED) device. Warfarin and metoprolol were used as the control substrates. To the receiver side was added 350 μL of phosphate buffered saline (pH 7.4, 1x). To the donor side was added 200 μL of plasma spiked with the test compound (5 μM). The same plasma/test compound solution (50 μL) was also used for the recovery sample. The plate was covered with Immunoware sealing tape and was incubated at 37 °C with shaking at 100 rpm for 5 h. After the incubation, both the receiver and donor sides were sampled (50 μL) and matched with the same volume of matrix from the other side. The recovery, donor, and receiver samples were extracted with 300 μL of cold ACN containing imipramine as the internal standard. After vortex and centrifugation, the supernatant (150 μL) was subjected to LC-MS quantitation. PPB (% bound) was calculated as %Bound = 100 × ([Donor]-[Receiver])/[Donor].[2]

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CYP Inhibition Assays. [2]
Studies were carried out in human liver microsomes (0.1 mg/mL for CYP 1A2/2C9/2D6/3A4; 0.5 mg/mL for CYP 2C19). Liver microsomes were purchased from BD Gentest. Aliquots of the compound stock solutions were diluted to 4 mM with acetonitrile, and then further diluted upon the addition of liver microsomes (0.2 mg/mL). An aliquot of 30 μL of diluted test compound solution was mixed with 15 μL of substrate solution. The plate was pre-warmed to 37 °C, then 15 μL of 8 mM NADPH (also pre-warmed to 37 °C) was added. The plate was incubated at 37 °C for the following incubation times: 5 min for 3A4, 10 min for 1A2/2C9/2D6, and 45 min for 2C19. The reaction was stopped by adding acetonitrile at the designated time point. The assay plates were shaken on a vibrator (IKA, MTS 2/4) for 10 min (600 rpm) and centrifuged at 5,594 g for 15 min (Multifuge × 3R). Aliquots of the supernatant were taken, diluted 1:3 into distilled water, and metabolite concentrations were analysed by LC-MS/MS compared to internal standards. Substrates used were Phenacetin (30 μM) for 1A2, Diclofenac (10 μM) for 2C9, S-Mephenytoin (35 μM) for 2C19, Midazolam (10 μM) or Testosterone (80 μM) for 3A4, and Bufuralol (10 μM) for 2D6. Metabolites measured were Acetaminophen for 1A2, 4’-Hydroxy-Diclofenac for 2C9, Hydroxy-Mephenytoin for 2C19, 1-Hydroxy-Midazolam or Osalmid for 3A4, and 1-Hydroxy-Bufuralol for 2D6. Positive control inhibitors used were α-Naphthoflavone for CYP1A2, Sulfaphenazole for CYP2C9, Omeprazole for CYP2C19, Quinidine for CYP2D6 and Ketoconazole for CYP3A4. The peak area response ratio (PARR) of metabolite to internal standard of the metabolites in test compound samples at desired time points was compared to the PARR in control samples to determine the percent of control sample (% control) at each time point. % inhibition was calculated as 100-% control.

A549 Cell Viability Assay. [2]
A549 cells were routinely maintained in filtered RPMI media supplemented with 10% dialyzed FBS using a humidified incubator (37 °C, 5% CO2, and ambient O2). In preparation for the viability assay, cells were harvested and resuspended in filtered RPMI media supplemented with 10% dialyzed FBS. Cells were seeded onto a 384-well black PerkinElmer tissue culture plate at a density of 1,000 cells/well in a volume of 40 μL. The tissue culture plate was incubated for 24 h at 37 °C with 5% CO2 and ambient O2. Stock solutions of the test compounds were prepared in 100% DMSO and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:40 in culture medium, and 10 μL/well were transferred to the tissue culture plate. Following compound addition, the microplate was incubated at 37 °C for 72 h. 10 μL of Promega’s CellTiter-Fluor reagent, GF-AFC substrate diluted in assay buffer, was added to the plate for a 1x final concentration. 0.5% DMSO and 20 μM etoposide were used as controls to define 100% and 0% viability, respectively. The plate was then shaken on an orbital shaker at 300 RPM for 15 min at RT followed by a 30 min incubation at 37 °C. The CellTiter-Fluor signal was measured using a PerkinElmer Envision plate reader: excitation-400 nm, emission-505 nm. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.[2]

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A549 Target Engagement Assay. [2]
A549 cells were routinely maintained in filtered RPMI media supplemented with 10% dialyzed FBS using a humidified incubator (37°C, 5% CO2, and ambient O2). In preparation for the target engagement assay, cells were harvested and resuspended in filtered RPMI media supplemented with 10% dialyzed FBS. Cells were seeded onto a 96-well tissue culture plate at a density of 15,000 cells/well in a volume of 100 μL. The tissue culture plate was incubated for 24 h at 37 °C with 5% CO2 and ambient O2. Stock solutions of the test compounds were prepared in 100% DMSO and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:200 in culture medium, and 200 μL/well were transferred to the tissue culture plate. Following compound addition, the microplate was incubated at 37 °C for 24 h. The L-glutamine and L-glutamate levels in the media were then measured using the YSI 2900 Biochemistry Analyzer. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.[2]

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Hepatocytes stability. [2]
Stock solutions were prepared at 10 mM in DMSO for the test compound. Aliquots of the stock solutions were diluted to 200 μM with DMSO and then further diluted to 2 μM with KHB buffer. The procedure was as follows: Count hepatocytes and then dilute the cell suspensions to the appropriate density (viable cell density = 2 × 106 cells/mL). Add 50 μL of pre-warmed (37 °C) 2 μM test compound to the wells designated for different time points. For 0 min, add 100 μL of ACN containing internal standard (IS) to the wells followed by 50 μL of hepatocytes solution and then seal the wells. Add 50 μL of pre-warmed hepatocytes solution to the wells designated for 15 min, 30 min, 60 min and 120 min, and start timing. Place the assay plate in an incubator at 37 °C. At 15 min, 30 min, 60 min and 120 min, add 100 μL of ACN to the wells, respectively, then seal the corresponding wells. After quenching, sonicate the plate for 5 min and then centrifuge at 5594 × g for 15 min (Thermo Multifuge × 3R). Transfer 50 μL of the supernatant from each well into a 96-well sample plate containing 120 μL of ultra-pure water for LC/MS analysis. The peak area response ratio (PARR) to IS of the compounds at 15 min, 30 min, 60 min, and 120 min was compared to the PARR at 0 min to determine the percent of the test compound remaining at each time point. Half-lives were calculated using Excel software, fitting to a single-phase exponential decay equation.[2]

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Caco-2 permeability. [2]
Caco-2 cells were obtained from American Tissue Culture Collection. The cells were maintained in Modified Eagle’s medium (MEM) containing 10% heat-inactivated FBS and 1% non-essential amino acids, in CO2 at 37 °C. Cells were seeded on polycarbonate filter inserts. The cells were cultured for 21–28 days prior to the transport experiments. The transepithelial electric resistance (TEER) and Lucifer Yellow permeability were checked before and after the assay. Compounds were dissolved at 10 mM in DMSO and diluted for studies in Hank’s Balanced Salt Solution (HBSS) with 25 mM HEPES, pH 7.4. Compounds were tested at 10 μM, in both the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions, and were conducted at 37 °C for 90 min. At the end of incubation, donor samples were diluted 10-fold by assay buffer, then 60 μL of receiver and diluted-donor samples were mixed with 60 μL of acetonitrile and concentrations were analyzed by LC-MS/MS compared to a standard curve.

Female CD-1 mice[2]
3 mg/kg for i.v.; 10 mg/kg for p.o. (Pharmacokinetic Analysis)
Intravenous injection and oral administration

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In vivo pharmacokinetics.[2]
Mouse: Female mice weighing 20–30 g were used for studies. Food and water were available to all animals ad libitum. The test article was dosed via tail vein (IV doses) or oral gavage (PO doses), respectively. Blood samples were collected from all animals at predose and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes containing the anticoagulant K2EDTA (3 animals per time point with 3 time points collected per animal). Plasma was separated from the blood by centrifugation at 4 °C and stored at −70 °C until analysis. Test article concentrations in plasma were quantified using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Rat: Male rats weighing 200–300 g were used for studies. Animals were fasted overnight and fed 4 h postdose. Water was available ad libitum for all animals. Test article was dosed via dorsal foot vein (IV doses) or oral gavage (PO doses). Blood samples were collected via tail vein from all animals at predose and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes containing the anticoagulant K2EDTA. Plasma was separated from the blood by centrifugation at 4 °C and stored at −70 °C until analysis. Test article concentrations in plasma were quantified using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Dog: Male Beagle dogs weighing 7–10 kg were used for studies. Animals were fasted overnight and fed 4 h postdose. Test article was administered to dogs via the cephalic vein (IV doses) or oral gavage (PO doses). Blood samples were collected via the saphenous vein or cephalic vein from all animals at predose and 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes containing the anticoagulant K2EDTA. Plasma was separated from the blood by centrifugation at 4 °C and stored at −70 °C until analysis. Test article concentrations in plasma were quantified using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Monkey: Male Cynomolgus monkeys weighing 3–5 kg were used for studies. Animals were fasted overnight and fed 4 h postdose. Test article was administered to monkeys via the cephalic vein (IV doses) or nasal gavage (PO doses). Blood samples were collected via the saphenous vein or cephalic vein from all animals at predose and 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose into tubes containing the anticoagulant K2EDTA. Plasma was separated from the blood by centrifugation at 4 °C and stored at −70 °C until analysis. Test article concentrations in plasma were quantified using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.[2]

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In Vivo Models.[2]
All experiments were conducted in compliance with institutional guidelines.

Glutamine and Glutamate Levels in H460 Xenograft Model. [2]
NSG female mice were implanted with H460 cells (5×105 cells/mouse diluted with matrigel 1:1). Mice were between 6–10 weeks old. All animals received LabDiet 5053 chow ad libitum. Tumors were allowed to grow to 300–400 mm3, and animals were treated with compound 27 (bis-hydrochloride) formulated in 0.5% methylcellulose in sterile water. Animals were euthanized via CO2 at 8 or 24 h after a single dose of compound 27 by oral gavage, and tumors were harvested. Each group contained 6 animals. Tumors were weighed and snap frozen. Tumor sections were homogenized using an OmniBEAD Ruptor 24 at 100 mg tissue/mL in MeOH/Water (80:20) containing 13C5-L-glutamine and 13C5-L-glutamate as internal standards. Homogenates were centrifuged at 15,000 rpm at 4 °C for 10 min An aliquot of 10 μL of the supernatant was diluted with 190 μL of 0.1% formic acid in ACN/water (50:50), vortexed for 15 sec, and centrifuged at 15,000 rpm at 4 °C for 5 min Samples were analyzed on an Agilent 1290 infinity LC system coupled with an Agilent 6460 triple quadrupole mass spectrometer operated at positive mode (ESI+). A Waters XBridge Amide column (3.5 μm; 4.6 × 100 mm) was used for analyte separation. The HPLC buffer A was 95% (v/v) water/ACN containing 20 mM ammonium hydroxide and 20 mM ammonium acetate. The HPLC buffer B was 100% ACN. The gradient was 80% B (0–1 min), 80–10% B (1–3 min), 10% B (3–5 min), 10–80% B (5–5.3 min), 80% B (5.3–10 min). The column temperature was 40 °C and the flow rate 0.5 mL/min. The sample injection volume was 2 μL. The detection conditions of the mass spectrometer were as follows: capillary voltage 4000 V, nebulizer pressure 35 psi, cell accelerate voltage 4 V, sheath gas temperature 400 °C, sheath gas flow 11 L/min, source gas temperature 300 °C, source gas flow 11 L/min, fragmenter voltage 80 V, collision energy 26 V (glutamate) and 14 V (glutamine). Metabolites were detected by compound specific multiple reaction monitoring transition (MRM) and retention time (RT): Glutamine (m/z 147>84, RT 4.93 min), 13C5-Glutamine (m/z 152>88, RT 4.93 min), Glutamate (m/z 148>84, RT 4.78 min), 13C5-Glutamate (m/z 153>88, RT 4.78 min). The method was validated with an analytical range of 10 – 5000 ng/mL for both glutamine and glutamate in ACN/Water (1:1). GraphPad Prism was used for generation of graphs, and data is expressed as the mean ± standard deviation.

Efficacy in Ru337 PDX model. [2]
8 week old NSG female mice were implanted with Ru337 (Memorial Sloan Kettering Cancer Center) patient derived xenografts (PDX) subcutaneously on the right flank. Tumors were allowed to grow to an average volume of 100 mm3 as monitored by caliper measurements. Animals were then randomized into groups of 8. All animals received chow ad libitum. Mice were treated with compounds on a 5 day-on/2 day-off schedule. Compound 27 was formulated in 0.5% methylcellulose in sterile water and dosed at 100 mg/kg, PO, BID (doses administered approximately 8 hours apart (8:00 and 16:00) each dosing day followed by a 16 hour gap before the next day’s dose). TAK-228 was formulated in 5% sucrose and 0.5% methylcellulose in sterile water and dosed at 1 mg/kg, PO, QD. Body weights were monitored twice per week. Tumor volume was calculated using the formula: V=l2*L/2 (l=length; L=width). GraphPad Prism was used for generation of graphs, and data is expressed as the mean ± standard deviation. For the combination arms, standard deviations were very low (bars smaller than the size of plotted datapoints).

The pharmacokinetic properties of compound 27 (IPN60090) in rat and dog are superior to its properties in mouse and monkey, with rat and dog showing lower in vivo clearances and 5–6x longer half-lives. Because of the low volumes of distribution across species, half-lives are short unless clearances are extremely low, as is the case for rat and dog. Clearances across species are qualitatively in agreement with in vitro metabolic stabilities. Given that the in vitro clearances in human microsomes and hepatocytes are very low (similar to or lower than values for rat and dog), we expect low in vivo clearances and favorable half-lives in humans, more similar to rat and dog than to mouse and monkey.[2]
compound 27 (IPN60090) was further tested in ascending single dose oral PK experiments in mouse, rat and dog (Figure 6). Across the dose ranges tested (up to 200 mg/kg in mouse, 100 mg/kg in rat and 10 mg/kg in dog), maximal concentrations and total exposures continually increased with dose, with high exposures achieved across species.

[1]. Gls1 inhibitors for treating disease. WO2016004404A2.

[2]. Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties. J Med Chem. 2020 Nov 12;63(21):12957-12977.

Glutaminase-1 Inhibitor IACS-6274 is an orally bioavailable inhibitor of the metabolic enzyme glutaminase-1 (GLS1), with potential antineoplastic and immunostimulating activities. Upon oral administration, IACS-6274 selectively targets, binds to and inhibits human GLS1, an enzyme that is essential for the conversion of the amino acid glutamine into glutamate. Blocking glutamine metabolism inhibits proliferation in rapidly growing tumor cells and leads to an induction of cell death. Unlike normal healthy cells, glutamine-dependent tumors heavily rely on the intracellular conversion of exogenous glutamine into glutamate and glutamate metabolites to provide energy and generate building blocks for the production of macromolecules, which are needed for cellular growth and survival.

C24H27F3N8O3

532.518194437027

532.22

C, 54.13; H, 5.11; F, 10.70; N, 21.04; O, 9.01

1853164-83-6

IPN60090 dihydrochloride;2102101-72-2; IPN60090;1853164-83-6; 2102101-78-8 (mesylate); 2102101-77-7 (sulfate); 2102101-80-2

118627280

Light yellow to yellow solid powder

1.6

2

11

11

38

804

1

CC1=CC(=CC(=N1)CC(=O)NC2=NN=C(C=C2)CC[C@H](CN3C=C(N=N3)C(=O)NC)F)OC4CC(C4)(F)F

GEHZIZWHNLQFAS-OAHLLOKOSA-N

InChI=1S/C24H27F3N8O3/c1-14-7-18(38-19-10-24(26,27)11-19)8-17(29-14)9-22(36)30-21-6-5-16(31-33-21)4-3-15(25)12-35-13-20(32-34-35)23(37)28-2/h5-8,13,15,19H,3-4,9-12H2,1-2H3,(H,28,37)(H,30,33,36)/t15-/m1/s1

1-[(2R)-4-[6-[[2-[4-(3,3-difluorocyclobutyl)oxy-6-methylpyridin-2-yl]acetyl]amino]pyridazin-3-yl]-2-fluorobutyl]-N-methyltriazole-4-carboxamide

IACS-6274; IACS6274; IACS 6274; IPN60090; CHEMBL4741924; 1-[(2R)-4-[6-[[2-[4-(3,3-difluorocyclobutyl)oxy-6-methylpyridin-2-yl]acetyl]amino]pyridazin-3-yl]-2-fluorobutyl]-N-methyltriazole-4-carboxamide; IACS6274; O6ZT4D087I; IPN 60090; IPN-60090

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 5~31.4 mg/mL (9.4~59.0 mM)

Solubility in Formulation 1: ≥ 3.14 mg/mL (5.90 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 31.4 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.)