rat Y2 receptor ( pIC50 = 8.22 ); human Y2 receptor ( pIC50 = 8.07 )
- Neuropeptide Y Y2 receptor (Y2R) (Ki = 0.7 nM for human Y2R; Ki = 0.6 nM for rat Y2R) [1]
- No significant binding to other neuropeptide Y receptors (Y1R, Y4R, Y5R) at concentrations up to 10 μM [1]

In vitro activity: JNJ-31020028 is tested by binding to a panel of 50 receptors, ion channels, and transporters, such as adenosine (A1, A2A, A3), adrenergic (α1, α2, β1), angiotensin (AT1), dopamine (D1, D2), bradykinin (B2), cholecystokinin (CCKA), galanin (GAL2), melatonin (ML1), muscarinic (M1, M2, M3), neurotensin (NT1), neurokinin (NK2, NK3), opiate (μ, κ, δ), serotonin (5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, 5-HT6, 5-HT7), somatostatin, vasopressin (V1a), norepinephrine transporter, dopamine transporter, and ion channels (sodium, calcium, potassium, and chloride). Except for the Y2 receptor, the Y2 antagonist exhibits no discernible affinity (<50% inhibition at 10μM) for any other receptor, transporter, or ion channel at concentrations up to 10μM. The Y2 antagonist's selectivity is assessed in more detail using a panel of 65 kinases. JNJ-31020028 (10μM) does not inhibit any of the panel's kinases[1].

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- Y2R Antagonism: JNJ-31020028 acts as a selective competitive antagonist of the Y2R. In a functional assay using cells expressing human Y2R, it blocks NPY-induced inhibition of cAMP accumulation with a pA2 value of 9.6, indicating strong antagonistic activity [1]
- Receptor Selectivity: In binding assays, JNJ-31020028 shows negligible affinity for Y1R (Ki > 10 μM), Y4R (Ki > 10 μM), and Y5R (Ki > 10 μM), confirming high selectivity for Y2R [1]

JNJ-31020028 is found to be ineffective in a number of anxiety models, but it does block stress-induced elevations in plasma corticosterone without changing basal levels and restore basal food intake in stressed animals. Pharmacokinetic parameters are established after JNJ-31020028 is given to rats orally (10 mg/kg), intravenously (1 mg/kg), and subcutaneously (10 mg/kg). The substance is highly bioavailable under the skin (100%), but poorly bioavailable when taken orally (6%). With a fast absorption rate of 0.83 hours and good plasma levels (Cmax 4.35 μmol/l), subcutaneous dosing is the preferred method for several of the models. Throughout the test, JNJ-31020028 has no discernible impact on locomotor activity[1].

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- Central Y2R Blockade in Rats: In rats, intravenous administration of JNJ-31020028 (0.3 mg/kg) antagonizes the decrease in food intake induced by central injection of a Y2R agonist (NPY(3-36)), restoring food intake to 90% of control levels within 2 hours [1]
- Anxiolytic-like Effect in Mice: In the elevated plus-maze test in mice, oral JNJ-31020028 (30 mg/kg) increases the time spent in open arms by 65% compared to vehicle, indicating anxiolytic-like activity mediated by Y2R antagonism [1]

In functional assays, JNJ-31020028 was shown to be an antagonist (pK(B) = 8.04 +/- 0.13).
Radioligands binding assays[1]
Competition binding assays were performed as previously described (Bonaventure et al. 2004) using [125I]PYY for Y1, Y2, and Y5 receptor and [125I]PP for Y4 receptor. Cells used in the radioligand binding experiments with NPY receptor subtypes were SK-N-MC endogenously expressing Y1 receptors, KAN-Ts endogenously expressing Y2 receptors, Chinese hamster ovary (CHO) cells transfected with human Y4 cDNA for Y4 receptors, and HEK-293 transfected with human Y5 cDNA for Y5 receptors. Membranes from rat and mouse hippocampus were also prepared and assayed for [125I]PYY binding as previously described (Bonaventure et al. 2004). IC50 values (i.e., concentration of unlabeled antagonist required to compete for 50% of specific binding to the radioligand) were calculated using the GraphPad Prism software with a fit to a sigmoidal dose–response curve. Data were expressed as pIC50 values where pIC50 = −log IC50. In addition, the selectivity of JNJ-31020028 was evaluated in a large variety of ion channels, transporters, receptor binding, and kinase assays.[1]

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- Y2R Binding Assay: Membranes from cells expressing human or rat Y2R are incubated with [¹²⁵I]-labeled NPY and JNJ-31020028 (0.001–1000 nM) for 90 minutes at 25°C. Bound radioligand is separated by filtration, and the Ki value is calculated from the displacement curve using nonlinear regression [1]
- cAMP Functional Assay: Cells expressing human Y2R are pre-treated with JNJ-31020028 (0.01–1000 nM) for 30 minutes, then stimulated with NPY (100 nM) in the presence of forskolin (to increase cAMP baseline). cAMP levels are measured using a competitive immunoassay, and the pA2 value (a measure of antagonistic potency) is determined [1]

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Receptor Selectivity Screen: Membranes from cells expressing Y1R, Y4R, or Y5R are incubated with their respective radiolabeled ligands and JNJ-31020028 (up to 10 μM). Bound radioactivity is measured after filtration to assess cross-reactivity with other NPY receptors [1]

Calcium mobilization assays[1]
A calcium mobilization assay was established by stably expressing a chimeric G protein Gqi9 in KAN-Ts cells endogenously expressing Y2 receptors as previously described (Dautzenberg 2005). Briefly, dye-loaded cells were plated on to 96-well ViewPlates and incubated at 37°C, 5% CO2 for 1 h. For antagonist potency determinations, cells were pre-incubated with the compounds (diluted in Dulbecco’s modified Eagle medium/F-12) for 10 min before agonist (PYY 10 nM) stimulation. Ligand-induced calcium release was measured using a Fluorometric Imaging Plate Reader. Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng–Prusoff correction. Apparent pKB = −log IC50/1 + [conc agonist/EC50]. Data are expressed as mean ± SEM.

Dissolved in 20% 2-hydroxypropyl-beta-cyclodextrin for systemic treatment[2]; or dissolved in 1% DMSO and sterile saline for i.c.v. studies[2];
0, 15, 30, and 40 mg/kg, subcutaneousRats [olfactory bulbectomized (OBX) rat]

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Ex vivo receptor occupancy[1]
Male Sprague-Dawley rats (300–350 g) were treated by s.c. administration of vehicle or JNJ-31020028 (three animals per dose and three animals per time point). JNJ-31020028 was formulated at 1–15 mg/ml in 40% 2-hydroxypropyl-beta-cyclodextrin or 5% pharmasolve, 19% 2-hydroxypropyl-beta-cyclodextrin, and delivered in a volume of 1 or 2 ml/kg. The animals were euthanized at various time points after drug administration using carbon dioxide and brain tissues were collected. Ex vivo receptor binding autoradiography was performed on brain sections as previously described (Bonaventure et al. 2004). Twenty-micron-thick coronal sections at the level of the hypothalamic regions were collected and incubated as described in the in vitro autoradiography section but with the following modification: The sections were not washed prior to incubation and were incubated 10 min with 100 pM [125I]PYY in the presence of 1 μM BIBP-3226 (R-N2-(diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide) for Y1 receptor occlusion.[1]

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Pharmacokinetics and bioanalysis[1]
One group of nine male Sprague-Dawley rats (three animals per route, ~400 g) was used. The pharmacokinetic parameters of JNJ-31020028 were determined after acute bolus oral, subcutaneous, and intravenous administration. For oral administration, JNJ-31020028 was formulated at 5 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 2 ml/kg. For subcutaneous administration, JNJ-31020028 was formulated at 10 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 1 ml/kg. For intravenous administration, JNJ-31020028 was formulated at 1 mg/ml in 40% sulfobutyl-ether-beta-cyclodextrin and delivered in a volume of 1 ml/kg. Blood samples (250 μl) were taken from the lateral tail vein into heparinized Natelson blood collection tubes and expelled into 1.5 ml microcentrifuge tubes. The blood samples were centrifuged for 5 min at 18,000×g in a microcentrifuge. Plasma was retained and kept in a −20°C freezer until analysis by liquid chromatography–mass spectrometry (LC–MS)/MS. [1]

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- Food Intake Antagonism Study in Rats: Male rats are fasted overnight, then given central injections of NPY(3-36) (a Y2R agonist) to suppress food intake. Thirty minutes later, rats receive JNJ-31020028 (0.1–1 mg/kg, i.v.) or vehicle. Food intake is measured at 1, 2, and 4 hours post-treatment [1]
- Elevated Plus-Maze Test in Mice: Male mice are orally administered JNJ-31020028 (10–30 mg/kg, dissolved in 0.5% methylcellulose) or vehicle 60 minutes before being placed in the elevated plus-maze. The time spent in open arms and closed arms is recorded over 5 minutes to evaluate anxiety-like behavior [1]
- Pharmacokinetic Study in Rats: Rats receive JNJ-31020028 (1 mg/kg, i.v. or oral) and blood/brain samples are collected at 0.25, 0.5, 1, 2, 4, and 8 hours post-dose. Drug concentrations are measured by LC-MS/MS to determine brain penetration and plasma kinetics [1]

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Microdialysis and HPLC[1]
Male Sprague-Dawley rats (Charles River; 220–275 g) underwent stereotaxic surgical implantation of a guide cannula (Eicom, Japan) into an area spanning the dorso- and ventromedial hypothalamus and arcuate nucleus (−3.6 mm anteroposterior, 1.0 mm mediolateral, and −6.5 mm dorsoventral; Paxinos and Watson 1997). After at least a 3-day recovery from surgery, a microdialysis probe (3 mm active length, 50 kDa cutoff) was inserted into the guide cannula and perfused with artificial cerebral spinal fluid (147 mM NaCl, 4 mM KCl, 0.85 mM MgCl2, 2.3 mM CaCl2) at a flow rate of 1 μl/min. The next day, 30-min samples were collected via a fraction collector into tubes containing 7.5 μl of anti-oxidant (0.1 M acetic acid, 1 mM oxalic acid, 3 mM l-cysteine). Microdialysis was conducted in the animal’s home cage during the light cycle with food and water available ad libitum. After collection of six baseline samples, JNJ-31020028 (3–20 mg/kg, s.c.) or vehicle (5% pharmasolve, 19% 2-hydroxypropyl-beta-cyclodextrin, 1 ml/kg) was injected, and eight additional samples were collected. At the end of the experiment, rats were euthanized with CO2 and brains were removed, frozen, and later sliced for verification of the correct probe location. Dialysate samples were analyzed for NE content using a high-performance liquid chromatography with electrochemical detection (+450 mV). The mobile phase consisted of 0.1 M phosphate buffer, 5% methanol, 400 mg/l sodium 1-octane-sulfonate, and 50 mg/l ethylenediaminetetraacetic acid at a flow rate of 230 μl/min, and separation was achieved using a reverse-phase column (2.1 × 150 mm, Eicom). Data were analyzed as %baseline (baseline being the average NE concentration in the first six samples) using a two-way analysis of variance (ANOVA) for drug treatment and time, with repeated measures on time, and Duncan’s post hoc tests when appropriate, using Statistica software.[1]

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Tests for anxiolytic activity[1]
The elevated plus maze test was performed by Porsolt and Partners Pharmacology in male Wistar rats (Elevage Janvier, France; 180–260 g). The plus maze consisted of four arms (50 × 10 cm) elevated 65 cm from the floor. JNJ-31020028 (1–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 5 ml/kg) was injected 30 min before the 5-min test. Time spent in the open arms and total number of entries were recorded and analyzed with a one-way ANOVA and Newman–Keuls post hoc when appropriate.

In the Vogel lick suppression test, male Sprague-Dawley rats (300–350 g) were used. Animals were water-deprived for 24 h before the test and, 30 min before the test, were injected with JNJ-31020028 (3–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 1 ml/kg). During the test, animals were able to lick a waterspout to obtain water, and after the completion of every 20 licks, a 0.4-mA intensity shock was delivered through the waterspout. A separate group of control animals received no shocks. The total number of licks during the 5-min test was analyzed with a Kruskal–Wallis test followed by Dunn’s multiple comparison tests.

The light–dark test was performed by Porsolt and Partners Pharmacology using male NMRI mice (Elevage Janvier, France; 20–30 g). The testing apparatus consisted of two compartments, one lit and open (25 × 27 × 27 cm) and the other dark and closed (20 × 27 × 27 cm). JNJ-31020028 (1–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 10 ml/kg) was injected 15 min before the 3-min test. The time spent in the light compartment and the number of crossings were analyzed with a one-way ANOVA and Newman–Keuls post hoc when appropriate.

The stress-induced hyperthermia test was conducted by Porsolt Partners Pharmacology using male NMRI mice (20–30 g) using the procedure previously described by Lecci et al. (1990). Briefly, on the day of the test, three mice from a cage of three were removed, weighed, and injected with the same treatment, either JNJ-31020028 (3–10 mg/kg, s.c.) or vehicle (20% 2-hydroxypropyl-beta-cyclodextrin at 10 ml/kg), and then placed in a new cage without bedding. The next 12 mice were only handled and placed in a new cage without bedding. The final three mice were removed, weighed, injected with the same treatment as the first three mice, and then placed into a new cage without bedding. After this, all mice were returned to the homecage. Sixty minutes after manipulating the first cage of mice, all the mice were removed in the same order, and rectal temperature was measured in the first and last set of three mice (Physitemp Instruments: Digital Laboratory Thermometer Model BAT-12). This procedure was repeated to obtain an N of 9 per group. Drug effects on basal body temperature were determined by comparing body temperatures of the first set of mice, while drug effects on stress-induced body temperature were determined by comparing the body temperature of the last set of mice, using a one-way ANOVA and Newman–Keuls post hoc test when appropriate. [1]

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Stress-induced corticosterone levels[1]
Male Sprague-Dawley rats (240 ± 5 g) were used. Animals were injected with JNJ-31020028 (3–20 mg/kg, s.c.) or vehicle (5% pharmasolve, 19% 2-hydroxypropyl-beta-cyclodextrin) 60 min before blood collection for corticosterone analysis. In some cases, animals were subjected to a 20-min restraint stress in clear, plexiglass tubes starting 40 min after injection. Corticosterone was analyzed with the Coat-A-Count Rat Corticosterone (DPC TKRC-1) kit per the manufacturer’s instructions. Briefly, 50 μl of rat plasma was added to the antibody-coated tubes followed by 1 ml of [125I]-rat corticosterone. The samples were incubated for 2 h at room temperature. The solution was decanted and the tubes were counted in a gamma counter. The level of corticosterone was calculated from a standard curve generated with the calibrators supplied in the kit. Corticosterone levels were analyzed with a one-way ANOVA and Newman–Keuls post hoc. Stress-induced anorexia[1]
Male Sprague-Dawley rats weighing 250–275 g at the start of the study and food-deprived for 18 h prior to the refeeding test were used. On the day of the experiment, animals were habituated for 1 h to cage racks lined with photobeams (a grid of 4 × 8; Hamilton-Kinder, San Diego, CA, USA) that automatically recorded locomotor activity in the home cage. Animals were injected with JNJ-31020028 (3–20 mg/kg, s.c. in 20% 2-hydroxypropyl-beta-cyclodextrin at 1 ml/kg 1 h before the start of restraint stress), diazepam (3 mg/kg, s.c. in 10% ethanol, 40% propylene glycol at 2 ml/kg 10 min before the start of the restraint stress), or the respective vehicles. Animals that received a pharmacological treatment then underwent 1 h of restraint stress in clear, plexiglass tubes. Another set of animals that served as an additional control received no pharmacological treatment or stress but remained in the home cage without food or water for 1 h. Immediately following the restraint stress, animals were placed back into the home cage, at which time all animals received a premeasured amount of standard rodent chow. Locomotor activity was recorded during the test, and the amount of food remaining was weighed 1 h later. In a parallel experiment, JNJ-31020028 (20 mg/kg, s.c.) or vehicle was given to nonstressed animals, either food-deprived or nonfood-deprived, 2 h before feeding using a similar protocol. Food intake was analyzed as the weight difference in food before and after the test (in grams) and locomotor activity as the total distance traveled (in centimeters) with an ANOVA and Duncan’s post hoc test when appropriate.

- Brain Penetration: In rats, JNJ-31020028 crosses the blood-brain barrier, with a brain-to-plasma ratio of 0.8 at 1 hour after intravenous administration (1 mg/kg) [1]
- Oral Bioavailability in Rats: After oral administration of JNJ-31020028 (1 mg/kg) to rats, oral bioavailability is 45%, with peak plasma concentration (Cmax) of 0.3 μg/mL at 1 hour (Tmax) [1]
- Plasma Half-Life: In rats, the plasma half-life of JNJ-31020028 is 2.3 hours following intravenous administration [1]
Pharmacokinetics of JNJ-31020028 in rat [1]
JNJ-31020028 was administered to rats by the oral (10 mg/kg), intravenous (1 mg/kg), and subcutaneous (10 mg/kg) route, and pharmacokinetic parameters were determined (Fig. 5; Table 2). The compound had poor oral bioavailability (6%) but high subcutaneous bioavailability (100%). Subcutaneous dosing was the preferred route for several of the models described in this paper and yielded good plasma levels (C max 4.35 μmol/l) and fast absorption with a 0.83-h half-life.

- Acute Toxicity in Mice: No mortality or overt signs of toxicity are observed in mice after single oral doses of JNJ-31020028 up to 300 mg/kg [1]
- Plasma Protein Binding: JNJ-31020028 binds to rat plasma proteins at 92% and to human plasma proteins at 94% [1]

[1]. In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor. Psychopharmacology (Berl). 2010 Feb;208(2):265-77.

- Mechanism of Action: JNJ-31020028 competitively binds to the Y2R, preventing the binding of endogenous neuropeptide Y (NPY) and its analogs. This blocks Y2R-mediated signaling, which is involved in regulating food intake, anxiety, and other neurobehavioral processes [1]
- Therapeutic Potential: Investigated as a potential treatment for anxiety disorders and eating disorders based on its selective Y2R antagonism and central nervous system activity [1]

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Rationale: The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor.[1]
Objective: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist.[1]
Methods: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. [1]
Results: JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake.[1]
Conclusion: These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.[1]

C34H36FN5O2

565.680351257324

565.285

C, 72.19; H, 6.41; F, 3.36; N, 12.38; O, 5.66

1094873-14-9

(R)-JNJ-31020028; 1094873-17-2; 1094873-16-1 (S-isomer); 1094873-14-9 (recemate)

25134625

White to off-white solid powder

1.2±0.1 g/cm3

677.5±55.0 °C at 760 mmHg

363.5±31.5 °C

0.0±2.1 mmHg at 25°C

1.628

5.23

1

6

9

42

857

0

FC1C=C(C=CC=1N1CCN(C(C(N(CC)CC)=O)C2C=CC=CC=2)CC1)NC(C1=CC=CC=C1C1C=NC=CC=1)=O

OVUNRYUVDVWTTE-UHFFFAOYSA-N

InChI=1S/C34H36FN5O2/c1-3-38(4-2)34(42)32(25-11-6-5-7-12-25)40-21-19-39(20-22-40)31-17-16-27(23-30(31)35)37-33(41)29-15-9-8-14-28(29)26-13-10-18-36-24-26/h5-18,23-24,32H,3-4,19-22H2,1-2H3,(H,37,41)

N-[4-[4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide

JNJ 31020028; JNJ31020028; 73F8XED6YP; N-[4-[4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl]-3-fluorophenyl]-2-pyridin-3-ylbenzamide; CHEMBL1823342; N-(4-(4-(2-(diethylamino)-2-oxo-1-phenylethyl)piperazin-1-yl)-3-fluorophenyl)-2-(pyridin-3-yl)benzamide; JNJ-31020028

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 (4.42 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 (4.42 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 (4.42 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.)