MAO-B; Monoamine oxidase B (MAO-B). IC₅₀ = 0.41 ± 0.11 μM. Selectivity Index (SI) > 24.4 (IC₅₀ MAO-A > 10 μM, 32.58% inhibition at 10 μM). [1]
Amyloid-β₁₋₄₂ (Aβ₁₋₄₂). Inhibition of self-induced Aβ₁₋₄₂ aggregation: 56.03% at 25.0 μM. Disaggregation of self-induced Aβ₁₋₄₂ fibrils: 81.91% at 25.0 μM. [1]
Cu²⁺-induced Aβ₁₋₄₂ aggregation: inhibition = 57.51% at 25.0 μM; disaggregation = 82.40% at 25.0 μM. [1]
Metal ions (Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Al³⁺). Forms 1:1 complex with Cu²⁺. [1]
MAO-B (monoamine oxidase B). MAO-B-IN-21 is an MAO-B inhibitor with antioxidant activity and anti-Abeta aggregation activity. The compound exhibits metal-ion chelating ability (particularly for Fe2+, Cu2+, and Zn2+), anti-neuroinflammatory activity (reduces NO and TNF-alpha production), neuroprotective effects, and BBB permeability. The specific IC50 value for MAO-B inhibition has not been fully published.

10x exhibited excellent MAO-B inhibitory activity with an IC₅₀ of 0.41 ± 0.11 μM and selectivity over MAO-A (SI > 24.4). [1]
10x showed good antioxidant activity with an ORAC-FL value of 1.16 Trolox equivalent. [1]
10x inhibited self-induced Aβ₁₋₄₂ aggregation (56.03% at 25.0 μM) and disaggregated self-induced Aβ₁₋₄₂ fibrils (81.91% at 25.0 μM). [1]
10x inhibited Cu²⁺-induced Aβ₁₋₄₂ aggregation (57.51% at 25.0 μM) and disaggregated Cu²⁺-induced Aβ₁₋₄₂ fibrils (82.40% at 25.0 μM). [1]
TEM images confirmed that 10x reduced Aβ₁₋₄₂ fibril formation and disaggregated pre-formed fibrils (both self- and Cu²⁺-induced). [1]
10x effectively chelated metal ions (Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Al³⁺) as shown by UV-Vis spectroscopy. Stoichiometry of 10x-Cu²⁺ complex was 1:1. [1]
10x blocked Cu²⁺-ascorbate-induced ROS production, reducing fluorescence to near-undetectable levels in a CCA-based assay. [1]
In PC-12 cells, 10x (1.0, 2.5, 10.0 μM) dose-dependently protected against H₂O₂-induced cell injury. Cell viability: model group 54.9%; 10x groups: 66.9%, 73.4%, 77.7%, respectively. No cytotoxicity was observed up to 10.0 μM. [1]
In LPS-induced BV-2 microglia cells, 10x (2.5, 10.0 μM) dose-dependently inhibited ROS production (DCFH-DA assay). [1]
10x (0.5, 2.5, 10.0 μM) dose-dependently inhibited NO release (25.9%, 37.9%, 76.3% inhibition) and TNF-α release (22.7%, 51.6%, 71.6% inhibition) in LPS-induced BV-2 cells. [1]
Molecular docking revealed that (S)-10x forms a strong hydrogen bond with Ile199 residue in MAO-B binding pocket (estimated binding energy: -11.78 kcal/mol), explaining its selective MAO-B inhibition. [1]
In silico docking with Aβ₁₋₄₂ pentamer showed (S)-10x binds to the β-sheet surface via hydrogen bonds with Gln15 (Chain A/B/D) and Lys16 (Chain B), with estimated binding energy of -9.54 kcal/mol. [1]

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Because of its metal chelating capabilities and/or antioxidant capability, MAO-B-IN-21 (37.5 μM in methanol) inhibits the generation of ROS caused by Cu2+, allowing free radicals to be extracted from the Cu2+-ascorbic acid redox system [1]. Auto-induced Aβ1-42 fibrils are disassembled and auto-induced Aβ1-42 aggregation is inhibited by MAO-B-IN-21 (25 μM; 24 h; 37 °C) [1]. In BV-2 microglia activated with LPS, MAO-B-IN-21 (2.5–25 μM; 24 hours) prevents the generation of ROS [1].

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In vitro, MAO-B-IN-21 inhibits MAO-B activity, reducing the oxidative deamination of monoamine neurotransmitters. It exhibits antioxidant activity, scavenging free radicals and reducing oxidative stress. The compound inhibits amyloid-beta (Abeta) aggregation, preventing the formation of toxic Abeta oligomers and fibrils. It demonstrates metal-ion chelating capability, binding to metal ions (Fe2+, Cu2+, Zn2+) that are implicated in Alzheimer's disease pathology. MAO-B-IN-21 reduces neuroinflammation markers (NO and TNF-alpha) in microglial cell cultures stimulated with LPS or Abeta. It exhibits neuroprotective effects in neuronal cell cultures exposed to oxidative stress (H2O2), excitotoxicity (glutamate), or Abeta toxicity. Detailed EC50/IC50 values are not fully published.

In Aβ₁₋₄₂-induced AD mice model (intracerebroventricular injection of Aβ₁₋₄₂), 10x (8 and 32 mg/kg, intragastric administration) significantly improved cognitive impairment in Morris water maze test. Treatment with 10x shortened escape latency time during training phase and increased time spent in target quadrant and number of platform crossings in probe trial compared to model group. High dose (32 mg/kg) showed better effects than donepezil (8 mg/kg). [1]
Biochemical analysis of mouse hippocampus revealed that 10x (8 and 32 mg/kg) significantly reduced Aβ₁₋₄₂ levels, decreased malondialdehyde (MDA) content, increased superoxide dismutase (SOD) activity, and reduced TNF-α levels compared to model group. [1]

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In Balb/C mice, MAO-B-IN-21 (30 mg/kg; ir; single dose) easily penetrates the blood-brain barrier [1]. MAO-B-IN-21 (8 mg/kg and 32 mg/kg; ir; single dose) decreases Aβ1-42 levels and shortens the escape latency (ELT), suggesting that the Aβ-induced AD mouse model's learning and memory capacities are enhanced. Enhance[1]. Balb/C mice were used for the pharmacokinetic investigation [1] Tissue T1/2 Tmax Cmax AUC0-t AUCbrain/plasma Plasma 1.15 h 0.5 h 1539 ng/mL 2941 ng·h/mL 0.69 Brain 2.07 h 1.0 h 922.7 ng/g 2032.5 ng·h/g

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In vivo, MAO-B-IN-21 has been evaluated in the Abeta1-42-induced Alzheimer's disease mouse model. The compound reduces Abeta aggregation and neuroinflammation, decreases oxidative stress markers, and improves cognitive function. It demonstrates neuroprotective effects and inhibits MAO-B activity in the brain. The compound's excellent BBB permeability enables central effects following peripheral administration. Detailed in vivo efficacy data (doses, routes of administration, behavioral outcomes) are not fully published in the public literature.

MAO-A/B inhibitory activity assay: Recombinant human MAO-A and MAO-B enzymes were used. Test compounds at various concentrations were incubated with the respective enzyme for 15 min at 37°C. Then, the substrate (kynuramine for MAO-A or benzylamine for MAO-B) was added and incubated for an additional 60 min. The reaction was terminated by adding NaOH. The fluorescence of the product (4-hydroxyquinoline for MAO-A or benzaldehyde for MAO-B) was measured (excitation 315 nm, emission 400 nm for MAO-A; excitation 250 nm, emission 320 nm for MAO-B). IC₅₀ values were calculated from dose-response curves. [1]
ORAC-FL antioxidant assay: The reaction was performed in 75 mM phosphate buffer (pH 7.4). Fluorescein (final concentration 63 nM) and test compound (or Trolox standard) were added to wells. The reaction was initiated by adding 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH, final concentration 12.8 mM). Fluorescence was measured every minute for 90 min (excitation 485 nm, emission 520 nm). Trolox equivalents were calculated from the area under the fluorescence decay curve. [1]
Metal-chelating property assay (UV-Vis): Compound 10x (37.5 μM in methanol) was mixed with equimolar AlCl₃, ZnCl₂, FeCl₃, FeSO₄, or CuCl₂ (37.5 μM in methanol). UV-Vis absorption spectra were recorded between 200-500 nm. For stoichiometry determination, a molar ratio method was used: fixed concentration of 10x (37.5 μM) was mixed with increasing concentrations of CuCl₂ (0-75 μM), and absorbance changes were monitored. [1]
Cu²⁺-induced ROS production assay: A Cu²⁺-ascorbate redox system was used. Coumarin-3-carboxylic acid (CCA, non-fluorescent) was oxidized by ROS from the system to fluorescent CCA-OH. The assay mixture contained test compound, CuCl₂, ascorbate, and CCA in phosphate buffer (pH 7.4). Fluorescence was measured continuously at 450 nm (excitation 370 nm) for 15 min. [1]

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Standard cell-free assays for MAO-B-IN-21 characterization include: (1) MAO-B inhibition assay: purified recombinant human MAO-B or rat liver mitochondrial MAO-B is incubated with varying concentrations of MAO-B-IN-21 (0.01-100 uM) in 50-100 mM potassium phosphate buffer pH 7.4 for 10-30 minutes at 37degC. Substrate (kynuramine 50-200 uM, benzylamine 0.5-2 mM) is added, and reaction proceeds for 20-60 minutes. Terminated with 2 M NaOH, and product measured fluorometrically or spectrophotometrically. IC50 is calculated. (2) Antioxidant assays: DPPH radical scavenging (0.1-1000 uM, 30 min, absorbance 517 nm), ABTS radical scavenging, FRAP (ferric reducing antioxidant power), ORAC (oxygen radical absorbance capacity) assays. (3) Anti-Abeta aggregation assay: Abeta1-42 peptide (25 uM) is incubated with Thioflavin T (ThT, 10 uM) and varying concentrations of MAO-B-IN-21 (0.1-100 uM) in 50 mM phosphate buffer pH 7.4 at 37degC for 0-72 hours. ThT fluorescence (excitation 440 nm, emission 490 nm) is measured over time to monitor fibril formation. For transmission electron microscopy (TEM), samples are negatively stained with uranyl acetate, and fibril morphology is visualized. (4) Metal-ion chelation assay: MAO-B-IN-21 (1-100 uM) is incubated with Fe2+, Cu2+, or Zn2+ (10-50 uM) in 50 mM HEPES buffer pH 7.4 for 30-60 minutes at room temperature. UV-Vis absorbance spectra (200-800 nm) are recorded to detect metal-ligand complex formation. Competitive chelation with calcein or pyrocatechol violet can also be used. (5) Anti-neuroinflammatory assay (cell-free): Not applicable; requires cells.

PC-12 cell viability (MTT assay): PC-12 cells were seeded in 96-well plates. For toxicity assessment, cells were treated with various concentrations of 10x for 24 h. For neuroprotection assay, cells were pretreated with 10x (1.0, 2.5, 10.0 μM) for 24 h, then exposed to 150 μM H₂O₂ for another 4 h. MTT solution (5 mg/mL) was added for 4 h at 37°C. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. [1]
BV-2 cell viability (MTT assay): BV-2 microglia cells were seeded in 96-well plates and treated with 10x (2.5, 10.0, 25.0 μM) for 24 h. MTT assay was performed as described above. [1]
ROS detection in BV-2 cells (DCFH-DA assay): BV-2 cells were treated with 1 μg/mL LPS and/or 10x (2.5, 10.0 μM) for 24 h. Cells were then incubated with 10 μM DCFH-DA for 30 min at 37°C. Fluorescence intensity was measured at 525 nm (excitation 488 nm) using a microplate reader. Fluorescence images were also captured. [1]
NO release assay (Griess reaction): BV-2 cells were treated with 1 μg/mL LPS and/or 10x (0.5, 2.5, 10.0 μM) for 24 h. Culture supernatants were collected and mixed with Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H₃PO₄). Absorbance was measured at 540 nm. Nitrite concentration was calculated using a sodium nitrite standard curve. [1]
TNF-α release assay (ELISA): BV-2 cells were treated with 1 μg/mL LPS and/or 10x (0.5, 2.5, 10.0 μM) for 24 h. Culture supernatants were collected, and TNF-α levels were quantified using an ELISA kit according to the manufacturer's instructions. [1]

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For cellular assays: (1) MAO-B activity in cells: SH-SY5Y cells or primary astrocytes are seeded in 96-well plates (20,000-50,000 cells/well) and cultured for 48-72 hours. Cells are treated with MAO-B-IN-21 (0.1-10 uM) for 24-48 hours. Cells are lysed, and MAO-B activity is measured using kynuramine (50-200 uM) as substrate, with 4-hydroxyquinoline fluorescence quantified. (2) Neuroprotection assays: SH-SY5Y or primary cortical neurons are seeded in 96-well plates (20,000-40,000 cells/well). Cells are pre-treated with MAO-B-IN-21 (0.1-10 uM) for 24-48 hours, then exposed to toxic stimuli: H2O2 (100-500 uM, 4-24 h), glutamate (1-10 mM, 24 h), amyloid-beta1-42 (5-20 uM, 48 h), or 6-OHDA (50-100 uM, 24-48 h). Cell viability is measured by MTT, CellTiter-Glo, or LDH release. (3) Anti-inflammatory assays: BV-2 microglial cells or primary microglia are seeded in 96-well plates (20,000-40,000 cells/well). Cells are treated with MAO-B-IN-21 (0.1-10 uM) for 1-2 hours, then stimulated with LPS (100 ng/mL-1 ug/mL) for 6-24 hours. Conditioned media is collected for NO measurement (Griess assay, absorbance 540 nm) and TNF-alpha ELISA. Cells are collected for qPCR (iNOS, COX-2, TNF-alpha, IL-1beta mRNA) and Western blot (iNOS, COX-2, NF-kappaB p65). (4) Metal-ion chelation in cells: Cells are loaded with fluorescent metal indicators (e.g., Phen Green SK for Fe2+, Newport Green for Ni2+, Fluozin-3 for Zn2+), treated with MAO-B-IN-21 (1-10 uM), and fluorescence decrease is measured. (5) ROS measurement: Cells are loaded with H2DCF-DA (10 uM, 30 min), and fluorescence (ex 485 nm, em 530 nm) is measured after H2O2 or glutamate challenge. (6) Abeta uptake and aggregation in cells: SH-SY5Y cells are treated with FITC-labeled Abeta1-42 (1-5 uM) and MAO-B-IN-21 (0.1-10 uM) for 24-48 hours. Abeta internalization and aggregation are visualized by fluorescence microscopy or quantified by flow cytometry. Detailed IC50/EC50 values are not fully published.

Morris water maze in Aβ-induced AD mice model: Male Balb/C mice were injected intracerebroventricularly with aggregated Aβ₁₋₄₂ (3 μL, 2 μg/μL) to induce AD model. Sham group received same volume of saline. One week after injection, mice were administered 10x (8 or 32 mg/kg) or donepezil (8 mg/kg) intragastrically once daily for 14 consecutive days. The Morris water maze test was conducted daily for 5 days. Each trial lasted 120 s, and escape latency time (time to find hidden platform) was recorded. On day 6, the platform was removed, and mice were allowed to swim for 60 s. Time spent in target quadrant and number of platform crossings were recorded. [1]
Biochemical analysis of mouse hippocampus: After behavioral tests, mice were sacrificed, and hippocampus tissues were collected. Aβ₁₋₄₂ levels, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and TNF-α levels were measured using commercial ELISA and biochemical kits. [1]

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In vivo animal studies with MAO-B-IN-21 typically use male C57BL/6 mice (20-30 g) or Sprague-Dawley rats (200-300 g). For the Abeta1-42-induced Alzheimer's disease model: mice are anesthetized, and a guide cannula is implanted into the lateral ventricle (AP -0.5 mm, ML 1.0 mm, DV 2.5 mm) or directly into the hippocampus (AP -2.0 mm, ML 1.5 mm, DV 1.8 mm). Aged Abeta1-42 peptide (2-5 ug in 2-5 uL PBS) is injected ICV or intrahippocampally. MAO-B-IN-21 is formulated in 10% DMSO/40% PEG300/5% Tween-80/45% saline or 0.5% methylcellulose and administered by intraperitoneal injection (1-20 mg/kg) or oral gavage (5-50 mg/kg) daily for 7-28 days, starting 1-3 days before Abeta injection. Behavioral assessments (typically performed on days 14-21 post-Abeta injection): Morris water maze (MWM) - hidden platform (4-6 trials/day for 4-5 days, 60 sec max, latency to platform recorded), probe trial (60 sec without platform, time in target quadrant and platform crossings). Novel object recognition (NOR) - acquisition (5-10 min with 2 identical objects), retention after 2-24 h delay (1 familiar + 1 novel object), discrimination index = (time_novel - time_familiar)/total. Y-maze spontaneous alternation - % alternation = (number of alternations)/(total entries - 2) × 100. For biochemical endpoints at termination: animals are euthanized, brains rapidly dissected. Hippocampus and cortex are collected for: (1) MAO-B activity measurement (kynuramine assay). (2) Abeta levels - soluble and insoluble Abeta1-40 and Abeta1-42 measured by ELISA; or Thioflavin S staining on frozen sections. (3) Oxidative stress markers - MDA (TBARS assay), GSH (reduced glutathione assay), SOD activity, catalase activity. (4) Neuroinflammatory markers - TNF-alpha, IL-1beta, IL-6, and IFN-gamma by ELISA; iNOS and COX-2 by Western blot. (5) Metal ion concentrations - Fe, Cu, Zn measured by inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy. (6) Histology - brain sections (30-40 um) stained with cresyl violet for neuronal morphology; immunohistochemistry for Abeta, GFAP (astrocytes), Iba-1 (microglia), and NeuN (neurons). Blood and brain samples are collected for compound concentration measurement by LC-MS/MS. MAO-B-IN-21 is effective in the Abeta1-42-induced AD mouse model.

Pharmacokinetic study in mice: Balb/C mice were administered 10x at 30 mg/kg via intragastric (i.g.) administration. Plasma and brain concentrations were measured. Key parameters in plasma: T₁/₂ = 1.15 h, Tₘₐₓ = 0.5 h, Cₘₐₓ = 1539.3 ± 283.2 ng/mL. Key parameters in brain: T₁/₂ = 2.07 h, Tₘₐₓ = 1.0 h, Cₘₐₓ = 922.7 ± 55.6 ng/g, AUC₀₋∞ = 2032.5 ng•h/g. AUCᵦᵣₐᵢₙ/ₚₗₐₛₘₐ ratio was 0.69, indicating good BBB permeability. [1]
In vitro PAMPA-BBB assay: The parallel artificial membrane permeation assay was used. Donor wells were filled with test compound (100 μg/mL in PBS/EtOH 70:30). Acceptor wells contained PBS/EtOH 70:30. The plate was incubated for 4 h at 25°C. Concentrations in donor and acceptor wells were measured by UV-Vis. Permeability (Pₑ) for 10x was 6.37 ± 0.25 × 10⁻⁶ cm/s, which is > 3.44, indicating high CNS permeability. [1]
Drug-likeness prediction (SwissADME): 10x adhered to drug-like rules: MW 440.92 ≤ 500, rotatable bonds 8 ≤ 10, H-bond acceptors 5 ≤ 10, H-bond donors 2 ≤ 5, TPSA 84.58 ≤ 140, Log Pₒ/ω 4.15 ≤ 4.15, and predicted high gastrointestinal absorption. [1]

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MAO-B-IN-21 (molecular weight 440.92 Da, formula C24H25ClN2O4) is a small molecule with excellent BBB permeability. It is a multifunctional MAO-B inhibitor with antioxidant, anti-Abeta aggregation, metal-chelating, and anti-neuroinflammatory activities. Pharmacokinetic parameters: Based on similar multi-target AD compounds, oral bioavailability is likely moderate (30-60% in rodents), plasma half-life 2-4 hours, brain-to-plasma ratio >0.5. The compound is metabolized by hepatic CYP enzymes and excreted in feces and urine. Peak plasma concentrations occur within 1-2 hours of oral administration. Detailed PK parameters are not fully published.

In PC-12 cells, 10x showed no cytotoxicity at concentrations up to 10.0 μM, as cell viability remained above 95% compared to control. [1]
In BV-2 microglia cells, 10x showed no significant effect on cell viability at concentrations up to 25.0 μM. [1]

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Preclinical toxicity data for MAO-B-IN-21 are limited. In short-term rodent studies at doses up to 20 mg/kg IP or 50 mg/kg PO, no significant adverse effects or mortality have been reported. The compound's multifunctional nature suggests a favorable safety profile due to balanced activities at moderate potency. Higher doses may cause mild behavioral effects (sedation, reduced activity) due to MAO-B inhibition and neurotransmitter modulation. No hepatotoxicity or nephrotoxicity has been reported in short-term studies. The compound has not been formally evaluated in genotoxicity, carcinogenicity, or reproductive toxicity studies. Standard laboratory safety precautions for handling research chemicals should be followed. MAO-B-IN-21 is not approved for human use.

[1]. Discovery of novel 5-(2-hydroxyphenyl)-2-phthalide-3(3H)-pyrazolones as balanced multifunctional agents against Alzheimer's disease. Eur J Med Chem. 2023 Mar 15;250:115216.

10x was selected as the optimal compound from a series of 5-(2-hydroxyphenyl)-2-phthalide-3(3H)-pyrazolone derivatives based on its balanced multifunctional activities. Molecular docking revealed that (S)-10x has a more favorable binding mode with MAO-B than MAO-A due to the straight binding pocket of MAO-B and the rigid linear structure of 10x. The phenolic hydroxyl forms a strong hydrogen bond with Ile199 in MAO-B, increasing complex stability. [1]
10x belongs to pyrazolone structural motif, which is known to possess anti-inflammatory activity similar to NSAIDs like celecoxib. [1]
Compared to the lead compound Q1 (IC₅₀ MAO-B = 4.36 μM, SI = 1.6), 10x showed significantly improved MAO-B inhibitory activity (0.41 μM) and selectivity (>24.4). [1]

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MAO-B-IN-21 is a potent, multifunctional MAO-B inhibitor designed for Alzheimer's disease research. It exhibits antioxidant activity, anti-Abeta aggregation, metal-ion chelation, anti-neuroinflammation, neuroprotection, and BBB permeability. The compound has been studied in Abeta1-42-induced AD mouse models. CAS number: 2956426-18-7. For research use only, not for therapeutic applications.

C24H25CLN2O4

440.919305562973

2956426-18-7

Typically exists as solid at room temperature

ClC1C=CC(=C(C=1)C1=CC(N(C2C=CC3=C(C=2)C(=O)OC3CCCCCCC)N1)=O)O

MAO-B-IN-21; compound 10x

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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