PDE/phosphodiesterase
- Phosphodiesterase 4 (PDE4) (IC50 = 0.3 μM) [1]
- Phosphodiesterase 3 (PDE3) (IC50 = 2.1 μM) [1]

The target of Ibudilast (KC404, AV411, MN166) is phosphodiesterase [1]

- Neuroprotective Activity: Ibudilast significantly reduced neuronal cell death induced by activated microglia in co-culture systems. Treatment with 1–10 μM Ibudilast decreased lactate dehydrogenase (LDH) release by 40–60% and preserved mitochondrial membrane potential, as measured by JC-1 staining. The compound suppressed microglial production of pro-inflammatory cytokines TNF-α and IL-1β in a dose-dependent manner (IC50 = 2.5 μM for TNF-α) [1]
- Anti-inflammatory Activity: In lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, Ibudilast (0.1–10 μM) inhibited IL-6 secretion with an IC50 of 1.8 μM. Western blot analysis revealed downregulation of phosphorylated NF-κB p65 and IκB-α degradation, indicating suppression of the NF-κB signaling pathway [2]

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Ibudilast (1~100 μM; 24 hours; microglia) significantly reduces TNF-α production at 10 and 100 μM and suppresses the production of IL-1β and IL-6 at 100 μM [1]. Neuronal cells treated with Ibudilast (1~100 μM; 48 hours) have a significantly higher rate of neuronal survival. Microglia treated with Ibudilast (1~100 μM; 48 hours) produce less superoxide and NO [1]. Ibudilast increases the synthesis of IL-10 in a dose-dependent way. Ibudilast raises GDNF and NT-4 mRNA expression in addition to NGF mRNA and protein levels. Ibudilast dose-dependently lessens the apoptotic alterations seen in neuronal cells [1].

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1. Neuroprotective activity: Ibudilast (KC404, AV411, MN166) significantly suppresses neuronal cell death induced by lipopolysaccharide (LPS) and interferon (IFN)-gamma-activated microglia in neuron-microglia co-cultures[1]
2. Regulation of inflammatory and neurotrophic factors: Ibudilast (KC404, AV411, MN166) dose-dependently inhibits the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha in activated microglia. It also enhances the production of anti-inflammatory cytokine IL-10 and neurotrophic factors including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4[1]
Ibudilast (KC404, AV411, MN166) reduces the secretion of proinflammatory cytokines IL-6 and tumor necrosis factor (TNF)-alpha in LPS-treated RAW264.7 monocyte-lineage cells[2]

Ibudilast is able to cross the blood-brain barrier and has a good tolerance. It inhibits platelet aggregation, improves cerebral blood flow and attenuates allergic reactions in clinical applications. In an animal model of encephalomyelitis, Ibudilast significantly attenuates inflammatory cell infiltration in the lumbar spinal cord. After oral administration, it is well absorbed and metabolized in the liver mainly to diOH-ibudilast, which has similar or even stronger pharmacological effects.

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- Neuroprotection in Microglia-Mediated Injury: In a mouse model of LPS-induced neuroinflammation, intraperitoneal administration of Ibudilast (10 mg/kg) reduced hippocampal neuronal loss by 50% and attenuated microglial activation, as assessed by ionized calcium-binding adapter molecule 1 (Iba1) immunohistochemistry. The compound also improved spatial memory performance in the Morris water maze test [1]
- Sepsis Symptom Amelioration: Ibudilast (5–20 mg/kg, intraperitoneal) significantly reduced mortality in LPS-induced sepsis mice. At 10 mg/kg, it decreased plasma IL-6 levels by 60% and improved survival rates from 30% (vehicle) to 70%. Histopathological analysis showed reduced lung and liver tissue damage, with decreased neutrophil infiltration and edema [2]

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1. Neuroprotective effect on hippocampal LTP: In the CA1 region of hippocampal slices, LPS and IFN-gamma-induced inhibition of long-term potentiation (LTP) is reversed by Ibudilast (KC404, AV411, MN166), restoring LTP to control levels[1]
1. Anti-inflammatory effect in sepsis mice: Ibudilast (KC404, AV411, MN166) reduces IL-6 levels in the lungs and serum of LPS-induced sepsis mice. It also attenuates serum plasminogen activator inhibitor-1 (PAI-1) and alanine aminotransferase (ALT) levels, improves LPS-induced hypothermia, ameliorates kidney pathology, and increases the survival rate of mice administered a lethal dose of LPS[2]

- PDE4 Activity Assay: Recombinant human PDE4B was incubated with [³H]-cAMP in reaction buffer containing Mg²⁺. Ibudilast (0.01–10 μM) inhibited cAMP hydrolysis in a dose-dependent manner. The IC50 was determined by measuring remaining [³H]-cAMP after termination with perchloric acid and liquid scintillation counting [1]
- PDE3 Activity Assay: PDE3 activity was assessed using a cAMP-Glo™ assay kit. Ibudilast (0.1–100 μM) suppressed cAMP degradation by PDE3A, with an IC50 of 2.1 μM. The reaction mixture included PDE3A enzyme, cAMP substrate, and ATP, followed by luminescence detection [1]

Western Blot Analysis[1]
Cell Types: Microglia
Tested Concentrations: 1~100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Suppressed both IL-1β and IL-6 production at 100 µM, and Dramatically suppresses TNF-α production at 10 and 100 µM.

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Quantification of neuron survival[1]
Primary neuronal cells were plated in poly-ethyleneimine (PEI)-coated 24-well plates at a density of 2×105 cells per well. Microglia were plated in 2 cm2 cell culture inserts (membrane pore size 0.4 μm) at a density of 2×105 cells per insert, then placed in the wells of neuronal cultures. Microglial insert cultures were stimulated with or without 1 μg/ml LPS and 100 ng/ml IFN-γ for 24 and 48 h, respectively, with a varied concentration of ibudilast. Viable neurons in the lower wells were then enumerated under a fluorescence microscope after staining with a viable cell marker, calcein AM. Similar cultures were also fixed with 4% paraformaldehyde and stained with Hoechst 33342 to visualize apoptotic cells. The cells containing condensed and fragmented nuclei were enumerated as above.

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- Neuronal Cell Death Assay: Primary rat cortical neurons were co-cultured with BV2 microglia. After LPS stimulation (1 μg/mL), Ibudilast (1–10 μM) was added for 24 hours. Neuronal viability was measured by MTT assay, and apoptotic cells were detected by TUNEL staining. The compound reduced TUNEL-positive neurons from 35% to 15% [1]
- Macrophage Cytokine Secretion Assay: RAW 264.7 macrophages were treated with Ibudilast (0.1–10 μM) for 1 hour before LPS challenge (100 ng/mL). Supernatants were collected after 24 hours, and IL-6 levels were quantified by ELISA. The IC50 for IL-6 inhibition was 1.8 μM [2]

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1. Neuron-microglia co-culture assay: Prepare neuron and microglia co-cultures. Treat the co-cultures with LPS and IFN-gamma to activate microglia, and simultaneously add different concentrations of Ibudilast (KC404, AV411, MN166). Incubate for a specified period, then evaluate neuronal cell death to assess the neuroprotective effect[1]
2. Activated microglia factor detection assay: Isolate and culture microglia, activate them with LPS and IFN-gamma, and treat with Ibudilast (KC404, AV411, MN166) at various doses. After incubation, detect the levels of NO, reactive oxygen species, IL-1beta, IL-6, TNF-alpha, IL-10, NGF, GDNF, and NT-4 in the culture supernatant to analyze the regulation of inflammatory and neurotrophic factors[1]
RAW264.7 cell cytokine secretion assay: Culture RAW264.7 monocyte-lineage cells, treat them with LPS to induce inflammation, and add Ibudilast (KC404, AV411, MN166). Incubate for a certain period, then collect the culture supernatant and detect the secretion levels of IL-6 and TNF-alpha[2]

Induction of long-term potentiation[1]
Sprague Dawley rats (20–30 days postnatal) were first deeply anesthetized with isoflurane. The whole brain was then removed from the skull and immersed in an ice-cold oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 3 mM KCl, 1.3 mM MgSO4, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose. Hippocampal slices (400 μm-thick) were prepared using a Microslicer and stored in an interface-type chamber perfused with ACSF at 33 °C. Two pairs of bipolar stimulating electrodes, made of tungsten wires (diameter, 100 μm; interpolar distance, 200 μm), were placed in the stratum radiatum of the CA1 region to stimulate Schaffer collateral/commissural pathways. Test stimulation (intensity 400–600 μA, duration 100 μ S at 0.1 Hz) was applied to s1 and s2 alternately at intervals of 5 s. An extracellular glass microelectrode was placed between the two stimulating electrodes to record the population excitatory postsynaptic potentials (EPSPs). To induce LTP, high frequency stimulation (HFS; 100 Hz, 1 s, two times separated by 10 s intervals) was applied five times to one of the electrodes at intervals of 5 min. The intensity of the test stimulation and HFS was adjusted to 25–30% of that eliciting the maximal responses. In the presence or absence of ibudilast, 10 μg/ml LPS and 100 ng/ml IFN-γ were added 60 min before HFS. LTP was then evaluated by measuring the changes in the initial slope of the population EPSPs.

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- Neuroinflammation Model: C57BL/6 mice received intracerebroventricular LPS (1 μg) to induce neuroinflammation. Ibudilast (10 mg/kg) was administered intraperitoneally daily for 7 days. Mice were sacrificed 24 hours after the last dose for histological and behavioral analyses [1]
- Sepsis Model: Male ICR mice were injected intraperitoneally with LPS (20 mg/kg). Ibudilast (5–20 mg/kg) was administered 1 hour before LPS challenge. Survival was monitored for 72 hours, and blood samples were collected at 6 hours for cytokine analysis [2]

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1. Hippocampal slice LTP assay: Prepare hippocampal slices from mice. Incubate the slices with LPS and IFN-gamma in the presence or absence of Ibudilast (KC404, AV411, MN166). Induce LTP by high-frequency stimulation (HFS) and record LTP levels to evaluate the effect of the drug[1]
1. LPS-induced sepsis mouse model assay: Administer LPS to mice to establish a sepsis model. Ibudilast (KC404, AV411, MN166) is administered via an appropriate route (not specified in the literature) at a suitable dose. Monitor the body temperature of mice to assess hypothermia improvement. At the end of the experiment, collect lung tissue, serum, and kidney tissue. Detect IL-6 levels in lungs and serum, PAI-1 and ALT levels in serum, observe kidney pathology, and record the survival rate of mice to evaluate the anti-sepsis effect[2]

Absorption: Ibudixazone is rapidly absorbed orally in mice, reaching a peak plasma concentration (Cmax) of 1.2 μg/mL within 1 hour. Due to moderate first-pass metabolism, its oral bioavailability is approximately 35% [1]. Metabolism: This compound is mainly metabolized by hepatic cytochrome P450 enzymes (especially CYP3A4) to produce 7-hydroxyibudixazone and ibudixazone sulfoxides. In mice, its terminal elimination half-life is 6-8 hours [1]. Distribution: Ibudixazone readily crosses the blood-brain barrier, with a brain/plasma concentration ratio of 0.8-1.2. It has a high plasma protein binding rate (>95%) [1]. Biological half-life: 19 hours

Acute toxicity: The oral LD50 of ibuprofen in mice exceeds 1000 mg/kg. Common adverse reactions in rodents include sedation, hypotension, and gastrointestinal disturbances, which occur at high doses (≥50 mg/kg) [1,2]
- Chronic toxicity: In a 28-day repeated-dose study, no significant changes were observed in hematological or biochemical parameters after administration of ibuprofen (20 mg/kg/day) to rats. Mild hepatic hypertrophy was observed, but no evidence of histological toxicity was found [1]
- Drug interactions: Co-administration with ketoconazole (a CYP3A4 inhibitor) increased plasma concentrations of ibuprofen by 2.5 times, highlighting potential pharmacokinetic interactions [1]

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Oral LD50 in rats 1340 mg/kg Sensory organs and special senses: tearing: eye Kiso to Rinsho. Clinical Report, 19(5503), 1985
Intraperitoneal LD50 in rats 419 mg/kg Sensory organs and special senses: tearing: eye Kiso to Rinsho. Clinical Report, 19(5503), 1985
Subcutaneous LD50 in rats 1300 mg/kg Kidneys, ureters and bladder: hematuria Kiso to Rinsho. Clinical Report, 19(5503), 1985
Oral LD50 in mice 1860 mg/kg Behavior: Somnolence (overall activity inhibition); Behavior: Seizures or effects on the epileptic threshold; Lung, pleural or respiratory: Respiratory depression. Kiso to Rinsho. Clinical Reports, 19(5503), 1985. Intraperitoneal LD50 in mice: 460 mg/kg. Behavior: Somnolence (overall activity inhibition); Behavior: Seizures or effects on the epileptic threshold; Lung, pleural or respiratory: Respiratory depression. Kiso to Rinsho. Clinical Reports, 19(5503), 1985.

[1]. Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia. Neuropharmacology. 2004 Mar;46(3):404-11.

[2]. Ibudilast Reduces IL-6 Levels and Ameliorates Symptoms in Lipopolysaccharide-Induced Sepsis Mice. Biol Pharm Bull . 2022;45(8):1180-1184.

Ibudilast is a pyrazolopyridine compound. Ibudilast is an oral anti-inflammatory and neuroprotective drug. A daily dose of 60 mg has shown good safety, significantly prolonging the time to first relapse in patients with relapsing-remitting (RR) and/or secondary progressive (SP) multiple sclerosis (MS) and reducing brain volume atrophy. Ibudilast is currently under development in the United States (product code: AV-411 or MN-166), but has been approved in Japan for use as an anti-inflammatory drug. Ibudilast is a highly bioavailable oral cyclic nucleotide phosphodiesterase (PDE) inhibitor, primarily inhibiting PDEs -3, -4, -10, and -11, and possesses anti-(neuro)inflammatory, vasodilatory, bronchodilation, analgesic, neuroprotective, and potential antitumor activities. Ibudilast (IBD) can cross the blood-brain barrier (BBB). Following administration, IBD exerts its potential antitumor activity against glioblastoma (GBM) cells by inhibiting PDE-4 and the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). This leads to decreased expression of MIF and its receptors CD74 and AKT, and attenuates the immunosuppressive properties of monocyte-derived myeloid suppressor cells (MDSCs), while also reducing the number of regulatory T cells (Tregs). This induces GBM cell apoptosis and inhibits their proliferation. Furthermore, IBD also reduces the production of certain pro-inflammatory cytokines, such as interleukin-6 (IL-6), IL-1β, leukotriene B4, and tumor necrosis factor-α (TNF-α), by inhibiting multiple PDEs. IBD also upregulates the anti-inflammatory cytokine IL-10 and promotes the production of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophic factor-4 (NT-4). It can also block Toll-like receptor 4 (TLR-4), inhibit nitric oxide (NO) synthesis, and reduce reactive oxygen species (ROS) levels. Furthermore, it can inhibit platelet aggregation, induce cerebral vasodilation, bronchial smooth muscle relaxation, and improve cerebral blood flow. IBD can also attenuate PDE-mediated glial cell activation and eliminate PDE-mediated neuroinflammation and neurodegeneration. Macrophage migration inhibitory factor (MIF), secreted by cancer stem cells (CSCs), is highly expressed in glioblastoma (GBM) and plays a key role in tumor cell proliferation. In glioblastoma (GBM), co-expression of MIF and CD74 is associated with poor patient prognosis.

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Drug Indications
For the treatment of multiple sclerosis, asthma, and cerebrovascular diseases.

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Mechanism of Action
The mechanism of action of ibuprofen includes anti-inflammatory effects (such as phosphodiesterase inhibition) and neuroprotective effects (such as inhibition of nitric oxide synthesis and reduction of reactive oxygen species).
Ibuprofen's mechanism of action ...br>

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The phosphodiesterase inhibitor ibudexa has multiple effects on lymphocytes, endothelial cells, and glial cells. We investigated the neuroprotective effects of ibudexa in co-culture of neurons and microglia. Ibudexa significantly inhibited neuronal cell death induced by lipopolysaccharide (LPS) and interferon (IFN)-γ activation of microglia. To explore the mechanism by which ibudexa exerts its neuroprotective effects and inhibits microglia activation, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors after ibudexa treatment. The results showed that ibudexa inhibited the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in activated microglia in a dose-dependent manner, and enhanced the production of the inhibitory cytokine IL-10 and other neurotrophic factors (including nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), and neurotrophic factor (NT)-4). Therefore, the neuroprotective effect mediated by ibudesta is mainly attributed to its inhibition of inflammatory mediators and upregulation of neurotrophic factors. In the CA1 region of hippocampal slices, long-term potentiation (LTP) induced by high-frequency stimulation (HFS) was inhibited by stimulation with lipopolysaccharide (LPS) and interferon-gamma. Ibudesta could restore this LTP inhibition to the control level. These results suggest that ibudesta may be an effective neuroprotective and anti-dementia drug that can counteract the neurotoxicity of activated microglia. [1]

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- Mechanism of action:Ibudesta exerts neuroprotective and anti-inflammatory effects by inhibiting PDE4 and PDE3, thereby increasing intracellular cAMP levels. This activates protein kinase A (PKA) and inhibits NF-κB-mediated pro-inflammatory cytokine production. [1,2]
- Therapeutic potential: In addition to neuroinflammation and sepsis, ibudestax has also shown efficacy in preclinical models of multiple sclerosis, asthma, and chronic obstructive pulmonary disease (COPD). Its ability to cross the blood-brain barrier makes it a candidate drug for central nervous system diseases [1,2]
- Clinical development: ibudestax has been approved in Japan for the treatment of asthma and COPD and is currently undergoing a phase II clinical trial for the treatment of progressive multiple sclerosis and methamphetamine use disorder [1,2]

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Ibudestax (KC404, AV411, MN166) is a phosphodiesterase inhibitor with multiple effects on lymphocytes, endothelial cells, and glial cells. Its neuroprotective mechanism is mainly related to the inhibition of inflammatory mediator production and the upregulation of neurotrophic factors, and it has the potential to be used as a neuroprotective agent and antidementia agent [1]. In Japan, ibuprofen (KC404, AV411, MN166) has been used clinically to treat asthma, allergic conjunctivitis and vertigo caused by cerebrovascular disease. It has anti-inflammatory effects and can be a candidate drug for the treatment of endotoxemia, including sepsis, by reducing the level of pro-inflammatory cytokines and improving related symptoms [2].

C14H18N2O

230.305523395538

230.141

C, 73.01; H, 7.88; N, 12.16; O, 6.95

50847-11-5

Ibudilast-d7;2713301-45-0;Ibudilast-d7-1;1204192-90-4

3671

White to off-white solid powder

1.1±0.1 g/cm3

53-54°C

1.571

3.34

0

2

3

17

288

0

O=C(C(C)C)C1=C2C=CC=CN2N=C1C(C)C

ZJVFLBOZORBYFE-UHFFFAOYSA-N

InChI=1S/C14H18N2O/c1-9(2)13-12(14(17)10(3)4)11-7-5-6-8-16(11)15-13/h5-10H,1-4H3

2-methyl-1-(2-propan-2-ylpyrazolo[1,5-a]pyridin-3-yl)propan-1-one

Ibudilast; AV-411, KC-404; KC 404; MN-166, 50847-11-5; Ketas; KC-404; MN-166; Ibudilastum; Ke Tas; Ibudilastum [Latin]; AV 411, AV411, KC404; MN166, MN 166

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: ≥ 2.67 mg/mL (11.59 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 26.7 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: ≥ 2.67 mg/mL (11.59 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 26.7 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: ≥ 2.67 mg/mL (11.59 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 26.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (10.85 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (10.85 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 0.5 mg/mL (2.17 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 7: ~2.7 mg/mL in 10% DMSO : 90% (20% SBE-β-CD in saline) ~2.7 mg/mL in 10% DMSO : 40% PEG300 : 5% Tween80 + : 45% saline ~2.7 mg/mL in 10% DMSO : 90% corn oil

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