PDE/phosphodiesterase

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].

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

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.

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.

Dissolved in saline; 0.3 and 1.0 mg/kg; i.v.

Carotid artery thrombosis model

Biological Half-Life
19 hours

rat LD50 oral 1340 mg/kg SENSE ORGANS AND SPECIAL SENSES: LACRIMATION: EYE Kiso to Rinsho. Clinical Report., 19(5503), 1985
rat LD50 intraperitoneal 419 mg/kg SENSE ORGANS AND SPECIAL SENSES: LACRIMATION: EYE Kiso to Rinsho. Clinical Report., 19(5503), 1985
rat LD50 subcutaneous 1300 mg/kg KIDNEY, URETER, AND BLADDER: HEMATURIA Kiso to Rinsho. Clinical Report., 19(5503), 1985
mouse LD50 oral 1860 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Kiso to Rinsho. Clinical Report., 19(5503), 1985
mouse LD50 intraperitoneal 460 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Kiso to Rinsho. Clinical Report., 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.

Ibudilast is a pyrazolopyridine.
Ibudilast is an anti-inflammatory and neuroprotective oral agent which shows an excellent safety profile at 60 mg/day and provides significantly prolonged time-to-first relapse and attenuated brain volume shrinkage in patients with relapsing-remitting (RR) and/or secondary progressive (SP) multiple sclerosis (MS). Ibudilast is currently in development in the U.S. (codes: AV-411 or MN-166), but is approved for use as an antiinflammatory in Japan.
Ibudilast is an orally bioavailable inhibitor of cyclic nucleotide phosphodiesterase (PDE), mainly PDE-3, -4, -10, and -11, with anti-(neuro)inflammatory, vasorelaxant, bronchodilator, analgesic, neuroprotective and potential anti-tumor activities. Ibudilast (IBD) is able to cross the blood-brain barrier (BBB). Upon administration, IBD exerts its potential anti-tumor activity against glioblastoma multiforme (GBM) cells by inhibiting PDE-4 and the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which results in a decrease in MIF, its receptor CD74, and AKT expression, and attenuates the immunosuppressive properties of monocytic myeloid-derived suppressor cells (MDSCs) and reduces T-regulatory cells (Tregs). This causes GBM cell apoptosis and inhibits GBM cell proliferation. In addition, IBD reduces, through its inhibitory effect on various PDEs, the production of certain pro-inflammatory cytokines, such as interleukin-6 (IL-6), IL- 1beta, leukotriene B4, and tumor necrosis factor-alpha (TNF-a). 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 neurotrophin-4 (NT-4). It also blocks toll-like receptor-4 (TLR-4), inhibits nitric oxide (NO) synthesis and reduces the level of reactive oxygen species (ROS). It also prevents platelet aggregation, causes cerebral vasodilation, bronchial smooth muscle relaxation, and improves cerebral blood flow. In addition, IBD attenuates the PDE-mediated activation of glial cells and abrogates PDE-mediated neuroinflammation and neurodegeneration. MIF is secreted by cancer stem cells (CSCs) and is highly expressed within GBM and plays a key role in tumor cell proliferation. Co-expression of MIF and CD74 in GBM is associated with poor patient survival.

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Drug Indication
For the treatment of multiple sclerosis, asthma, and cerebrovascular disease.

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Mechanism of Action
Ibudilast has mechanisms that include anti-inflammatory effects, such as phosphodiesterase inhibition, and neuroprotective effects, such as inhibition of [nitric oxide] synthesis and reduction in reactive oxygen species.

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The phosphodiesterase inhibitor, ibudilast, has many effects on lymphocytes, endothelial cells, and glial cells. We examined the neuroprotective role of ibudilast in neuron and microglia co-cultures. Ibudilast significantly suppressed neuronal cell death induced by the activation of microglia with lipopolysaccharide (LPS) and interferon (IFN)-gamma. To examine the mechanisms by which ibudilast exerts a neuroprotective role against the activation of microglia, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors following ibudilast treatment. In a dose-dependent manner, ibudilast suppressed the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha and enhanced the production of the inhibitory cytokine, IL-10, and additional neurotrophic factors, including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4 in activated microglia. Thus, ibudilast-mediated neuroprotection was primarily due to the inhibition of inflammatory mediators and the upregulation of neurotrophic factor. In the CA1 region of hippocampal slices, long-term potentiation (LTP) induced by high frequency stimulation (HFS) could be inhibited with LPS and interferon-gamma stimulation. Ibudilast returned this LTP inhibition to the levels observed in controls. These results suggest that ibudilast may be a useful neuroprotective and anti-dementia agent counteracting neurotoxicity in activated microglia.[1]

C14H18N2O

230.31

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

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.)