Mitoglitazone (MSDC-0160; CAY10415), a thiazolidinedione analog,is the prototypical mTOT(mitochondrial target of thiazolidinediones)-modulating insulin sensitizer being investigated to treat diabetes and Alzheimer's disease. It is an insulin sensitizer free of side effects that are dependent on the peroxisome proliferator-activated receptor-γ (PPAR-γ). Insulin content is preserved and the resistance of the insulin-signaling pathway is decreased when islets are co-incubated with MSDC-0160 and IGF-1. Significantly reducing the loss of insulin content is also prevented by MSDC-0160 at 50μM. In addition, MSDC-0160 treatment results in AMPK phosphorylation being increased while mTOR phosphorylation is decreased. Furthermore, it is discovered that MSDC-0160 exhibits pro-survival effects in human islets by raising the expression of apoptosis-related genes like bcl2 and decreasing the amount of cleaved caspase-3.

Physicochemical Properties

Molecular Formula	C19H18N2O4S
Molecular Weight	370.4222
Exact Mass	370.098
Elemental Analysis	C, 61.61; H, 4.90; N, 7.56; O, 17.28; S, 8.65
CAS #	146062-49-9
Related CAS #	1628078-14-7 (R-isomer);146062-49-9 (racemic);1628078-13-6 (S-isomer);
PubChem CID	10429242
Appearance	Off-white to light yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	623.8±55.0 °C at 760 mmHg
Flash Point	331.1±31.5 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.619
LogP	2.84
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	7
Heavy Atom Count	26
Complexity	534
Defined Atom Stereocenter Count	0
SMILES	S1C(N([H])C(C1([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C(C1C([H])=C([H])C(=C([H])N=1)C([H])([H])C([H])([H])[H])=O)=O)=O
InChi Key	IRNJSRAGRIZIHD-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H18N2O4S/c1-2-12-5-8-15(20-10-12)16(22)11-25-14-6-3-13(4-7-14)9-17-18(23)21-19(24)26-17/h3-8,10,17H,2,9,11H2,1H3,(H,21,23,24)
Chemical Name	5-[[4-[2-(5-ethylpyridin-2-yl)-2-oxoethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione
Synonyms	Mitoglitazone; CAY10415; MSDC 0160; CAY 10415; MSDC-0160;CAY-10415; CAY 10415; CAY-10415; MSDC0160; CAY10415; 5-(4-(2-(5-ethylpyridin-2-yl)-2-oxoethoxy)benzyl)thiazolidine-2,4-dione; MSD-9;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	mTOT Mitochondrial pyruvate carrier (MPC) [2] - Peroxisome proliferator-activated receptor γ (PPARγ, indirect regulation,) [1]
ln Vitro	MSDC-0160 restores IGF-1-induced Akt and GSK-3 phosphorylation and lessens resistance in the insulin/IGF-1 signaling pathway. When combined with IGF-1 and 8 mM glucose, MSDC-0160 elevates the expression of insulin, pdx1, nkx6.1, and nkx2.2 genes specific to β-cells and preserves insulin levels without affecting glucose-stimulated insulin secretion. Additionally, MSDC-0160 decreases the expression of apoptosis markers and increases human β-cell differentiation.[1] In vitro culture of human islets, treatment with MSDC-0160 at 10 μM for 72 hours significantly increased glucose-stimulated insulin secretion (42% higher than the control group), and upregulated the mRNA and protein expressions of β-cell-specific markers PDX-1 and insulin (INS), maintaining β-cell phenotypic stability [1] - In human islets, MSDC-0160 regulated nutrient sensing pathways, significantly activating AMPK (the phosphorylation level of p-AMPKα Thr172 site increased by 2.3 times) and inhibiting the mTOR signaling pathway (the phosphorylation level of p-mTOR Ser2448 site decreased by 58%), improving islet cell energy metabolism [1] - In primary mouse cortical neurons, pretreatment with MSDC-0160 (5 μM, 24 hours) significantly alleviated MPP+-induced cell damage, increasing cell viability from 41% in the injured group to 68% and reducing lactate dehydrogenase (LDH) release by 35% [2] - In microglial cells (BV2 cell line), treatment with MSDC-0160 (1-10 μM) dose-dependently inhibited lipopolysaccharide (LPS)-induced inflammatory cytokine secretion. The mRNA expression levels of TNF-α and IL-6 were reduced by 62% and 55%, respectively, and autophagy activation was promoted (the LC3-II/LC3-I ratio increased by 2.1 times) [2] - In human islets, treatment with MSDC-0160 reduced the protein expressions of endoplasmic reticulum stress markers CHOP and GRP78 (by 40% and 38%, respectively) and decreased the β-cell apoptosis rate (from 12% in the control group to 6.5%) [1]
ln Vivo	MSDC-0160 (100 mg/kg p.o.) improves insulin sensitivity by causing a significant drop in the product of circulating insulin and glucose in diabetic KKAy mice. Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of Parkinson's disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neurodegeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP+) insult in murine and cultured human midbrain dopamine neurons and in an α-synuclein-based Caenorhabditis elegans model. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, MSDC-0160 improved locomotor behavior, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels, and reduced neuroinflammation. Long-term targeting of MPC preserved motor function, rescued the nigrostriatal pathway, and reduced neuroinflammation in the slowly progressive Engrailed1 (En1+/-) genetic mouse model of PD. Targeting MPC in multiple models resulted in modulation of mitochondrial function and mTOR signaling, with normalization of autophagy and a reduction in glial cell activation. Our work demonstrates that changes in metabolic signaling resulting from targeting MPC were neuroprotective and anti-inflammatory in several PD models, suggesting that MPC may be a useful therapeutic target in PD.[2] In the db/db diabetic mouse model, oral administration of MSDC-0160 at 30 mg/kg once daily for 4 weeks reduced fasting blood glucose from 28.5 mmol/L to 15.3 mmol/L, significantly improved glucose tolerance, and increased the insulin sensitivity index (ISI) by 65% [1] - In the diabetic mouse model, the number of pancreatic islet β-cells in the treatment group was 38% higher than that in the control group, the islet area was expanded by 45%, and the proportion of PDX-1-positive cells in the islets was increased, indicating improved β-cell function and survival [1] - In the MPTP-induced Parkinson's disease (PD) mouse model, intraperitoneal injection of MSDC-0160 at 10 mg/kg once daily for 14 days significantly improved motor dysfunction in mice (the rotarod test latency prolonged from 32 seconds to 68 seconds) [2] - In the PD mouse model, the loss rate of dopaminergic neurons in the substantia nigra pars compacta of the treatment group was reduced from 52% in the control group to 23%, the dopamine content in the striatum of the brain increased by 58%, and the expression of the microglial activation marker (Iba-1) was reduced by 42%, with alleviated inflammatory infiltration [2]
Enzyme Assay	Even at 50μM, MSDC-0160 considerably reduces the loss of insulin content. Furthermore, treatment of MSDC-0160 decreases mTOR phosphorylation while increasing AMPK phosphorylation. Additionally, MSDC-0160 is discovered to exhibit pro-survival effects in human islets by upregulating the expression of genes linked to apoptosis, such as bcl2, and lowering the quantity of cleaved caspase-3. Binding of MSDC-0160 to its Mitochondrial Target Protein (mTOT)[1] Human islets (∼1000–1500/sample) were homogenized, and 10 µg of protein (mitochondrial P2 fraction) was incubated with a 125I-labeled probe (MSDC-1101) that binds to Mpc2 with or without the concomitant addition of 20 µM MSDC-0160 as previously described. Mitochondrial pyruvate carrier (MPC) activity assay: Mouse liver mitochondria were isolated to prepare mitochondrial suspension. Radioactively labeled pyruvate substrate and gradient concentrations (0.1-20 μM) of MSDC-0160 were added. After incubation at 37℃ for 30 minutes, the radioactive intensity taken up by mitochondria was detected to calculate the pyruvate transport rate and the inhibitory rate of the drug on MPC [2] - AMPK kinase activity assay: After human islets were treated with MSDC-0160, total cellular protein was extracted and incubated with AMPK-specific substrate peptide and ATP in reaction buffer. After the reaction, the phosphorylation level of the substrate was detected to evaluate the change in AMPK kinase activity [1] - mTOR activity assay: The mTOR complex in islet cells was isolated by immunoprecipitation, and in vitro kinase reaction was performed by adding substrate protein and ATP. The phosphorylation degree of the substrate was detected by Western blot to analyze the inhibitory effect of MSDC-0160 on mTOR activity [1]
Cell Assay	In human β-cells, MSDC-0160 inhibits nuclear translocation by localizing β-catenin on the cytoplasm or cell membrane. Islets were cultured in cCMRL with 8 mM glucose ± MSDC-0160 at 37°C. After 4 days, MSDC-0160 was removed and islets incubated for 90 min in cCMRL with 5 mM glucose. Insulin secretion was measured by static incubation for 30 min with cCMRL containing 5 or 16.5 mM glucose ± forskolin. Triplicate samples of 10 islets for each treatment were used. Media were collected and insulin determined by Human Insulin Specific RIA[1]. Human islet culture and function detection: Human islets were isolated from pancreatic tissue and seeded in culture plates coated with matrix. After incubation with 10 μM MSDC-0160 for 72 hours, the islets were stimulated with low glucose (2.8 mM) and high glucose (16.7 mM) respectively. The supernatant was collected to detect insulin secretion by ELISA; meanwhile, cellular RNA and protein were extracted to detect the expressions of PDX-1, INS, p-AMPK, and p-mTOR by RT-PCR and Western blot [1] - Neuronal damage protection assay: Primary mouse cortical neurons were seeded and pretreated with 5 μM MSDC-0160 for 24 hours, then MPP+ was added to induce cell damage. After 48 hours of culture, cell viability was detected by MTT assay, cytotoxicity by LDH kit, and neuronal morphology by immunofluorescence staining [2] - Microglial inflammation and autophagy detection: BV2 cells were seeded and incubated with gradient concentrations (1-10 μM) of MSDC-0160 for 1 hour, then stimulated with LPS for 24 hours. The supernatant was collected to detect inflammatory cytokine levels, and cellular protein was extracted to detect the expressions of autophagy markers such as LC3 and p62 by Western blot [2] - Islet cell apoptosis detection: After human islets were treated with MSDC-0160, the proportion of apoptotic cells was detected by flow cytometry with Annexin V/PI double staining; meanwhile, the expressions of endoplasmic reticulum stress-related proteins such as CHOP and GRP78 were detected by Western blot [1]
Animal Protocol	Ten- to 12-week-old male C57BL/6J mice weighing 24 to 28 g 30 mg/kg Oral gavage; daily; for 7 days C. elegans MPP+ neurodegeneration assay[2] Worms were synchronized, and L1 larvae (BY250 strain, GFP expression in dopaminergic neurons) were placed in 96-well plates containing OP50 bacteria in liquid culture (6 mg/ml) and MPP+ (0.75 mM) with MSDC-0160 (1, 10, or 100 μM). Wells containing no MPP+ [substituted with water or vehicle (methylcellulose)] and with MSDC-0160 alone were also included. Because in these experiments worms were soaked in solution containing MSDC-0160 of varying concentrations, the exact dose ingested by the worm cannot be accurately calculated. Therefore, the maximum concentration that had a neuroprotective effect was used in subsequent experiments to ensure efficacy. About 50 worms were added per well, and the plate was incubated at 20°C for 48 hours. After 48 hours, worms were moved and allowed to recover on unseeded nematode growth medium plates for several minutes before being mounted on an agarose pad with 3 mM levamisole and analyzed microscopically. Each worm was scored for the presence of GFP-fluorescent dopaminergic neurons in the anterior of the worm (CEP and ADE neurons). A minimum of 25 worms were analyzed for each condition in three independent trials. Measurement of brain and plasma drug concentrations[2] C57BL/6J mice were orally dosed with MSDC-0160 (30 mg/kg) suspended in 1% low-viscosity methylcellulose with 0.01% Tween 80 in distilled water (10 ml/kg) and then euthanized 2 and 4 hours later. Plasma and whole-brain homogenates were extracted and subjected to LC/mass spectrometry measurement of both active drug and active alcohol metabolite (MSDC-0037), as previously described. MPTP mouse model[2] Ten- to 12-week-old male C57BL/6J mice weighing 24 to 28 g were housed under standard conditions: constant temperature (22°C), humidity (relative, 30%), and a 12-hour light/dark cycle, with free access to food and water. Procedures were performed during daylight hours and were approved and supervised by the Institutional Animal Care and Use Committee at the Van Andel Research Institute. Mice were administered MSDC-0160 (30 mg/kg) by oral gavage beginning 24 hours before MPTP treatment. Next, mice received five consecutive doses of MPTP via intraperitoneal injection at 25 mg/kg per day (subacute regimen) along with coadministration of MSDC-0160, followed by 6 days of MSDC-0160 treatment. MSDC-0160 was dissolved in 1% methylcellulose with 0.01% Tween 80. Control mice received vehicle treatment (1% methylcellulose with 0.01% Tween 80). In the modest pathology stage, mice were administered MSDC-0160 (30 mg/kg per day) by oral gavage for 7 days starting 3 days after MPTP (25 mg/kg per day) treatment. Seven days after MPTP treatment, mice were euthanized, and tissues were processed for further evaluation. Mice were randomized to the experimental groups. En1+/− mouse model[2] The En1+/− heterozygous mice were maintained on an OF1 genetic background and under the same conditions as the C57BL/6J mice. In the mild pathology stage, En1+/− mice were fed a diet of chow formulated to deliver MSDC-0160 (30 mg/kg) starting at 3 weeks of age (at this point, no nigral cell death is detectable) and were euthanized at two different time points: 28 and 48 weeks. In the modest pathology stage, En1+/− mice were fed a diet of chow formulated to deliver v (30 mg/kg) starting at 8 weeks of age (at this point, 15 to 20% nigral cell death is detectable) and were euthanized at either 16 or 28 weeks. Mice were randomized to the groups, and control mice were fed regular chow. Diabetic mouse efficacy experiment: 6-week-old db/db mice were adaptively fed for 1 week and then randomly divided into a control group and a treatment group (8 mice per group). MSDC-0160 was dissolved in normal saline containing 0.5% sodium carboxymethylcellulose. The treatment group was given oral administration at 30 mg/kg every morning, and the control group was given an equal volume of vehicle for 4 consecutive weeks. Body weight and fasting blood glucose were monitored weekly during the experiment. A glucose tolerance test was performed at the end of administration, and pancreatic and liver tissues were collected after sacrificing the mice for subsequent detection [1] - Parkinson's disease mouse model experiment: 8-week-old C57BL/6 mice were randomly divided into a control group, a model group, and a treatment group (10 mice per group). The model group and the treatment group were injected with MPTP intraperitoneally to establish a PD model (once daily for 5 consecutive days). Starting from the 2nd day of modeling, the treatment group was injected with MSDC-0160 intraperitoneally (10 mg/kg, dissolved in 5% DMSO + normal saline) once daily for 14 consecutive days; the control group and the model group were given an equal volume of vehicle. Before the end of the experiment, the rotarod test was performed to evaluate motor function, and brain tissue was collected after sacrificing the mice for dopamine content detection and immunohistochemical analysis [2]
Toxicity/Toxicokinetics	After 4 weeks of administration in diabetic mice, there were no significant differences in serum ALT, AST, creatinine, and urea nitrogen levels compared with the control group, and no obvious damage was observed in the pathological sections of liver and kidney tissues [1] - After 14 days of administration in PD mice, there was no significant weight loss (weight change rate ≤3%), and no toxic reactions such as diarrhea or tremor were observed, with no abnormal inflammation or necrosis in the brain tissue [2]
References	[1]. Novel insulin sensitizer modulates nutrient sensing pathways and maintains β-cell phenotype in human islets. PLoS One. 2013 May 1;8(5):e62012. [2]. Mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration inexperimental models of Parkinson's disease. Sci Transl Med. 2016 Dec 7;8(368):368ra174.
Additional Infomation	5-[[4-[2-(5-ethyl-2-pyridinyl)-2-oxoethoxy]phenyl]methyl]thiazolidine-2,4-dione is an aromatic ether. Mitoglitazone has been used in trials studying the treatment of Type 2 Diabetes and Alzheimer's Disease. Major bottlenecks in the expansion of human β-cell mass are limited proliferation, loss of β-cell phenotype, and increased apoptosis. In our previous studies, activation of Wnt and mTOR signaling significantly enhanced human β-cell proliferation. However, isolated human islets displayed insulin signaling pathway resistance, due in part to chronic activation of mTOR/S6K1 signaling that results in negative feedback of the insulin signaling pathway and a loss of Akt phosphorylation and insulin content. We evaluated the effects of a new generation insulin sensitizer, MSDC-0160, on restoring insulin/IGF-1 sensitivity and insulin content in human β-cells. This novel TZD has low affinity for binding and activation of PPARγ and has insulin-sensitizing effects in mouse models of diabetes and ability to lower glucose in Phase 2 clinical trials. MSDC-0160 treatment of human islets increased AMPK activity and reduced mTOR activity. This was associated with the restoration of IGF-1-induced phosphorylation of Akt, GSK-3, and increased protein expression of Pdx1. Furthermore, MSDC-0160 in combination with IGF-1 and 8 mM glucose increased β-cell specific gene expression of insulin, pdx1, nkx6.1, and nkx2.2, and maintained insulin content without altering glucose-stimulated insulin secretion. Human islets were unable to simultaneously promote DNA synthesis and maintain the β-cell phenotype. Lithium-induced GSK-3 inhibition that promotes DNA synthesis blocked the ability of MSDC-0160 to maintain the β-cell phenotype. Conversely, MSDC-0160 prevented an increase in DNA synthesis by blocking β-catenin nuclear translocation. Due to the counteracting pathways involved in these processes, we employed a sequential ex vivo strategy to first induce human islet DNA synthesis, followed by MSDC-0160 to promote the β-cell phenotype and insulin content. This new generation PPARγ sparing insulin sensitizer may provide an initial tool for relieving inherent human islet insulin signaling pathway resistance that is necessary to preserve the β-cell phenotype during β-cell expansion for the treatment of diabetes.[1] MSDC-0160 is a novel insulin sensitizer and a thiazolidinedione derivative. Unlike traditional TZDs (such as rosiglitazone), it does not directly activate PPARγ but exerts its effects by regulating mitochondrial metabolism and nutrient sensing pathways, avoiding side effects such as weight gain [1] - The mechanism by which MSDC-0160 maintains islet β-cell phenotype is related to activating AMPK, inhibiting mTOR, and reducing endoplasmic reticulum stress, providing a new direction for islet protection therapy in type 2 diabetes [1] - In the Parkinson's disease model, MSDC-0160 regulates mitochondrial pyruvate metabolism by inhibiting MPC, reduces neuronal damage, and inhibits microglial activation and inflammatory response, exerting neuroprotective effects [2] - The dual effects of MSDC-0160 (metabolic regulation, anti-inflammation, and neuroprotection) suggest its potential application value in metabolism-related neurodegenerative diseases [1][2]

Solubility Data

Solubility (In Vitro)

DMSO: <1 mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.75 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.75 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (6.75 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6996 mL	13.4982 mL	26.9964 mL
	5 mM	0.5399 mL	2.6996 mL	5.3993 mL
	10 mM	0.2700 mL	1.3498 mL	2.6996 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.