Alk3 (activin-like kinase 3) – predominant target; also binds to Alk2 to some extent, but no competition with Alk6 [1]

- THR-123 inhibited TNF-α induced IL-6 production in human renal tubular epithelial cells (HK-2) in a dose-dependent manner [1]
- THR-123 inhibited TNF-α induced IL-8 and ICAM-1 production in HK-2 cells [1]
- THR-123 exhibited anti-apoptotic activity similar to BMP7 in TGF-β-induced apoptosis of tubular epithelial cells (Annexin V labeling) [1]
- THR-123 inhibited hypoxia-induced apoptosis of tubular epithelial cells [1]
- THR-123 inhibited cisplatin-induced apoptosis of tubular epithelial cells [1]
- THR-123 inhibited TGF-β induced epithelial-to-mesenchymal transition (EMT) program: restored TGF-β-suppressed E-cadherin to normal levels; inhibited TGF-β induced expression of snail (Sna) and CTGF; associated with Smad1/5 phosphorylation (p-Smad1/5) [1]
- After 48 hours of incubation with TGF-β and epidermal growth factor (EGF), tubular epithelial cells exhibited mesenchymal features indicative of EMT; THR-123 reversed TGF-β-induced EMT, associated with restoration of E-cadherin expression [1]

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THR-123 (0-100 μM, 60 min) inhibited TNF-α-induced expression of IL-6, IL-8 and ICAM-1 in HK-2 cells in a concentration-dependent manner [1]. THR-123 (250 μM) significantly inhibited TGF-β1, hypoxia or cisplatin-induced apoptosis in HK-2 cells [1]. THR-123 (10 μM) restored the expression of E-cadherin inhibited by TGF-β1, reduced the expression of CTGF and Snail1, and reversed the cell morphology transition to mesenchymal form [1].

- Ischemic reperfusion injury (IRI) model in mice: THR-123 treated mice displayed significantly less tubular damage in IRI kidney compared to control mice (7 days after IRI) [1]
- Unilateral ureteral obstruction (UUO) model: Oral administration of THR-123 (5 mg/kg or 15 mg/kg) inhibited interstitial volume expansion in UUO kidneys at 5 days; both intraperitoneal and oral administration of THR-123 inhibited fibrosis at 7 days; decreased expression of fibronectin (Fn1) and type I collagen (Col4a1) [1]
- Nephrotoxic nephritis (NTN) model: THR-123 treatment (initiated at six weeks following NTN induction) improved glomerular lesion (sclerosis), tubular atrophy and fibrosis; decreased blood urea nitrogen; significantly decreased number of cells exhibiting EMT program (FSP1 and E-cadherin double-positive); inhibited accumulation of Mac-1 and F4/80 positive macrophages; increased accumulation of p-Smad1/5 [1]
- Alport syndrome model (COL4A3KO mice): THR-123 treatment significantly inhibited tubular atrophy and interstitial fibrosis; significantly improved blood urea nitrogen level; inhibited EMT program acquisition; inhibited macrophage infiltration; associated with increase in p-Smad1/5 [1]
- Diabetic nephropathy (DN) model in CD-1 mice (streptozotocin-induced): THR-123 treatment (5-6 months) substantially reversed mesangial matrix expansion; inhibited tubular atrophy and interstitial volume increase; substantially reversed tubular atrophy and interstitial volume expansion; significantly reversed renal dysfunction (blood urea nitrogen); inhibited induction of EMT program; reduced macrophage infiltration; associated with increased accumulation of p-Smad1/5 [1]
- Combination therapy with captopril (ACE inhibitor) in advanced diabetic nephropathy: Combination of CPR with THR-123 significantly reduced mesangial expansion and substantially reversed it; completely inhibited tubular atrophy and interstitial volume expansion; significantly inhibited progressive loss of renal function; combination therapy exhibited additive anti-apoptotic effects; associated with increased accumulation of p-Smad1/5 [1]
- In Alk3 deleted mice subjected to IRI or NTN: THR-123 did not exhibit therapeutic effect; no inhibition of macrophage accumulation, EMT program, or apoptosis; no restoration of renal function [1]

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THR-123 (5 mg/kg, face, once daily for 3 weeks) restored renal fibrosis in mice with chronic tubular fibrosis (NTN) induced by kidney injury [1]. THR-123 (5 mg/kg, face, once daily for 7 days) significantly reduced renal tubular injury tissue and damage in a renal reperfusion injury (IRI) model [1]. THR-123 (5–15 mg/kg, lateral wall or intraperitoneal injection, once daily for 5–7 days) inhibited interstitial volume expansion and collagen deposition in a unilateral ureteral obstruction (UUO) model [1]. THR-123 (5 mg/kg, lateral wall, once daily from 8 to 16 weeks of age) did not alter glomerular abnormalities and significantly inhibited renal tubular apoptosis and interstitial fibrosis observed in a COL4A3KO model [1]. THR-123 (5 mg/kg, oral, once daily for 3 months) focused on the progression of fibrosis associated with advanced diabetic nephropathy [1].

- Radio-ligand receptor competitive binding assays: Highly purified extra-cellular domain (ECD) of Alk3 or Alk6 (expressed as fusion protein with Fc domain) was immobilized on each well. Peptide analog or unlabeled BMP7 was added, followed by 125I-labeled BMP7. Radiolabeled BMP7 complex was counted in an auto-gamma counter. Unlabeled BMP7 served as positive control. Binding of cold BMP7 to immobilized receptor ECDs was determined by competition with 125I-labeled BMP7 and analyzed by Scatchard analysis to determine effective dissociation constants of BMP7 for each receptor ECD. To estimate the effective dissociation constant of THR-123 for a particular receptor ECD, the dissociation constant for cold BMP7 was multiplied by the ratio of the ED50 of THR-123 to the ED50 for cold BMP7. Data demonstrated that THR-123 competes with BMP7 for Alk3 and to some extent with Alk2, whereas absolutely no competition was observed with Alk6 [1]

- Anti-inflammatory efficacy in vitro cell-based assay using human renal tubular epithelial cell line (HK-2): The assay tested the ability of compounds to reverse the increase in production of cytokine IL-6 that resulted from stimulation of cells with tumor necrosis factor (TNF)-α [1]
- TGF-β-induced apoptosis in tubular epithelial cells was analyzed by Annexin V labeling [1]
- Hypoxia-induced apoptosis of tubular epithelial cells was analyzed [1]
- Cisplatin-induced apoptosis was analyzed [1]
- TGF-β induced EMT program: Cells were incubated with TGF-β and epidermal growth factor (EGF) for 48 hours; E-cadherin expression analyzed by western blot; gene expression of snail (Sna) and CTGF analyzed by quantitative PCR; Smad1/5 phosphorylation (p-Smad1/5) analyzed [1]

Animal/Disease Models: Nephrotoxic serum nephritis, NTN model established in CD1 mice[1]
Doses: 5 mg/kg
Route of Administration: Oral administration (p.o.), once daily for 3 weeks
Experimental Results: Significantly reversed the already formed renal fibrosis, and improve glomerular sclerosis, renal tubular atrophy and interstitial fibrosis. Significantly reduced renal function indicators. Inhibited epithelial-mesenchymal transition (EMT) and reduce the number of E-cadherin and FSP1 double-positive renal tubular cells. Reduced macrophage infiltration and activate the BMP-Smad signaling pathway (p-Smad1/5).

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Animal/Disease Models: IRI model established in C57Bl/6 mice (8 weeks)[1]
Doses: 5 mg/kg
Route of Administration: Oral administration (p.o.), once daily for 7 days
Experimental Results: Significantly reduced the proportion of renal tubular necrosis and lowered the blood urea nitrogen level. Reduced macrophage infiltration and alleviated cell apoptosis.

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Animal/Disease Models: UUO model established in CD1 mice (8-12 week)[1]
Doses: 5 and 15 mg/kg (p.o.) or 5 mg/kg (i.p.)
Route of Administration: Oral administration (p.o.) or intraperitoneal injection (i.p.), once daily for 5-7 days
Experimental Results: Inhibited interstitial volume expansion and collagen deposition. Reduced the expression of fibrosis markers (fibronectin and type I collagen).

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Animal/Disease Models: Alport syndrome model established in eight weeks-old COL4A3KO mice on C57Bl/6 background[1]
Doses: 5 mg/kg
Route of Administration: Oral administration (p.o.), once daily from 8 weeks of age to 16 weeks of age
Experimental Results: Improved glomerular sclerosis, tubular damage and interstitial fibrosis. Restored the expression of E-cadherin and reduce the fibroblast marker FSP1.

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Animal/Disease Models: Streptozotocin induced Diabetic nephropathy, DN model established in male CD1 mice (8 week)[1]
Doses: 5 mg/kg
Route of Administration: Oral administration (p.o.), once daily for 3 months
Experimental Results: Reduced mesangial matrix expansion, macrophage infiltration and cell apoptosis. Delayed the deterioration of renal function in combination of Captopril.

- In PBS-mannitol buffer: THR-123 was stable for over 400 minutes [1]
- In rat plasma: THR-123 slowly degraded with half-life of 358 minutes [1]
- In whole blood: THR-123 degraded rapidly with half-life of 70 minutes [1]
- Alpha-phase assessment: THR-123 levels immediately decreased within 5 minutes (by almost 90%) after intravenous administration, suggesting very short half-life in alpha-phase [1]
- Beta-phase assessment of 125I-THR-123: half-life of 55-58 minutes [1]
- Six hours after intravenous administration of 125I-THR-123: majority of radioactivity still localized in kidney and bladder, suggesting THR-123 accumulates in kidney and is excreted via bladder into urine [1]
- Orally administered 125I-labeled THR-123: localized primarily to kidney cortex within one hour after ingestion and peaked at about 3 hours; twenty-four hours after ingestion, most radioactivity cleared from kidney [1]

- No osteogenic activity was induced by THR-123 treatment [1]
- Blood sugar level and body weight were not altered in all groups analyzed when compared to untreated diabetic mice at similar age [1]

[1]. https://pubmed.ncbi.nlm.nih.gov/22306733/

- THR-123 is a 16-residue long cyclic peptide of approximately 2 kDa molecular weight, cyclized via a disulfide bond between the first and 11th residue positions to stabilize the loop and preserve a 3D conformation similar to the finger 2 loop of BMP7 [1]
- THR-123 was designed by identifying regions of the 3D structure of TGF-β and BMP most likely involved in receptor interactions, using structure-variance analysis (SVA) program that weighs physical-chemical residue properties at each position based on correlation with activity [1]
- THR-123 suppresses progression of kidney disease and substantially reverses established kidney fibrosis without inducing osteogenic activity [1]
- THR-123 inhibits inflammation, apoptosis, EMT program and reverses renal fibrosis [1]
- Combination of THR-123 and captopril (angiotensin converting enzyme inhibitor) had additive therapeutic effect in controlling renal fibrosis associated with diabetic kidney disease [1]

C83H124N22O27S2

1926.13

Cys-Tyr-Phe-Asp-Asp-Ser-Ser-Asn-Val-Leu-Cys-Lys-Lys-Tyr-Arg-Ser (disulfide bridge: Cys1-Cys11)CYFDDSSNVLCKKYRS (disulfide bridge: Cys1-Cys11)

CYFDDSSNVLCKKYRS (disulfide bridge: Cys1-Cys11)

Typically exists as solids at room temperature

THR-123; THR123; THR 123

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.)