| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| Other Sizes |
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| Targets |
Synthetic version of an active region of thymosin β₄
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| ln Vitro |
In vitro biotransformation studies [1]
In order to identify possible metabolites of TB-500 (N-acetylated LKKTETQ) in horses, the peptide was incubated with homogenised horse liver. The much cleaner extracts from the in vitro studies would facilitate identification of metabolites. Portions of the mixtures were removed throughout the incubation process for LC/MS analyses. Various in vitro metabolites were identified for the N-acetylated LKKTETQ peptide (Fig. 3). The metabolites observed were mainly from a sequential loss of amino acid residues from the C-terminus. Trace amounts of metabolites M1a, M2a, M3, M4 and M5 were also detected in the control sample without homogenised liver, indicating that these metabolites could be formed from non-enzymatic hydrolysis of the peptide bonds. In the presence of the liver enzymes, additional metabolites M0, M1b, M2b, M2c and M2d generated from isomerisation were detected. They could be stereo- or region-isomers of the parent peptide and the C-terminus truncated metabolites N-acetylated LKKTET and N-acetylated LKKTE. The observed masses of these isomers were very close to their respective calculated masses since the mass tolerance window was only ±3 ppm. Fig. 4 shows the product-ion scan of individual metabolites and the corresponding assignment of the fragment ions. The respective isomers gave highly similar mass spectra, suggesting that they should be stereoisomers with identical amino acid sequences. All experimental masses of the fragment ions from the metabolites were very similar to their theoretical values with deviations of no more than 2 ppm (except for the low abundant ion of metabolite M1b at 2.9 ppm), which further substantiated our postulation. |
| ln Vivo |
A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin β(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses [1].
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| Enzyme Assay |
In vitro biotransformation studies [1]
For the in vitro metabolism study of TB-500, a mixture of homogenised liver (from 1-cm cube) and 2 mL of β-NAD (1.5 mM), glucose-6-phosphate (7.65 mM), magnesium chloride (4.4 mM), EDTA (1 mM) and glucose-6-phosphate dehydrogenese (1 U/mL) in phosphate buffer (pH 7.4, 5 mL) was incubated with TB-500 (N-acetylated LKKTETQ, 1 mg) at 37 °C with shaking. Portions (200 μL each) of the mixture were taken out before and 1, 2, 4 and 20 h after incubation for LC/MS analysis. The reaction was terminated by incubating at 80 °C for 30 min. All portions of incubation mixture were immediately stored at 4 °C prior to analysis. Portions of the incubation mixture were each centrifuged through an Ultrafree-CL centrifugal filter device with 0.45 μm membrane pore size at 13,000 × g for 5 min. The filtrate was then diluted ten-fold with aqueous acetic acid (2%). The diluted filtrate obtained was ready for UHPLC/MS analysis. Control experiments in the absence of either (a) TB-500 or (b) homogenised liver were performed in parallel. Unless otherwise stated, all labware used was plasticware. |
| Animal Protocol |
Administration studies [1]
A single dose of TB-500 (containing 10 mg of N-acetylated LKKTETQ) was administered subcutaneously to each of two thoroughbred geldings. Blood samples were collected before administration, and 0.5, 1, 2, 4, 6, 8, 10, 11, 26, 34, 51, 58, 75, 100 h post administration. Urine samples were collected prior to administration and for 4 days after administration. |
| References |
[1]. Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry. J Chromatogr A. 2012 Nov 23;1265:57-69.
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| Additional Infomation |
TB-500 is an emerging veterinary preparation for the racing industry, and claims to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. The key ingredient in TB-500 is a synthetic peptide N-acetylated LKKTETQ, with an amino acid sequence matching an active region (17LKKTETQ23) of the thymosin β4 protein. Although peptide LKKTETQ could be a natural degradation product of thymosin β4, acetylation of the N-terminus ensures this peptide is exogenous in the equine. In this paper, sensitive and selective methods were presented for the identification of N-acetylated LKKTETQ in equine urine and plasma. The identities of the metabolites were suggested from mass spectral interpretation and accurate mass measurement. N-acetylated LKKTETQ peptide and its metabolites could be simultaneously detected after solid-phase extraction using ion-exchange cartridges and analysis by LC/MS. Detection of the peptide N-acetylated LKKTETQ could be achieved at less than 0.02 ng/mL in equine plasma and less than 0.01 ng/mL in equine urine. The methods gave adequate recoveries and precision, and the absence of interference from biological matrices. The peptide N-acetylated LKKTETQ and its metabolites were successfully identified in post-administration samples collected from two horses after having been treated with a single dose of TB-500 (containing 10 mg of N-acetylated LKKTETQ). Administration of TB500 could be detected for up to 11 h based on the detection of the metabolite N-acetylated LK (M5). To our knowledge, this is the first report of a method to control the misuse of TB-500 (N-acetylated LKKTETQ) in horses, and is the first identification of TB-500 and its metabolites in biological fluids collected from treated horses. [1]
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| Molecular Formula |
C38H68N10O14
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|---|---|
| Molecular Weight |
889.005129814148
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| Exact Mass |
888.491
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| Elemental Analysis |
C, 51.34; H, 7.71; N, 15.76; O, 25.19
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| CAS # |
885340-08-9
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| Related CAS # |
885340-08-9; TB500 TFA; 75591-33-4;
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| PubChem CID |
62707662
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| Sequence |
N-Acetyl-Leu-Lys-Lys-Thr-Glu-Thr-Gln;
N-acetyl-L-leucyl-L-lysyl-L-lysyl-L-threonyl-L-alpha-glutamyl-L-threonyl-L-glutamine
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| SequenceShortening |
Ac-LKKTETQ
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
-8.6
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| Hydrogen Bond Donor Count |
14
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| Hydrogen Bond Acceptor Count |
16
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| Rotatable Bond Count |
32
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| Heavy Atom Count |
62
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| Complexity |
1530
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| Defined Atom Stereocenter Count |
9
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| SMILES |
C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)C)O
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| InChi Key |
ADKDNDYYIZUVCZ-ZQNQAVPYSA-N
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| InChi Code |
InChI=1S/C38H68N10O14/c1-19(2)18-27(42-22(5)51)35(58)44-23(10-6-8-16-39)32(55)43-24(11-7-9-17-40)33(56)47-30(20(3)49)36(59)45-25(13-15-29(53)54)34(57)48-31(21(4)50)37(60)46-26(38(61)62)12-14-28(41)52/h19-21,23-27,30-31,49-50H,6-18,39-40H2,1-5H3,(H2,41,52)(H,42,51)(H,43,55)(H,44,58)(H,45,59)(H,46,60)(H,47,56)(H,48,57)(H,53,54)(H,61,62)/t20-,21-,23+,24+,25+,26+,27+,30+,31+/m1/s1
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| Chemical Name |
(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-3-hydroxybutanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxybutanoyl]amino]-5-amino-5-oxopentanoic acid
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| Synonyms |
TB500; 885340-08-9; UNII-QHK6Z47GTG; QHK6Z47GTG; TB-500; (2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-6-aminohexanoyl]amino]-6-aminohexanoyl]amino]-3-hydroxybutanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxybutanoyl]amino]-5-amino-5-oxopentanoic acid; TB 500; L-Glutamine, N-acetyl-L-leucyl-L-lysyl-L-lysyl-L-threonyl-L-alpha-glutamyl-L-threonyl-;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~250 mg/mL (~281.21 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1248 mL | 5.6242 mL | 11.2485 mL | |
| 5 mM | 0.2250 mL | 1.1248 mL | 2.2497 mL | |
| 10 mM | 0.1125 mL | 0.5624 mL | 1.1248 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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