TB-500

What TB-500 is

TB-500 is a synthetic peptide of seven amino acids with an acetyl group on its N-terminus: Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln, usually written Ac-LKKTETQ. The sequence corresponds to residues 17 to 23 of thymosin β4 (Tβ4), a 43-residue protein found in nearly every mammalian cell type and in plasma and wound fluid [1, 2]. Those residues span the region of Tβ4 mapped in the 1990s as its principal actin-binding motif [3], and it is on that basis that the fragment has been studied and, separately, marketed.

The most important thing to establish at the outset is that TB-500 and thymosin β4 are not the same molecule. Thymosin β4 is a 43-residue, N-acetylated, acidic peptide of roughly 4.96 kDa [1]. TB-500 is a 7-residue fragment of about 889 Da, roughly one sixth of the parent's length and mass, which retains one functional motif and none of the others. Much of what is written about TB-500 online quotes animal and clinical data generated with full-length Tβ4, including the ophthalmic product RGN-259, and presents it as data on the fragment. This monograph keeps the two apart.

The name originated in the veterinary and racing supply trade rather than in a laboratory. When a product sold as "TB-500" was seized by Belgian customs and analysed by the Ghent doping-control laboratory, its active content was identified by high-resolution mass spectrometry as the N-acetylated 17–23 fragment of Tβ4, and the identification was confirmed by synthesis of the same sequence [4]. That paper remains the best primary description of what the compound in the vial is.

The research material is a white lyophilised powder. The reference identifiers are in the table below; the values on any batch certificate should agree with them within the precision of the method used. Note that CAS 77591-33-4, which some suppliers print on TB-500 products, belongs to full-length thymosin β4, not the fragment.

How the literature describes its mechanism

The biology of TB-500 is inseparable from the biology of its parent, so the parent comes first.

Thymosin β4 and G-actin sequestration. Thymosin β4 was originally isolated from thymus and assumed to be a thymic hormone. Its real intracellular function was identified in the early 1990s: it is the main G-actin-sequestering peptide of the cell. It binds monomeric actin in a 1:1 complex and holds it in a pool that cannot polymerise, acting as a buffer that suppresses filament formation while keeping a reservoir of monomer available when filaments are needed [1]. Because Tβ4 is present at concentrations comparable to actin itself, this buffering is a major determinant of the balance between free and polymerised actin, and so of cell shape and motility [1, 2].

Tβ4 also occurs outside cells, in plasma and wound fluid, and the extracellular peptide has been associated with a different set of activities: induction of matrix metalloproteinases, chemotaxis of endothelial cells and keratinocytes, angiogenesis and anti-inflammatory effects. Huff and colleagues' 2001 review notes that the molecular mechanisms behind these extracellular effects were unknown, and the question is not fully resolved since [1]. Goldstein, Hannappel and Kleinman's 2005 review summarises the case that Tβ4 "moonlights" as a repair factor in skin, cornea and ischaemic organs [2].

The LKKTET motif. Van Troys and colleagues mapped the actin-binding site of Tβ4 by synthesising full-length variants with point substitutions. They identified two structural elements: an N-terminal segment (residues 1–16) that appears to require an α-helix for binding, and a hexapeptide motif at residues 17–22, LKKTET, in which charged and hydrophobic residues contribute to the actin contact. The motif is conserved across the β-thymosin family and present in the C-terminal headpiece of villin and dematin [3]. LKKTET has since become shorthand for the β-thymosin actin-binding motif. TB-500 contains it, plus the following glutamine, with the N-terminal acetylation of the native protein carried over.

What a fragment can and cannot do. Native Tβ4 needs both the N-terminal helix and the LKKTET motif for full actin affinity [3], so a seven-residue peptide carrying only the central motif should be expected to bind actin weakly in isolation. That is consistent with how it has been used experimentally: not as an actin-sequestering agent, but as a tool for asking which of Tβ4's extracellular activities depend on the actin-binding region.

The clearest experiment is that of Philp and colleagues, who compared full-length Tβ4, its fragments and synthetic peptides in endothelial cell migration and vessel-sprouting assays. The seven-residue actin-binding motif was required for angiogenic activity, the motif peptide alone reproduced the effect of the full protein, peptides lacking part of the motif were inactive, and added soluble actin inhibited both adhesion and sprouting, which the authors took to mean that the activity is mediated through actin binding [5]. A 2010 review by Sosne and colleagues gathers this and related work into a map of Tβ4's functional segments: the N-terminal tetrapeptide Ac-SDKP, which is anti-inflammatory and anti-fibrotic; an N-terminal 15-residue segment with anti-apoptotic activity; and the central LKKTETQ sequence, residues 17–23, to which the authors assign migration, angiogenesis and wound-healing activity [6]. That map is the strongest rationale for studying the 17–23 fragment on its own. It is also the limit of what the mapping shows: it localises an activity within the parent protein, and does not establish that the fragment is equivalent to the parent beyond the systems tested.

How extracellular Tβ4, or its fragment, acts on cells remains open. The parent has been reported to form a complex with integrin-linked kinase (ILK) and PINCH that activates the survival kinase Akt in cardiac cells [7], and the same pathway has been proposed in other tissues [8]. Whether the fragment engages the same machinery has not been shown directly. For an in-vitro programme the relevant readouts are cell migration (scratch or Boyden chamber assays), endothelial tube formation or aortic ring sprouting, and actin-binding assays, run alongside full-length Tβ4 as the reference compound.

The research record

Full-length thymosin β4, preclinical. Malinda and colleagues reported that Tβ4, applied topically or given intraperitoneally, accelerated re-epithelialisation of full-thickness dermal wounds in rats by 42% at day 4 and up to 61% at day 7 compared with saline, with greater wound contraction, collagen deposition and angiogenesis, and that it stimulated keratinocyte migration two- to three-fold in a Boyden chamber [9]. Comparable effects on corneal re-epithelialisation and inflammation after injury were the starting point for the ophthalmic programme [8, 12]. In the heart, Bock-Marquette and colleagues reported that Tβ4 promoted migration and survival of cardiomyocytes and endothelial cells, formed a complex with ILK and PINCH that activated Akt, and, in mice after coronary artery ligation, improved early cardiomyocyte survival and cardiac function [7]. None of these experiments used the 17–23 fragment.

Full-length thymosin β4, clinical. Tβ4 has been taken into human trials chiefly by RegeneRx Biopharmaceuticals, under the code RGN-259 for the ophthalmic formulation. The 2012 review by Goldstein, Hannappel, Sosne and Kleinman summarises the programme as it then stood [8]. The published clinical results concern the eye.

Sosne, Dunn and Kim reported a small, multicentre, randomised, double-masked, placebo-controlled phase 2 trial in severe dry eye, including dry eye associated with graft-versus-host disease. Nine patients received 0.1% RGN-259 or vehicle six times daily for 28 days, with follow-up to day 56. The treated group showed a 35.1% greater reduction in ocular discomfort and a 59.1% greater reduction in total corneal fluorescein staining than the vehicle group at day 56 [10]. The trial is informative but very small.

A larger phase 2 trial by Sosne and Ousler used the controlled adverse environment (CAE) model: 72 patients with moderate to severe dry eye were randomised equally to 0.1% Tβ4 drops or placebo for 28 days. Neither primary endpoint, ocular discomfort and inferior corneal staining, was met at the pre-specified time point. Several secondary measures favoured the active arm, including about 27% lower discomfort during the CAE challenge at day 28 and greater improvement in central and superior corneal staining [11]. Sosne's 2018 review traces the ophthalmic programme from bench to bedside and describes the subsequent phase 3 trials in dry eye and the study of neurotrophic keratopathy [12]. As of this writing, no Tβ4 product holds a marketing authorisation in the UK, the EU or the US.

Trials of topical Tβ4 in epidermolysis bullosa and chronic ulcers were run in the same period and are referred to in the reviews [8, 12]; peer-reviewed efficacy reports from them are sparse. The cardiac work has not progressed to published efficacy trials in humans.

The 17–23 fragment itself. The fragment's own published record is small, and it is worth being exact about it. The best-characterised experiments are the in-vitro angiogenesis and migration assays of Philp and colleagues, discussed above [5]. Shah, Reyes-Gordillo and Rojkind, working on hepatic stellate cells as a model of liver fibrosis, reported that Tβ4 inhibited PDGF-BB-induced activation, proliferation and migration of the cells, that this activity resided in the actin-binding domain, and that a peptide corresponding to residues 17–23 reproduced the inhibition at nanogram-per-millilitre concentrations [13]. This is the only recent primary paper we have identified that tests the TB-500 sequence as such in a defined cell system. Esposito and colleagues' 2012 paper is a chemical characterisation rather than a biological study: it identifies the seized product as Ac-LKKTETQ and reports its synthesis and mass-spectrometric fingerprint for anti-doping detection [4].

There are no published controlled trials of TB-500 in humans, and the animal studies of the fragment that circulate online are mostly unpublished, uncontrolled, or are in fact studies of the full protein. The honest summary is that the fragment carries a well-mapped motif with reproducible activity in a handful of cell assays, and that nearly everything else attributed to it was measured with a molecule six times its size.

Regulatory status in the United Kingdom

TB-500 is not an ingredient of any licensed medicine in the UK, and it is not a controlled drug under the Misuse of Drugs Act 1971. BuyUK Peptides supplies it as a chemical for in-vitro laboratory use under the Terms of Sale. It is not a medicine, is not manufactured or presented as one, carries no therapeutic claim, and is not for human or veterinary administration. Purchasers must be 18 or over and are responsible for ensuring that their use of the material complies with the laws and institutional rules that apply to them. Our guide to the UK rules on research compounds sets this out in more detail.

Separately from medicines law, TB-500 is named on the World Anti-Doping Agency's Prohibited List. The 2026 List places "thymosin-β4 and its derivatives e.g. TB-500" in section S2.3, Growth factors and growth factor modulators, within class S2, Peptide hormones, growth factors, related substances and mimetics. Everything in S2 is prohibited at all times, in and out of competition [14]. Anti-doping laboratories have validated detection methods for the fragment specifically [4].

Handling, storage and stability

TB-500 is supplied lyophilised in a glass vial with a rubber stopper and an aluminium flip-off crimp. A short, acetylated, hydrophilic peptide of this kind is among the easier research peptides to handle.

Sealed vial. Store at −20 °C for long-term storage; 2–8 °C is acceptable for material in routine use over a few months. Protect from light. Supplier stability data for the acetate salt indicate the lyophilised solid is stable for several years at −20 °C. Allow a cold vial to reach room temperature before opening, so that moisture does not condense onto the cake, and avoid repeated freeze–thaw cycles.

Reconstitution. The peptide is readily soluble in water and in phosphate-buffered saline at pH 7.2, at 10 mg/ml or more, and can be dissolved directly in aqueous assay buffers without an organic co-solvent. Add the solvent down the side of the vial and swirl gently; dissolution is rapid. The reconstitution calculator gives the volume to add for a target concentration, and the figures should be run against the net peptide content on the certificate rather than the nominal vial mass.

Reconstituted solution. Keep at 2–8 °C and protect from light. The sequence has no cysteine, methionine or tryptophan, so the common oxidative degradation routes do not apply, but the C-terminal glutamine is liable to deamidation in solution over time. Supplier guidance does not recommend keeping aqueous solutions beyond a day; for cell-based work the practical approach is to prepare a concentrated stock, divide it into single-use aliquots and freeze them at −20 °C or below, thawing each once. Discard any solution that becomes cloudy.

Solubility and compatibility. With two lysines against one glutamate and a free C-terminal carboxylate, the peptide is close to neutral at physiological pH but binds readily to charged surfaces at low concentration. Use low-binding plasticware for dilute solutions, and if a solution must be filtered, prefer a PES or PVDF membrane over nylon.

What a sound certificate for TB-500 looks like

Every batch we release carries an independent certificate of analysis, and the figures from it are transcribed on the product page and in the certificate register. For TB-500 the certificate should contain the following.

Purity by RP-HPLC. Reversed-phase HPLC on a C18 column with UV detection at 214 or 220 nm reports purity as the area of the main peak as a percentage of all integrated peaks. The peptide has no aromatic residues, so detection at 280 nm is uninformative and a certificate reporting purity at that wavelength should prompt questions. A good batch shows a single dominant peak at 98% or above. The main impurities to look for are deletion sequences missing one lysine or threonine, and the des-acetyl peptide, which elutes close to the parent.

Identity by LC-MS. Electrospray mass spectrometry confirms the main peak has the expected mass. For TB-500 the average molecular weight is 889.0 Da and the monoisotopic mass is 888.5 Da. A peptide of this size usually appears as the singly charged [M+H]⁺ ion at m/z 889.5 or the doubly charged [M+2H]²⁺ at m/z 445.3; the certificate should say which it reports. The mass distinguishes the fragment unambiguously from full-length Tβ4 at 4963 Da, which is one reason to insist on seeing it.

Net peptide content. Lyophilised peptides contain bound water and counter-ions, usually trifluoroacetate or acetate from purification. Net peptide content, where reported, expresses the fraction of the powder that is peptide and is needed to prepare solutions of accurate concentration.

Endotoxin. Where the batch has been tested, a limulus amoebocyte lysate (LAL) result is reported in endotoxin units per milligram. This matters for the cell-based assays in which this compound is most often used, because endotoxin contamination can itself alter migration and inflammatory readouts.

Our guide to reading a certificate of analysis goes through a complete report line by line.

Related compounds

TB-500 is most often considered alongside two other short peptides studied in tissue-repair models. BPC-157 is a 15-residue fragment of a gastric protein with its own, largely rodent, literature, and the two are frequently combined in research; our BPC-157 and TB-500 blend is a co-lyophilised preparation at a fixed ratio. GHK-Cu is a copper-binding tripeptide released from collagen that has been studied in wound and skin models for longer than either. None of the three shares a mechanism with the others, and the combinations in circulation are empirical rather than mechanistically derived. For the two side by side, including the shape of each evidence base, see BPC-157 vs TB-500.

Frequently asked questions

Is TB-500 the same thing as thymosin β4? No. Thymosin β4 is a 43-residue protein of about 4.96 kDa; TB-500 is a 7-residue fragment of about 889 Da. The fragment retains the actin-binding motif and reproduces some of the parent's activity in cell migration assays, but the clinical trials of RGN-259 and the animal studies of wound and cardiac repair used the full protein [7, 8, 10, 11].

Why does the certificate show a mass around 889 Da rather than 4,963 Da? Because it is the fragment. A certificate for TB-500 reporting a mass near 4,963 Da would indicate full-length thymosin β4, and the product would be mislabelled one way or the other.

Why do some suppliers list CAS 77591-33-4? That number is registered to full-length thymosin β4. The CAS number for the acetylated 17–23 fragment is 885340-08-9.

Is TB-500 prohibited in sport? Yes. It is named explicitly on the WADA Prohibited List under S2.3, and class S2 is prohibited at all times [14].

How long does a sealed vial last? A lyophilised short peptide of this kind is stable for years at −20 °C and for many months at 2–8 °C. The batch certificate reports the test date; the storage guidance on this page applies from receipt.

References

1. Huff T, Müller CSG, Otto AM, Netzker R, Hannappel E. β-Thymosins, small acidic peptides with multiple functions. International Journal of Biochemistry and Cell Biology. 2001;33(3):205–220. doi:10.1016/S1357-2725(00)00087-X. PMID 11311852.
2. Goldstein AL, Hannappel E, Kleinman HK. Thymosin β4: actin-sequestering protein moonlights to repair injured tissues. Trends in Molecular Medicine. 2005;11(9):421–429. doi:10.1016/j.molmed.2005.07.004. PMID 16099219.
3. Van Troys M, Dewitte D, Goethals M, Carlier MF, Vandekerckhove J, Ampe C. The actin binding site of thymosin β4 mapped by mutational analysis. EMBO Journal. 1996;15(2):201–210. doi:10.1002/j.1460-2075.1996.tb00350.x. PMID 8617195.
4. Esposito S, Deventer K, Goeman J, Van der Eycken J, Van Eenoo P. Synthesis and characterization of the N-terminal acetylated 17–23 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential. Drug Testing and Analysis. 2012;4(9):733–738. doi:10.1002/dta.1402. PMID 22962027.
5. Philp D, Huff T, Gho YS, Hannappel E, Kleinman HK. The actin binding site on thymosin β4 promotes angiogenesis. FASEB Journal. 2003;17(14):2103–2105. doi:10.1096/fj.03-0121fje. PMID 14500546.
6. Sosne G, Qiu P, Goldstein AL, Wheater M. Biological activities of thymosin β4 defined by active sites in short peptide sequences. FASEB Journal. 2010;24(7):2144–2151. doi:10.1096/fj.09-142307. PMID 20179146.
7. Bock-Marquette I, Saxena A, White MD, DiMaio JM, Srivastava D. Thymosin β4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature. 2004;432(7016):466–472. doi:10.1038/nature03000.
8. Goldstein AL, Hannappel E, Sosne G, Kleinman HK. Thymosin β4: a multi-functional regenerative peptide. Basic properties and clinical applications. Expert Opinion on Biological Therapy. 2012;12(1):37–51. doi:10.1517/14712598.2012.634793. PMID 22074294.
9. Malinda KM, Sidhu GS, Mani H, et al. Thymosin β4 accelerates wound healing. Journal of Investigative Dermatology. 1999;113(3):364–368. doi:10.1046/j.1523-1747.1999.00708.x. PMID 10469335.
10. Sosne G, Dunn SP, Kim C. Thymosin β4 significantly improves signs and symptoms of severe dry eye in a phase 2 randomized trial. Cornea. 2015;34(5):491–496. doi:10.1097/ICO.0000000000000379. PMID 25826322.
11. Sosne G, Ousler GW. Thymosin beta 4 ophthalmic solution for dry eye: a randomized, placebo-controlled, Phase II clinical trial conducted using the controlled adverse environment (CAE) model. Clinical Ophthalmology. 2015;9:877–884. doi:10.2147/OPTH.S80954.
12. Sosne G. Thymosin beta 4 and the eye: the journey from bench to bedside. Expert Opinion on Biological Therapy. 2018;18(sup1):99–104. doi:10.1080/14712598.2018.1486818. PMID 30002412.
13. Shah R, Reyes-Gordillo K, Rojkind M. Thymosin β4 inhibits PDGF-BB induced activation, proliferation, and migration of human hepatic stellate cells via its actin-binding domain. Expert Opinion on Biological Therapy. 2018;18(sup1):177–184. doi:10.1080/14712598.2018.1478961. PMID 30063851.
14. World Anti-Doping Agency. The Prohibited List 2026. Section S2.3, Growth factors and growth factor modulators: "Thymosin-β4 and its derivatives e.g. TB-500". In force 1 January 2026. wada-ama.org/en/prohibited-list.

This monograph summarises the published literature for laboratory researchers. It is not medical advice, and nothing on this site is for human or veterinary use.