TB-500: what it is, how it works and what studies have found

TB-500 is searched far more often than it is studied. Much of what is written about it online is borrowed from research on thymosin beta-4, a 43-amino-acid protein, while the molecule anti-doping laboratories actually find in TB-500 products is a seven-residue fragment of that protein. Getting that distinction right is the first step in reading the literature, and in knowing what a vial labelled TB-500 contains.

This overview covers both molecules, keeps their evidence separate, and states the evidence level for each finding. It is written for researchers and quality teams and gives no guidance on human use.

What is TB-500?

TB-500 is a trade-style common name, not an INN or a United States Adopted Name. FDA’s 2026 evaluation states that TB-500 free base “is understood today to be a seven amino acid synthetic fragment of thymosin beta-4 (β4) from amino acids 17 to 23 and an acetyl group at the N-terminal leucine amino acid (Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln-OH)” [1]. The same document warns that FDA “has encountered multiple salts, and derivatives, including different active moieties, sold commercially under the same common name” [1].

The fragment identity comes from anti-doping chemistry. In 2012, a Ghent University laboratory detected and identified N-terminally acetylated LKKTETQ in a TB-500 product by high-resolution mass spectrometry and synthesised it by solid-phase synthesis as a reference standard [2]. A Hong Kong equine doping-control laboratory described TB-500 as a veterinary preparation whose key ingredient is LKKTETQ with artificial N-terminal acetylation [3].

TB-500 vs thymosin beta-4

Property	TB-500 (as described by FDA)	Thymosin beta-4
What it is	Synthetic fragment, residues 17-23, N-acetylated [1]	Natural 43-residue protein, N-terminally acetylated [4]
Sequence	Ac-LKKTETQ-OH [1]	Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES [5]
Molecular formula	C38H68N10O14 (free base) [1] [6]	C212H350N56O78S [7]
Molecular weight	889.01 g/mol free base; 949.1 g/mol acetate [1]	4963 g/mol [7]
CAS number	885340-08-9 (free base) [1]	77591-33-4 [7]
INN	None [1]	Timbetasin [7]
UNII	QHK6Z47GTG (free base) [1]	2D5MRE3SSY [7]
Methionine	None	One, at position 6 of the mature chain [5] [24]
Human clinical studies	None found by FDA [1]	Phase 1 to 3 trials (see below)
WADA	S2 [8]	S2 [8]

FDA states plainly that “thymosin beta-4 and TB-500 are not the same substance”, although several websites use the terms interchangeably [1]. The confusion is visible even in regulatory filings. In the withdrawn 503A nomination, FDA found that the nominated substance was TB-500 free base, the certificate of analysis was for the acetate, the molecular formula matched neither form, and a second CAS number given matched neither [1]. FDA also notes that several supplier websites use the free-base CAS number for the acetate [1]. Products that contain full-length thymosin beta-4 and are sold as TB-500 should carry the CAS number 77591-33-4 and a mass near 4963 g/mol [7]; products containing the fragment should show 889 g/mol for the free base [1].

Structure and chemistry of thymosin beta-4

Thymosin beta-4 was sequenced in 1981 from calf thymus: 43 amino acid residues, a molecular weight of 4982, an isoelectric point of 5.1, and an N-terminus “blocked by an acetyl group” [4]. It was isolated from thymosin fraction 5 and was first thought to be a thymic hormone [1] [4].

The human protein is encoded by TMSB4X [5]. UniProt records a 44-residue precursor whose initiator methionine is removed, leaving the 43-residue chain with N-acetylserine at the start, and notes that the N-terminal tetrapeptide AcSDKP is released as a separate hemoregulatory peptide [5]. UniProt annotates the whole chain as a disordered region [5]. Residues 17-23 of the mature chain, LKKTETQ, are the sequence used in TB-500 [1] [2].

Mechanism of action: actin sequestration

The best-established function of thymosin beta-4 is cytoskeletal. In 1991, Safer and colleagues showed that “Fx”, the 5 kDa peptide that holds much of the unpolymerised actin in resting platelets, is identical in sequence to thymosin beta-4 [9]. Thymosin beta-4 forms a 1:1 complex with actin monomers and inhibits their polymerisation [9]. UniProt summarises its role as binding and sequestering G-actin and thereby inhibiting actin polymerisation [5].

Two further findings shape the research literature:

• Cardiac signalling (animal). In mouse embryonic and postnatal cardiomyocytes, thymosin beta-4 formed a functional complex with PINCH and integrin-linked kinase (ILK) and activated Akt; after coronary artery ligation in mice it improved early myocyte survival and cardiac function [10].
• Wound repair (animal). In a rat full-thickness wound model, topical or intraperitoneal thymosin beta-4 increased re-epithelialisation by 42% over saline at day 4 and by up to 61% at day 7, and stimulated keratinocyte migration in vitro [11].

These are findings for the full-length protein. Whether a seven-residue fragment reproduces them is a separate question, and the direct evidence is thin, as the next section shows.

What is known about TB-500 itself

The research on Ac-LKKTETQ as a molecule is mostly analytical and metabolic:

Study	Evidence level	Model	Main finding	Ref
Esposito 2012	Analytical	TB-500 product	Identified Ac-LKKTETQ by HPLC-HRMS; synthesised reference standard by SPPS	[2]
Ho 2012	Animal (horse)	Equine plasma and urine	Detection at 0.02 ng/mL (plasma) and 0.01 ng/mL (urine); metabolites identified after administration	[3]
Rahaman 2024	In vitro and animal (rat)	Human serum, enzyme systems, rat urine, fibroblasts	Ac-LK main early metabolite; Ac-LKK detected up to 72 h; only Ac-LKKTE showed wound-healing activity	[12]
Biçer 2026	Animal (rat)	Achilles tendon repair, n = 32	TB-500 group had significantly higher load to failure and lower Bonar and Movin scores than controls; sequence of test material not reported	[13]

Metabolism

In horses, plasma TB-500 peaked 60 to 120 minutes after subcutaneous administration and became unquantifiable within 6 to 10 hours, according to FDA’s summary of the Hong Kong study [1]. Equine liver homogenate removed residues one at a time from the C-terminus, producing Ac-LKKTET, Ac-LKKTE, Ac-LKKT, Ac-LKK and Ac-LK, and also produced stereoisomers of the parent peptide [1]. In rats, Ac-LK was the main metabolite in the first six hours and Ac-LKK was a long-lived metabolite detectable for up to 72 hours [12].

Activity

The Korean doping-control study tested the parent peptide and its metabolites in a fibroblast scratch assay. Only Ac-LKKTE showed significant wound-healing activity, leading the authors to suggest that “the previously reported wound-healing activity of TB-500 in literature may be due to its metabolite Ac-LKKTE rather than the parent form” [12]. FDA’s reading of the same study is that TB-500 free base did not induce wound closure, that the study lacked a concentration-response analysis, and that it remains unknown whether TB-500 has wound-healing properties in vivo [1].

The 2026 rat Achilles study is the only in vivo efficacy study of TB-500 identified for this review. Compared with untreated controls, the TB-500 group showed higher maximum load to failure (p < 0.05) and lower total Bonar (p = 0.016) and Movin (p = 0.017) scores; adding BPC-157 conferred no additional benefit [13]. The authors describe it as an exploratory study needing longer-term work [13]. The paper describes TB-500 only as a synthetic peptide derived from thymosin beta-4 and does not report the sequence or molecular weight of the material tested, so it cannot be confirmed whether the fragment or the full-length protein was used [13].

Published thymosin beta-4 clinical research

All of the human trials below used full-length thymosin beta-4, either synthetic or recombinant, or an ophthalmic solution of it (RGN-259). None tested the TB-500 fragment, and FDA found no clinical data on TB-500 [1].

Study	Phase / design	Population	n	Main finding	Ref
Ruff 2010	Phase 1, randomised, placebo-controlled, IV, synthetic thymosin beta-4	Healthy volunteers	4 cohorts of 10	No dose-limiting toxicity or serious adverse events over single and 14-day daily dosing; half-life rose with dose	[14]
Wang 2021	Phase 1, randomised, double-blind, IV, recombinant (NL005)	Healthy Chinese volunteers	54 single-dose, 30 multiple-dose	Adverse events mild to moderate; no serious adverse events; no accumulation	[15]
Zhang 2025	Randomised, double-blind, placebo-controlled, recombinant	STEMI after primary PCI	96	Overall infarct area not significantly different; smaller infarcts in the subgroup dosed within 8 h of PCI (n = 43)	[16]
Sosne 2015	Phase 2, randomised, double-masked, eye drops	Severe dry eye	9	Day 56: 35.1% less ocular discomfort and 59.1% less corneal staining than vehicle	[17]
ARISE-3	Phase 3, randomised, placebo-controlled, eye drops	Dry eye	700 enrolled	Primary endpoints similar to placebo (corneal staining −0.41 vs −0.46; discomfort −0.4 vs −0.4)	[18]
SEER-1	Phase 3, terminated (business decision), eye drops	Neurotrophic keratopathy	18	Complete healing at day 29 in 6 of 10 vs 1 of 8 on placebo	[19]
SEER-2	Phase 3, eye drops	Neurotrophic keratopathy	70 (estimated)	Recruiting; no results posted	[20]
Guarnera 2007	Randomised, double-blind, placebo-controlled, dose-escalation	Venous leg ulcers	72 planned	Design report; 21 enrolled in the first group at time of writing	[21]

Ophthalmic studies

All three registered phase 3 trials of thymosin beta-4 identified for this review are ophthalmic [18] [19] [20]. A nine-patient phase 2 trial of thymosin beta-4 eye drops (RGN-259) in severe dry eye reported, at day 56, a 35.1% reduction in ocular discomfort (p = 0.0141) and a 59.1% reduction in total corneal fluorescein staining (p = 0.0108) compared with vehicle [17]. The authors called it a “small trial” [17]. The much larger ARISE-3 phase 3 trial, with 351 patients on RGN-259 and 348 on placebo analysed, reported mean changes in inferior corneal staining of −0.41 versus −0.46 and in ocular discomfort of −0.4 versus −0.4 at day 15 [18]. In neurotrophic keratopathy, SEER-1 was terminated after 18 patients for a business decision, with complete healing at day 29 in 6 of 10 treated patients versus 1 of 8 on placebo [19]. A second phase 3 study, SEER-2, is listed as recruiting [20].

Cardiac studies

Cardiac clinical studies followed the 2004 mouse work [10]. A first phase 1 study of intravenous synthetic thymosin beta-4 in healthy volunteers found no dose-limiting toxicity, and the authors suggested further development for cardiac ischaemia [14]. A Chinese recombinant form, NL005, completed a phase 1 study in 84 healthy volunteers with no serious adverse events [15]. In the subsequent randomised trial in 96 patients with ST-elevation myocardial infarction, the overall difference in infarcted area versus placebo was not significant; a reduction was reported in the 43 patients who received the first dose within 8 hours after PCI [16]. That is a subgroup finding, and the authors called for further randomised studies [16].

Wound healing

A European randomised, double-blind study in venous leg ulcers was designed to randomise 72 patients 3:1 to thymosin beta-4 or placebo over 84 days, with complete closure of the index ulcer as the efficacy measure [21]. The 2007 report describes the design and early enrolment rather than results [21].

Safety findings reported in studies

• Thymosin beta-4 (human). In intravenous phase 1 studies, adverse events were infrequent and mild or moderate, with no serious adverse events or dose-limiting toxicities [14] [15]. The dry-eye phase 2 trial described the eye drops as safe and well tolerated [17].
• TB-500 (human). No human safety data. FDA identified no acute or repeat-dose toxicity studies of TB-500 free base or acetate [1].
• Quality and immunogenicity. FDA states that TB-500 “may pose risk for immunogenicity for certain routes”, that it “has not identified any human exposure data”, and that it “lacks important information regarding any safety issues raised by this drug” [22]. Its 2026 evaluation adds that publicly available certificates did not include tests for impurities, aggregates, microbial quality or bacterial endotoxin [1].

TB-500 regulatory status

Region / body	Status	Ref
United States (FDA)	Not approved; no USP or NF monograph; not a component of an approved drug. Listed by FDA as nominated but withdrawn, under the name thymosin beta-4 fragment (LKKTETQ). FDA’s 2026 evaluation proposes not adding TB-500 free base or acetate to the 503A bulks list	[1] [22]
Advisory committee	Discussed by the Pharmacy Compounding Advisory Committee on 23 July 2026; members reportedly voted 8 to 6, with one abstention, in favour of listing for wound healing; non-binding; no minutes, vote or final FDA determination posted on FDA’s meeting page at the time of writing	[23] [27] [28]
Thymosin beta-4 (any region)	No marketing authorisation identified in this review; ophthalmic phase 3 development ongoing	[20]
Sport (WADA)	TB-500 and thymosin beta-4 prohibited under S2; FDA cites section S2.3	[1] [8]

FDA evaluated TB-500 for wound healing, the use given in the nomination, and concluded that the evaluation criteria “weigh against” adding it to the 503A bulks list because it is not well characterised, it raises immunogenicity concerns for injectable routes, and there is no clinical or adequate nonclinical evidence for the proposed use [1]. The Pharmacy Compounding Advisory Committee discussed TB-500 on 23 July 2026 [23]. Trade press reported that members voted 8 to 6, with one abstention, in favour of adding TB-500 to the 503A bulks list, the same tally reported for BPC-157 and KPV, and that the vote does not constitute FDA approval or demonstrated clinical benefit [27]. ABC News listed TB-500 among six peptides the committee recommended and noted that the vote is non-binding and still needs FDA sign-off [28]. The vote ran counter to the conclusion of FDA’s own evaluation [1]. FDA’s meeting page posts no minutes or voting results, and any listing would require FDA rulemaking [23] [27]. A favourable vote does not make TB-500 an approved drug and adds nothing to the clinical evidence described above, which contains no human studies of TB-500 itself [1]. FDA notes that WADA approved a project in 2013 to determine detection limits for TB-500 metabolites [1]. The US Anti-Doping Agency’s 2027 list advisory lists “TB-500 (Prohibited under S2)” and “Thymosin-beta 4 (Prohibited under S2)” [8].

TB-500 in laboratory research

TB-500 research is dominated by analytical method development, which has produced validated detection methods even though its biology is little studied and FDA considers the substance not well characterised [1]:

• Identity work: HPLC with high-resolution Orbitrap mass spectrometry for identification, and triple-quadrupole LC-MS for detection in plasma and urine [2].
• Doping control: solid-phase extraction on ion-exchange cartridges with LC-MS, confirming the parent and metabolites in equine samples [3].
• Metabolite quantification: UHPLC-Q-Exactive Orbitrap MS/MS with synthesised metabolite standards in human serum, enzyme systems and rat urine [12].
• Cell assays: fibroblast scratch-wound and cytotoxicity assays comparing parent and metabolites [12].
• Full-length protein models: rat full-thickness wounds and keratinocyte migration [11]; mouse coronary ligation with ILK and Akt activity [10].

Because the metabolites are C-terminal truncations of the parent (Ac-LKKTET down to Ac-LK) [1], a research method for TB-500 should resolve these from the intact heptapeptide; truncated sequences are also among the impurity classes FDA lists for solid-phase synthesis [1].

Synthesis, purity and impurities

The TB-500 heptapeptide is made by Fmoc solid-phase peptide synthesis. FDA’s summary of the published route starts with Fmoc-Gln(Trt)-OH on Wang resin, uses HBTU and DIPEA couplings in DMF, removes Fmoc groups with piperidine, acetylates the N-terminal leucine with acetic anhydride in pyridine, then cleaves with Reagent K and precipitates the peptide in methyl tert-butyl ether [1]. The N-terminal acetyl group is part of the molecule’s identity: the unacetylated heptapeptide is a different compound and an obvious process impurity to look for.

FDA lists the generic impurity classes for a solid-phase peptide: incomplete couplings, truncations, side reactions, isomeric impurities and free amino acids from starting materials, residual solvents, coupling reagents, activators, catalysts and scavengers, and aggregates [1]. The acetate certificate of analysis supplied with the nomination reported total impurities by HPLC of no more than 2.0% and a largest single impurity of no more than 1.0%, but no identification, assay, aggregate or endotoxin results; a public free-base certificate FDA reproduced covered only appearance, LC-MS and HPLC identity, and purity [1]. Specification setting for research and API peptides is covered in peptide API impurities and specifications, and route selection in SPPS vs LPPS.

Full-length thymosin beta-4 is a different manufacturing problem. Clinical material has been produced both synthetically [14] and recombinantly [15]. Its single methionine (Met6) can oxidise to the sulfoxide, which binds actin with about 20-fold lower affinity under polymerising conditions and almost lacks the polymerisation-inhibiting activity of the native protein [24]. Methionine sulfoxide is therefore a critical impurity for thymosin beta-4 but not for the TB-500 fragment, which contains no methionine.

Storage and stability of lyophilized TB-500

FDA’s evaluation cites product data reporting that TB-500 free base powder should be kept sealed, away from moisture and light, under nitrogen, at −80 °C for up to two years or −20 °C for one year; for the acetate, public sources recommend below −15 °C, protected from light, in a tightly closed container [1]. FDA adds that peptides such as TB-500 are sensitive to pH, temperature, concentration and excipients, which can drive aggregation and degradation [1]. Laboratory practice for lyophilized peptides is set out in lyophilised peptide stability and storage.

TB-500 vs BPC-157

	TB-500	BPC-157
Parent molecule	Thymosin beta-4, residues 17-23 [1]	A gastric juice protein (BPC) [25]
Length	7 residues, N-acetylated [1]	15 residues [25]
Proposed biology	Described as the actin-binding region of thymosin beta-4 [1] [3]	VEGFR2 activation in angiogenesis models [26]
Head-to-head rat tendon study	Significant biomechanical gain [13]	Not significant on total scores [13]
WADA class	S2 [8]	S0 [8]

BPC-157’s evidence base, human reports and regulatory history are reviewed in BPC-157: structure, mechanism and research evidence.

How Winhycare Pharma supplies TB-500

Winhycare Pharma supplies TB-500 as research-use-only material at a purity of at least 99%, as stated in our brochure. Because the name TB-500 is used for more than one molecule, every certificate of analysis states the sequence, molecular weight and salt form, so laboratories can confirm exactly what they are receiving. Custom synthesis is available. Product details are on the TB-500 page; to discuss supply, contact our pharma team.

For laboratory research use only. Not for human or veterinary use.