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Thymosin beta-4

Also known as TB4, Tbeta-4, Tß4, TMSB4X gene product, RGN-259 (ophthalmic formulation), RGN-352 (intravenous formulation)

Endogenous 43-amino-acid actin-sequestering protein; the compound behind almost all TB-500 marketing claims.

early-clinical Recovery & tissue endogenous peptide

At a glance

Category
Recovery & tissue
Status
endogenous peptide
Route
In trials: intravenous, topical (gel) and ophthalmic (eye drops). Grey-market use is subcutaneous or intramuscular.
Half-life
Short. In a first-in-human single- and multiple-dose phase 1 study of recombinant human thymosin beta-4 given intravenously at 0.25-25 mcg/kg, terminal half-lives across dose cohorts ranged from 0.5 to 2.08 hours, with Cmax and AUC increasing proportionally with dose 1. No published pharmacokinetics exist for subcutaneous dosing in humans.
Onset
In the ophthalmic trials, healing endpoints were assessed at 28-43 days.
Molecular weight
4921 g/mol
Sequence
SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES (43 amino acids, N-terminally acetylated in vivo)

A naturally occurring human protein, not an approved medicine anywhere. Synthetic and recombinant thymosin beta-4 has been through phase 1 1, phase 2 and phase 3 trials 2 (mainly ophthalmic, by RegeneRx Biopharmaceuticals) without gaining marketing authorisation in any jurisdiction. The material sold to consumers is generally the seven-residue fragment TB-500, which the FDA lists separately among bulk drug substances that may present significant safety risks 6, not this protein.

Doping status: Prohibited (WADA S2.3, growth factors - "Thymosin-ß4 and its derivatives e.g. TB-500")

Checked against the WADA 2026 Prohibited List. For a specific product and country, Global DRO is the lookup athletes are expected to use — the List names substances, not brand names.

Mechanism of action

Thymosin beta-4 is the dominant intracellular G-actin-sequestering protein in most mammalian cells, present at concentrations that can approach 0.5 mM. It binds monomeric actin in a 1:1 complex and acts as a buffer, allowing the cytoskeleton to be rapidly disassembled and rebuilt. This is not a signalling role in the classical sense: it is a structural regulator of how quickly a cell can change shape and move.

That cytoskeletal function underlies the migration effects that dominate the literature: keratinocytes, corneal epithelial cells and endothelial cells move faster in its presence. Downstream and less well characterised effects that are repeatedly described include upregulation of VEGF and angiogenesis, suppression of NF-kB signalling and pro-inflammatory cytokines, and anti-apoptotic activity via Bcl-2 and laminin-5. No specific cell-surface receptor for thymosin beta-4 has been definitively identified, which remains a real gap: an extracellularly administered protein producing effects through a mechanism defined intracellularly is a mechanistic loose end, not a solved question.

A separate and important pathway runs through proteolysis. The N-terminal tetrapeptide Ac-SDKP (residues 1-4, also known as goralatide) is released from the intact protein by prolyl oligopeptidase and meprin alpha, and is itself an antifibrotic and haematopoietic-regulating peptide degraded by ACE. Part of the antifibrotic activity attributed to thymosin beta-4 is attributable to Ac-SDKP rather than to the parent protein.

This is the point on which the distinction from TB-500 turns. TB-500 is the acetylated heptapeptide Ac-LKKTETQ, residues 17-23, the actin-binding motif only. It contains neither the N-terminal region that yields Ac-SDKP nor the remainder of the 43-residue chain. Retaining the actin-binding motif is not the same as reproducing the protein's biology, and the two should not be treated as interchangeable.

What the research shows

Thymosin beta-4 has a genuine, if modest and unsuccessful, clinical development history: phase 1 safety data, phase 2 signals in dry eye and dermal ulcers, and a phase 3 in neurotrophic keratopathy that missed its primary timepoint. None of it has produced an approval. Crucially, all of that human evidence concerns this 43-residue protein, and none of it concerns the heptapeptide fragment sold commercially as TB-500. Anyone citing 'the clinical trials' as support for an injected fragment is transferring evidence across molecules.

Research in humans

Recombinant human thymosin beta-4 was given intravenously to healthy volunteers in a single- and multiple-dose phase 1 study without dose-limiting toxicity 1. The ophthalmic programme (RGN-259, 0.1% eye drops) is the most developed: a phase 2 in severe dry eye reported improvement in signs and symptoms, the ARISE-1 through ARISE-3 dry eye trials produced mixed results across endpoints, and the SEER-1 phase 3 in neurotrophic keratopathy terminated early at n=18 with 6/10 treated patients achieving complete corneal healing at day 29 versus 1/8 on placebo (p=0.0656, not significant at that timepoint; significance was reached at day 43). Earlier phase 2 work in pressure ulcers, venous stasis ulcers and epidermolysis bullosa reported acceptable tolerability with limited efficacy signals. The intravenous cardiac programme (RGN-352) in acute myocardial infarction was suspended without published results. All human dosing has been intravenous, topical or ophthalmic; there is no published human trial of subcutaneous thymosin beta-4.

Animal and lab research

The preclinical base is substantial and spans cardioprotection after myocardial ischaemia, corneal epithelial healing, dermal wound healing, and functional recovery in rodent models of stroke, traumatic brain injury, spinal cord injury and demyelination. Doses and routes vary widely between laboratories and are often local rather than systemic.

Caveats. The much-publicised finding that thymosin beta-4 activates adult epicardial progenitor cells to regenerate cardiac muscle has proved difficult to reproduce in independent laboratories, and the field has substantially retreated from that claim. The ophthalmic trials are small and the pivotal phase 3 was stopped early with 18 patients, which makes both the positive and negative readings fragile. No long-term toxicology or carcinogenicity dataset has been published. Finally, the market itself is a source of error: vials sold as 'TB-500' sometimes contain the full protein and sometimes the heptapeptide, and users frequently cannot tell which they have.

What it is used for

  • Corneal and ocular surface disease (neurotrophic keratopathy 2, dry eye 3) - the only setting with phase 3 data
  • Chronic wounds and ulcers, in early clinical work
  • Grey-market injection for tendon, ligament and muscle injury, without any human trial support for that route or indication
  • Cited as the scientific justification for TB-500 products, which are a different molecule

Dosing

Dose
Clinical: RGN-259 eye drops 0.1%, dosed topically to the eye. Intravenous phase 1 dosing was weight-based and escalating. Grey-market injection protocols quote 2-5 mg per administration, but these are protocols written for TB-500 and simply reused.
Frequency
Ophthalmic trials dosed multiple times daily. Injection protocols quote once or twice weekly.
Route
Ophthalmic and intravenous in trials; subcutaneous or intramuscular in grey-market use
Duration
28-42 days in the ophthalmic trials
  • There is no established subcutaneous dose for thymosin beta-4 in humans. Every published human exposure has been intravenous 1, topical or ophthalmic 23. Any figure quoted for injection is extrapolated, usually from TB-500 protocols that were themselves never validated - the FDA states it has identified no human exposure data for that fragment at all 6.
  • The dose scales are not comparable between routes: an eye drop delivers a local concentration to an avascular surface, and cannot be reasoned from to a systemic milligram dose.
  • Because the protein is 4921 g/mol and the fragment is roughly 889 g/mol, a milligram of one is not molar-equivalent to a milligram of the other. Check the stated molecular mass on any vial.

These figures describe what the literature and published protocols report. They are not advice and not a dosing instruction.

Protocols

RGN-259 ophthalmic solution in neurotrophic keratopathy (phase 3)

published trial

Source: International Journal of Molecular Sciences, 2022

Treatment period0.1% RGN-259 ophthalmic solution instilled into the affected eye, several times daily
Lengththe ophthalmic trials ran 28-42 days

The only phase 3 schedule for full thymosin beta-4 is an eye drop, delivered to an avascular surface. It cannot be reasoned from to a systemic milligram dose, and the 2-5 mg injection figures circulating under this compound's name were not derived from it — they are TB-500 protocols reused under a different molecule's name.

Grey-market injection for recovery (user-reported)

user protocol — not validated

Source: The pattern repeated across bodybuilding and peptide forums together with vendor labelling of vials sold as 'thymosin beta-4' or 'TB-500'

This is not a validated schedule. It is a pattern that circulates among users and sellers, reproduced because it is what people actually follow — not because it has been tested. No trial established these doses, this interval or this duration, and nobody is checking what is in the vial. Treat every number below as an assertion, not a finding.

Reported loading2-5 mg per injection, roughly twice weekly for the first weeks
Reported maintenancea similar amount once weekly, or once every one to two weeks

Every published human exposure to the full protein was intravenous, topical or an eye drop; there is no subcutaneous human dose for thymosin beta-4 at all. The figures that circulate — 2-5 mg injected for tendon, ligament and muscle recovery — are not derived from any trial of this molecule. They are TB-500 protocols reused under this name, and TB-500 is a different, smaller molecule (roughly 889 g/mol against 4921), so a milligram of one is not molar-equivalent to a milligram of the other. Users frequently cannot tell whether a vial labelled 'TB-500' or 'Tβ4' contains the heptapeptide fragment or the full protein, which makes the reused number doubly unreliable. Injection-site reactions are reported; immunogenicity from repeated injection of a protein is a real theoretical concern that has not been characterised; and the pro-migratory, pro-angiogenic biology makes active malignancy the standing reason for caution. The material is unverified for identity and purity.

Schedules are reproduced as their source states them. Units, IU and milligrams explains why the figures are not interchangeable between products.

Reconstitution

Vial sizes
2 mg, 5 mg, 10 mg
Solvent
bacteriostatic water (0.9% benzyl alcohol)
Storage
Reconstituted at 2-8 °C; the customary 3-4 week window is convention rather than a published stability figure for this protein. Lyophilised powder frozen or refrigerated, protected from light.

Worked example

5 mg vial + 2 ml bacteriostatic water = 2.5 mg/ml. 2.5 mg = 1 ml, i.e. 100 units on a U100 insulin syringe.

Do not shake. A 43-residue largely unstructured protein is prone to aggregation on agitation; let the diluent run down the vial wall and swirl.

Work it out for Thymosin beta-4

Safety

Side effects

  • Intravenous phase 1 dosing in healthy volunteers reported no serious adverse events and no dose-limiting toxicities; all adverse events were mild or moderate and resolved without treatment 1
  • Ophthalmic use: transient ocular discomfort, burning or stinging on instillation
  • Injection site reactions with grey-market subcutaneous use
  • Immunogenicity is a theoretical concern for any injected recombinant or synthetic protein and has not been characterised for chronic dosing
  • There is no controlled safety dataset for repeated subcutaneous administration in humans

Do not use if

  • Active or previous malignancy. This is the best-substantiated concern for this molecule: elevated thymosin beta-4 expression has been associated with tumour cell migration, invasiveness and metastatic potential in colorectal, melanoma and other tumour types, and the combination of pro-angiogenic and pro-migratory activity is unfavourable in an oncological setting.
  • Pregnancy and breastfeeding: no data
  • Children and adolescents: no data outside of trial settings
  • Competitive athletes under the WADA code: prohibited at all times under S2.3, growth factors and growth factor modulators, listed as "Thymosin-ß4 and its derivatives e.g. TB-500" and classed as a non-Specified substance 5

Interactions

Not systematically studied. One mechanistically real interaction is worth noting: Ac-SDKP, the N-terminal fragment released from thymosin beta-4, is degraded by angiotensin-converting enzyme, so ACE inhibitors raise Ac-SDKP levels. Co-administration of an ACE inhibitor with thymosin beta-4 could therefore amplify the Ac-SDKP-dependent portion of its activity. This has not been studied clinically. The interaction does not apply to TB-500, which contains no Ac-SDKP sequence.

Sources