Thymosin Alpha-1 10mg Peptide

Thymosin Alpha-1 is a 28-residue acetylated peptide first purified from calf thymus and sequenced by Goldstein and colleagues in 1977, who described it as a heat-stable, highly acidic molecule present in the crude preparation then known as thymosin fraction 5. The international non-proprietary name for the synthetic version is thymalfasin, and the peptide is sold as a medicine under the trade name Zadaxin in a substantial number of countries outside North America.

The molecule is not synthesised as a standalone gene product. It corresponds to the acetylated N-terminal 28 residues of prothymosin alpha, a 111-residue nuclear protein involved in chromatin remodelling. For decades the field assumed Thymosin Alpha-1 was an extraction artifact created by proteolysis during purification. Work published in 2003 showed otherwise: a lysosomal asparaginyl endopeptidase identified as legumain cleaves the Asn28-Gly29 bond in prothymosin alpha, and Thymosin Alpha-1 is found free in the cytosol of several mammalian tissues at concentrations comparable to the parent protein.

Two things make it unusual in a research peptide catalogue. It has a genuine regulatory history, with marketing authorisations in more than 30 countries and 41 studies currently registered on ClinicalTrials.gov, and it has a mechanism that runs through innate immune pattern recognition receptors rather than a classical hormone receptor. Both facts are frequently garbled in secondary sources, and the peptide is routinely confused with two unrelated compounds: TB-500, which is a fragment of Thymosin Beta-4, and Thymalin, which is a thymus extract rather than a defined sequence.

Thymosin Alpha-1 has no dedicated receptor of its own. The best-characterised route of action is agonism at Toll-like receptors on antigen-presenting cells. In fungus-pulsed murine dendritic cells, the peptide drove functional maturation and interleukin-12 production through a p38 MAPK and NF-kB dependent pathway that required MyD88, and mice deficient in the relevant Toll-like receptors lost the effect (Romani et al., Blood 2004). A follow-up study in murine cytomegalovirus infection localised the activity to plasmacytoid dendritic cells and traced it through TLR9, MyD88 and IRF7 to an interferon-alpha and interferon-gamma dependent effector arm (Bozza et al., International Immunology 2007).

Downstream of that innate signal the peptide acts on T cells. Across preclinical and clinical work it increases the proportion of mature CD4-positive and CD8-positive cells, raises interleukin-2 and interferon-gamma output, and pushes differentiation toward a Th1 phenotype. It also increases surface expression of MHC class I on infected cells and of monocyte HLA-DR, which is why HLA-DR was chosen as the mechanistic secondary endpoint in the ETASS sepsis trial rather than a cytokine panel.

The effect is bidirectional rather than uniformly stimulatory, and the cleanest demonstration of that is a 2015 in vitro study in human monocyte-derived dendritic cells. Adding Thymosin Alpha-1 alongside the viral-sensing agonists for TLR3 and TLR7/8, or alongside pandemic H1N1 influenza A infection, raised HLA class I and II expression and interferon output. Adding it alongside the bacterial agonists for TLR2 and TLR4, or alongside Bacillus Calmette-Guerin infection, lowered the same readouts sharply (Giacomini et al., Expert Opinion on Biological Therapy 2015). The peptide reads the context set by the other receptor rather than imposing a fixed direction.

A third strand of the mechanism concerns tolerance. In the dendritic cell work the peptide induces indoleamine 2,3-dioxygenase, an enzyme that restrains effector T cell expansion and supports regulatory T cell function. That is the mechanistic reason the same molecule can be studied as an immune activator in hepatitis B and as a brake on immune-related adverse events during checkpoint inhibitor therapy, which is what NCT06178146 is testing.

Goldstein and colleagues isolated the peptide from calf thymus and published the sequence in the Proceedings of the National Academy of Sciences in 1977, naming it for its position within thymosin fraction 5. That fraction was a crude acid-extracted mixture, and the alpha, beta and gamma prefixes in the thymosin family are an isoelectric-focusing classification from that era, not a statement of structural or functional relatedness. This is the single most consequential piece of nomenclature history for anyone comparing peptides in this class.

The 28 residues match exactly the N-terminus of prothymosin alpha, a highly acidic 111-residue nuclear protein. Human prothymosin alpha, UniProt P06454, begins MSDAAVDTSSEITTKDLKEKKEVVEEAENGRDAP. After removal of the initiator methionine and N-terminal acetylation, residues 1 to 28 of the mature chain are the peptide, and residue 29 is the glycine that follows the Asn28-Gly29 bond.

That bond turned out to be the answer to a long-standing objection. Because neither Thymosin Alpha-1 nor the related 35-residue Thymosin Alpha-11 could be recovered from extracts prepared with protease inhibitors, the field assumed both were generated by proteolysis during purification. Sarandeses and colleagues showed in the Journal of Biological Chemistry in 2003 that mammalian legumain, a lysosomal asparaginyl endopeptidase, cleaves Asn28-Gly29 and Asn35-Gly36 in prothymosin alpha with comparable efficiency in vitro, and that Thymosin Alpha-1 is the dominant product detected free in the cytosol in vivo, at concentrations similar to the parent protein. The peptide is a real cellular species, not a purification ghost.

Thymalfasin is a licensed medicine in more than 30 countries, sold as Zadaxin, with chronic hepatitis B as the anchor indication. China granted marketing authorisation in the mid-1990s and remains the largest market. Italy and a group of other national regulators registered it in the narrower role of an adjuvant to influenza vaccine in elderly and immunocompromised recipients, including chronic hemodialysis patients who failed to reach protective antibody titres on prior immunisation. Other national approvals across Asia, the Middle East and South America cover hepatitis B, hepatitis C, and use as an immune adjunct in oncology, with the exact wording varying by country.

The United States is the conspicuous exception. Thymalfasin has never received a US marketing authorisation for any indication. What it holds instead are orphan drug designations, granted between 1991 and 2006, covering chronic active hepatitis B, hepatocellular carcinoma, malignant melanoma, and DiGeorge anomaly with immune defects. An orphan designation is a development incentive, not a finding of efficacy, and the two are conflated constantly in secondary material about this peptide.

The European position is similarly specific. There is no centralised European marketing authorisation, and one was never pursued. The European Medicines Agency did grant orphan designation EU/3/02/110 to thymalfasin for the treatment of hepatocellular carcinoma on 30 July 2002. National registrations within Europe, such as the Italian vaccine-adjuvant indication, sit outside the centralised route.

A separate and often-missed point: the US clinical development programme that produced the largest sepsis dataset was industry-sponsored, and the sponsor is named in the competing-interests statement of the phase 3 trial that read out negative. That is worth knowing when weighing the literature, because a majority of the randomised evidence for this peptide originates from a single country and a small number of sponsoring relationships.

The trial that established the indication randomised 98 patients with clinicopathologically proven chronic hepatitis B to a 26-week course, a 52-week course, or 18 months of observation. Complete virological response, defined as clearance of both serum HBV DNA and HBeAg and assessed 18 months after entry, was 40.6% in the 26-week arm, 26.5% in the 52-week arm and 9.4% in the untreated arm. The result the authors emphasised was the shape of the curve rather than the endpoint alone: response rates were similar across all three groups at the end of therapy and separated only during follow-up, which fits an immunomodulator that hands the response back to the host rather than a direct antiviral. Blinded histology showed improvement in lobular necroinflammation. No responder cleared HBsAg.

A later randomised study in 56 anti-HBe-positive patients compared the peptide with interferon-alpha against a historical control group. Complete response at the end of treatment favoured interferon, 46.7% against 30.8%, but the ordering reversed by the end of a six-month follow-up, 42.3% against 23.3%. Delayed responses were significantly more common in the peptide arm and post-treatment flares significantly more common in the interferon arm. Tolerability differed sharply: no adverse effects were recorded in the peptide group.

The modern question is whether adding the peptide to a nucleoside analogue is worth it. A 2020 systematic review pooling seven randomised trials and 1,144 patients with HBV-related cirrhosis found that adding it to entecavir raised complete response, and at 24 weeks raised both the HBV DNA undetectable rate and HBeAg loss. By weeks 48 and 52 the differences had closed, and HBsAg loss at week 52 showed no difference at all. Adverse event counts were lower in the combination arm. Every included trial came from mainland China, which the authors state as the principal limitation.

ETASS, published in Critical Care in 2013, randomised 361 patients with severe sepsis across six Chinese teaching hospitals, single-blind, with 1.6 mg given subcutaneously twice daily for five days then once daily for two. The 28-day all-cause mortality primary endpoint was 26.0% against 35.0%, relative risk 0.74, and the p value landed at 0.062 on the nonstratified analysis and 0.049 on the log-rank test. In-hospital mortality, a secondary endpoint, was 28.7% against 39.4% with p equal to 0.032. Read honestly, ETASS missed its primary endpoint and hit a secondary one, and the monocyte HLA-DR result gave the mechanistic case its strongest support.

TESTS was designed to settle the question and published in the BMJ in January 2025. It randomised 1,106 adults meeting sepsis-3 criteria across 22 Chinese centres, double-blind and placebo-controlled, with study drug given every 12 hours for seven days. 28-day all-cause mortality was 23.4% against 24.1%, hazard ratio 0.99, and a log-rank p of 0.93. No secondary or safety outcome separated the groups. The trial found no evidence that the peptide reduces 28-day mortality in sepsis, and it is roughly three times the size of ETASS.

The prespecified subgroups are the part worth arguing about. TESTS reported a significant interaction by age, with a hazard ratio of 1.67 in patients under 60 and 0.81 in patients 60 and over, and by diabetes status, 0.58 with diabetes against 1.16 without. A 2025 systematic review pooling 11 randomised trials and 1,927 patients found an overall odds ratio of 0.73 for 28-day mortality, but that signal disappeared when the analysis was restricted to high-quality trials, 0.82, or to multicentre trials, 0.86. The same review's trial sequential analysis concluded the accumulated sample is still inadequate to settle the question.

The plain reading is that the pooled positive result is driven by smaller, lower-quality, single-centre studies, and that the largest and best-controlled trial is null overall. Whether a real effect exists in an older or metabolically compromised subgroup is an open question with a specific shape, which is exactly the sort of hypothesis a subgroup interaction is good for generating and bad for confirming.

The adjuvant work is the least-discussed and most regulatorily consequential part of the file, since it is the basis of the Italian registration. A pilot trial in 94 hemodialysis patients gave an adjuvanted pandemic H1N1v influenza vaccine alone or together with Thymosin Alpha-1 at two dose levels, with hemagglutination inhibition, microneutralisation and single radial hemolysis measured on days 0, 21, 42, 84 and 168. Both peptide groups beat the vaccine-only group on geometric mean titre and geometric mean ratio at day 21, and both met all three CHMP licensing criteria for seroconversion, seroprotection and titre increase. The vaccine-only group did not. No adverse event was attributed to either the peptide or the vaccine.

The rationale is that uremic and elderly immune systems fail to convert vaccine antigen into durable antibody because of the same antigen-presentation deficit the peptide acts on, so the peptide is being used to fix the host rather than the antigen. That logic carried into the COVID era. A randomised study in 194 end-stage renal disease patients across five dialysis centres in Kansas City compared 1.6 mg twice weekly for eight weeks against no peptide, followed for a further four months, with interim counts of 3 deaths against 7 and 5 serious COVID-19 adverse events against 7. The numbers are small and the trial was reported as preliminary.

Interest in the mechanism has not faded. NCT06821100 is a currently recruiting Phase 1 trial of thymalfasin as an enhancer of vaccine response in older adults receiving COVID-19 booster doses, which is the same hypothesis applied to a different antigen.

The largest oncology study is a 488-patient randomised trial in metastatic melanoma comparing dacarbazine plus interferon-alpha against the same backbone with the peptide at 1.6, 3.2 or 6.4 mg, plus a dacarbazine-plus-peptide arm without interferon. Median overall survival was 9.4 months in patients receiving the peptide against 6.6 months in the control arm, hazard ratio 0.80 with a p of 0.08, and progression-free survival showed a comparable non-significant trend. Response counts in the 3.2 mg arms were sufficient to reject the null of a 5% response rate. Adding the peptide produced no additional toxicity, which is the finding that keeps it in combination protocols.

That tolerability is why ClinicalTrials.gov currently lists 41 registered studies against this compound, weighted heavily toward oncology combinations. Active examples include NCT05086614, a Phase 3 adjuvant trial after radical resection of high-risk stage II and III colorectal cancer, NCT06461910 and NCT07277439 in neoadjuvant gastric and gastroesophageal junction adenocarcinoma alongside PD-1 blockade, and NCT06178146, a Phase 4 study using the peptide against immune-related adverse events secondary to checkpoint inhibitors rather than against the tumour.

On the antiviral side, the COVID-19 literature is the largest recent block and its quality is uneven. The most-cited study is a retrospective review of 76 severe cases from two Wuhan hospitals reporting mortality of 11.11% against 30.00%, with treated patients showing recovered CD4-positive and CD8-positive counts, reduced PD-1 and Tim-3 on CD8-positive T cells, and rising T-cell receptor excision circles. It is retrospective and unblinded. The only randomised US trial, a 49-patient open-label study in hospitalised patients with hypoxemia and lymphocytopenia, found no significant difference in clinical recovery but did show 3.84 times the day-1-to-day-5 CD4-positive T cell increase in treated patients on low-flow oxygen. The immunological effect replicates; the clinical benefit has not been demonstrated in a properly controlled trial.

The confusion with TB-500 is the most common error in circulation, and it is purely a naming accident. Thymosin Beta-4 is a 43-residue actin-sequestering protein from a different gene, and TB-500 is a 7-residue fragment of it, Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln, weighing roughly 889 g/mol. Thymosin Alpha-1 is 28 residues from prothymosin alpha at 3,108.3 g/mol. The alpha and beta labels came from where the two peptides landed on an isoelectric-focusing gel of thymosin fraction 5 in the 1970s, not from any shared structure, receptor or pathway. Their mechanisms have no overlap: one engages Toll-like receptors on dendritic cells, the other binds monomeric G-actin and regulates cytoskeletal dynamics and cell migration. This catalogue stocks both, and they answer different research questions. Secondary sources get even the residue counts wrong in both directions, describing TB-500 as 43 residues, which is Thymosin Beta-4, or Thymosin Beta-4 as 7 residues, which is TB-500.

Thymalin is a different kind of error. It is a polypeptide fraction extracted from calf thymus tissue, one of the original Khavinson bioregulator preparations, and it is a mixture rather than a single defined molecule. No CAS number, molecular formula or exact mass applies to it, and lot-to-lot characterisation is a real problem for anyone using it. Thymosin Alpha-1 is one defined 28-residue sequence with an exact mass, a CAS number and a PubChem record. Historically the two overlap, since Thymosin Alpha-1 was itself purified out of a crude thymic extract, but describing Thymalin as being in the same family is a category error: one is a chemical entity, the other is a preparation.

Thymulin is a third neighbour worth keeping straight. It is a zinc-dependent nonapeptide of thymic epithelial origin, unrelated in sequence to either alpha or beta thymosins, and it is the compound most often meant when older literature refers to a thymic hormone with a metal requirement.

The peptide is conformationally unusual. Circular dichroism shows no preferred conformation in water. In the presence of small unilamellar vesicles of dimyristoylphosphatidylcholine and dimyristoylphosphatidic acid, in sodium dodecyl sulphate micelles, or with zinc ions present, it becomes partly structured. In water and trifluoroethanol, two-dimensional NMR resolves two elements: a beta-turn spanning residues 5 to 8 and an alpha helix spanning residues 17 to 24. The working interpretation is that folding is triggered by contact with the lymphocyte membrane rather than being intrinsic, which is consistent with an intrinsically disordered fragment of an intrinsically disordered parent protein.

For handling purposes the practical consequences are favourable. There is no cysteine, so no disulfide bond exists to scramble or reduce. The molecule is strongly acidic, with nine glutamate and four aspartate residues against four lysines, so it is freely soluble in aqueous buffer and carries a large net negative charge at neutral pH. The N-terminal serine is acetylated, which blocks the free amine and removes one route to N-terminal degradation. Lyophilised material stored at minus 20 degrees Celsius, dark and dry, is the standard condition; in solution at 2 to 8 degrees Celsius the working window quoted across suppliers ranges from a few days to 30 days, and that spread reflects an absence of published stability-indicating data for research-grade material rather than a genuine consensus.

One analytical check is worth doing on every lot and is almost never mentioned. Acetylation contributes 42 Da. A correctly acetylated peptide gives a monoisotopic-consistent average mass near 3,108.3, while the des-acetyl form lands near 3,066. If a mass spectrum on a certificate of analysis reports a principal species around 3,066 rather than 3,108, the N-terminal acetyl group is missing and the material is not the compound described here, regardless of what the HPLC purity percentage says. HPLC area percent measures homogeneity, not identity, and the two are routinely reported as if the first proved the second.

Pharmacokinetically the peptide is short-lived. Peak serum concentration is reached within about two hours of a subcutaneous dose, the serum half-life is approximately two hours, and blood levels return to baseline within 24 hours. The absolute subcutaneous bioavailability figures of 90% to 95% that circulate in secondary sources do not trace back to a published human study, and are omitted here for that reason.