Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro.
Rahaman. Khandoker Asiqur KA; Muresan. Anca Raluca AR; Min. Hophil H; Son. Junghyun J; Han. Hyung-Seop HS; Kang. Min-Jung MJ; Kwon. Oh-Seung OS
Key Findings
- TB‑500 and its breakdown products can be measured accurately with a new UHPLC‑Orbitrap method.
- The main early metabolite in rats is Ac‑LK; a longer‑lasting metabolite is Ac‑LKK, detectable up to 72 hours.
- No cytotoxic (cell‑killing) effects were seen for TB‑500 or any of its metabolites.
- Only the metabolite Ac‑LKKTE showed a significant increase in wound‑healing activity in fibroblast cells.
Practical Outcomes
- For biohackers interested in using TB‑500 for skin repair, the benefit likely comes from the metabolite Ac‑LKKTE rather than the parent peptide, so timing and dosing strategies that favor its formation may be more effective. The lack of observed toxicity supports a relatively safe safety profile at studied doses, but the study does not provide specific dosing recommendations.
Summary
The study shows that the peptide TB-500 itself isn’t toxic and doesn’t speed up wound healing, but one of its breakdown products, called Ac‑LKKTE, does help wounds close faster. The researchers also mapped how TB‑500 is broken down in the body, finding that another fragment, Ac‑LK, appears quickly and another, Ac‑LKK, sticks around for days.
Abstract
TB-500 (Ac-LKKTETQ), derived from the active site of thymosin β4 (Tβ4), has various biological functions in its unacetylated form, LKKTETQ. These functions include actin binding, dermal wound healing, angiogenesis, and skin repair. The biological effects of TB-500, however, have not been documented. And the analysis of TB-500 and its metabolites have been neither simultaneously quantified nor structurally identified using synthesized authentic standards. This study was aimed to investigating simultaneous analytical methods of TB-500 and its metabolites in in-vitro and urine samples by using UHPLC-Q-Exactive orbitrap MS, and to comparing the biological activity of its metabolites with the parent TB-500. The metabolism of TB-500 was investigated in human serum, various in-vitro enzyme systems, and urine samples from rats treated with TB-500, and their biological activities measured by cytotoxicity and wound healing experiments were also evaluated in fibroblasts. The simultaneous analytical method for TB-500 and its metabolites was developed and validated. The study found that Ac-LK was the primary metabolite with the highest concentration in rats at 0-6 h intervals. Also, the metabolite Ac-LKK was a long-term metabolite of TB-500 detected up to 72 hr. No cytotoxicity of the parent and its metabolites was found. Ac-LKKTE only showed a significant wound healing activity compared to the control. The study provides a valuable tool for quantifying TB-500 and its metabolites, contributing to the understanding of metabolism and potential therapeutic applications. Our results also 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.
Study Information
pubmed
2024
2024-02-07T00:00:00.000Z
10.1016/j.jchromb.2024.124033
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