In vitro
This study developed and validated a stability-indicating High-Performance Liquid Chromatography (HPLC) method for the simultaneous quantification of two GLP-1 receptor agonists — Semaglutide (SEM) and Tirzepatide (TIR) — used in the treatment of type 2 diabetes and obesity. The method employed a C18 column with an isocratic mobile phase of 0.1% formic acid and acetonitrile (30:70), achieving rapid separation with retention times of 1.42 min for SEM and 1.68 min for TIR. The method was validated per ICH guidelines, demonstrating strong linearity (1–500 µg/mL, r > 0.9999), sensitivity (LOD: 10 ng/mL for TIR; 16 ng/mL for SEM), accuracy, and precision. The method successfully resolved both compounds from degradation products generated under acidic, basic, oxidative, and photolytic stress conditions. It was also applied to bulk drug, pharmaceutical dosage forms, and spiked rat plasma. A comprehensive six-pronged sustainability assessment was performed using nine analytical greenness, whiteness, blueness, and violet innovation tools. A key limitation is that the plasma work used spiked rat samples rather than real patient samples, meaning no clinical or pharmacokinetic conclusions about humans can be drawn.
BMC chemistry · Jan 2026DOI ↗ In vitro
This study investigated the chemical and physical stability of afamelanotide (melanotan-1), a synthetic 13-amino acid peptidomimetic of α-melanocyte stimulating hormone (α-MSH) approved as an orphan drug for erythropoietic protoporphyria. Researchers subjected the compound to a range of stress conditions — acidic, basic, neutral, oxidative, UV light exposure, and elevated temperature (60°C) — following International Council for Harmonisation (ICH) guidelines Q1A(R2) and Q5C. Using gradient reversed-phase HPLC coupled with ultra-high-performance liquid chromatography–high resolution tandem mass spectrometry (UHPLC-HRMS/MS), the team identified and structurally characterized 14 distinct degradation products. Collision-induced dissociation fragmentation patterns enabled detailed elucidation of each product's structure. Key degradation pathways identified included truncation, methylation, deacetylation, and oxidation. The analytical method was validated per ICH Q2(R1) guidelines. This work is purely analytical and pharmaceutical in nature — it does not involve human subjects, animals, or cell-based experiments. Its primary value lies in establishing a comprehensive stability profile of afamelanotide to inform rational drug formulation design. No clinical outcomes, efficacy, or safety data in biological systems were assessed.
Journal of pharmaceutical and biomedical analysis · Jan 2026DOI ↗ In vitro
This review/research paper examines two naturally occurring peptides — carnosine (β-alanyl-L-histidine) and GHK (glycyl-L-histidyl-L-lysine) — and explores strategies to improve their stability and bioavailability through glycoconjugation. A key challenge addressed is the rapid degradation of carnosine by the enzyme carnosinase and the inherent low stability of GHK. The authors report that conjugating these peptides to either trehalose (a disaccharide) or hyaluronan (a polysaccharide) inhibits carnosinase activity and increases tripeptide stability, while also protecting the saccharide components from degradation. The study further investigates copper-binding properties of these glycoconjugates, finding that the saccharide components potentiate the Cu,Zn-superoxide dismutase-like (antioxidant) activity of the resulting copper(II) complexes. The glycoconjugates are reported to act as copper ionophores in cell culture, increasing intracellular copper levels and stimulating copper-driven signaling pathways, leading to enhanced expression of trophic and angiogenic proteins including BDNF, BMP-2, and VEGF. Copper chaperones CCS and Atox-1 are implicated as transcription factors in these pathways. Limitations include reliance on in vitro cell culture models and biochemical assays, with no human clinical data presented.
Antioxidants (Basel, Switzerland) · Dec 2025DOI ↗ In vitro
This study describes the development of a high-yield Chinese hamster ovary (CHO) cell manufacturing platform for a biosimilar of dulaglutide, a GLP-1/IgG4-Fc fusion protein approved for type 2 diabetes. Researchers used apoptosis-resistant CHO 4BGD cells and sequentially transfected two expression plasmids encoding dulaglutide, employing a two-step transgene amplification strategy using methotrexate (MTX) followed by methionine sulfoximine (MSX) selection. The dual-selection approach resulted in approximately 30% higher titers in polyclonal populations compared to MTX amplification alone. Through a clonal cell line selection pipeline, the top clone (4BGD/Dul #73) achieved a product titer of 1.05 g/L in a 3-week fed-batch process, with a specific productivity of up to 22 pg·cell⁻¹·day⁻¹ and stable expression over 69 days without selective pressure. Purity assessed by size-exclusion HPLC showed ≥95% monomer content. Biological activity testing in a GLP-1 receptor/CRE-luciferase reporter assay yielded an EC₅₀ of 52 pM for the biosimilar candidate versus 76 pM for the reference drug. Limitations include the absence of in vivo or clinical data, with all findings limited to cell culture and in vitro bioassay systems.
Pharmaceuticals (Basel, Switzerland) · Dec 2025DOI ↗ In vitroPreprint
This paper proposes a molecular mechanism for BPC-157, a synthetic 15-amino-acid peptide previously studied in preclinical models for regenerative and cytoprotective effects. The authors hypothesize — based on computational structural modeling and in silico docking — that BPC-157 adopts a polyproline II (PPII) helix conformation and engages the SH3 domains of Src family kinases (c-Src, Yes, Fyn). The proposed interaction is suggested to relieve autoinhibition of these kinases, potentially activating downstream FAK-ERK and PI3K-Akt signaling pathways. To build a tool for future experimental testing, the authors engineered an mCherry-BPC157₂ fusion protein, encoded it in a baculovirus vector, and expressed it in insect (Sf9) cells. Expression was confirmed by fluorescence imaging and western blot at the expected ~31 kDa size. Importantly, this study does not include human subjects, animal experiments, or in vitro binding assays — the core mechanistic claims rest entirely on computational modeling. The fusion protein work is a proof-of-concept for a future experimental reagent. Findings should be interpreted as hypothesis-generating only.
Unknown journal · Dec 2025DOI ↗ In vitro
This study investigated how Thymosin β4 (Tβ4), a 43-amino-acid secreted peptide, may protect against hypoxia-induced blood-brain barrier (BBB) disruption using human brain microvascular endothelial cells (hBMVECs) as an in vitro model. The researchers exposed hBMVECs to hypoxic conditions to simulate aspects of ischemic injury and traumatic brain injury (TBI), then examined whether Tβ4 pretreatment could reverse resulting damage. They measured gene expression of tight junction proteins, Sphingosine 1-phosphate receptor 1 (S1PR1), endothelial cell permeability, and tight junction dynamics. The study found that Tβ4 pretreatment appeared to reverse hypoxia-induced impairment of BBB components, and identified S1PR1 as a potential mechanistic target. Notably, when S1PR1 was pharmacologically inhibited, Tβ4 lost its protective effect, suggesting S1PR1 signaling is required for Tβ4's action. The authors conclude that S1PR1 pathway modulation is central to hypoxia-induced BBB pathophysiology and propose Tβ4 as a candidate therapeutic agent warranting further investigation. Key limitations include the exclusive use of cell culture models, absence of animal or human data, and lack of in vivo validation of the proposed mechanism.
Scientific reports · Dec 2025DOI ↗ In vitroPreprint
This paper proposes a molecular mechanism for BPC-157, a synthetic pentadecapeptide previously studied in preclinical settings for regenerative and cytoprotective properties. The authors use structural modeling and in silico docking to hypothesize that BPC-157 adopts a polyproline II (PPII) helix conformation that enables it to bind the SH3 domains of Src family kinases (SFKs), including c-Src, Yes, and Fyn. According to this model, such binding would relieve SH3-mediated autoinhibition of these kinases, triggering downstream FAK-ERK and PI3K-Akt signaling cascades associated with cell survival and repair. To build a tool for future experimental validation, the researchers engineered an mCherry-BPC157₂ fluorescent fusion protein, encoded it in a baculovirus vector, and expressed it in Sf9 insect cells. Expression was confirmed by fluorescent imaging and western blot at the predicted ~31 kDa molecular weight. Notably, no binding interaction or functional activity in mammalian systems was experimentally demonstrated; the SH3 engagement hypothesis remains computational. Limitations include the absence of human or animal data, reliance on in silico docking, and use of an insect cell expression system solely for protein production validation.
Unknown journal · Nov 2025DOI ↗ In vitro
This study investigated whether Tβ4-17, a small bioactive peptide derived from the precursor protein thymosin β4 and identified via iTRAQ technology, could enhance the sensitivity of cisplatin (DDP)-resistant ovarian cancer cells to chemotherapy. Using in vitro cell line models of DDP-resistant ovarian cancer, the researchers tested the combination of Tβ4-17 with DDP on cell proliferation, migration, and apoptosis. Results indicated that the combination significantly inhibited proliferation and migration of resistant cells while promoting apoptosis compared to either treatment alone. Mechanistically, the study found that NF-κB (specifically the p65 subunit) was highly expressed in DDP-resistant ovarian cancer cells, and that Tβ4-17 appeared to downregulate NF-κB p65 protein expression. These findings were supported by qRT-PCR, Western blot, CCK-8 assays, EDU fluorescence proliferation assays, and scratch migration assays, as well as experiments using NF-κB inhibitors and activators. Key limitations include reliance solely on in vitro cell line models with no animal or human data, and the absence of pharmacokinetic or safety assessments. The study suggests a potential mechanism by which Tβ4-17 may overcome chemoresistance in ovarian cancer but requires substantial further validation.
Medical oncology (Northwood, London, England) · Nov 2025DOI ↗ In vitro
This study investigated PapB, a radical S-adenosyl-l-methionine (rSAM) enzyme involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Classically, RiPP maturase enzymes require an N-terminal leader sequence on the precursor peptide to guide substrate recognition and modification. The researchers discovered that PapB can function in a "leader-independent" manner — meaning it can process peptide substrates that entirely lack canonical leader sequences. To demonstrate the practical utility of this finding, the team applied PapB to three analogues of glucagon-like peptide (GLP-1) pathway agonists — a therapeutically relevant class of peptides — and showed that the enzyme achieved complete conversion of each linear peptide into a thioether-macrocyclized (C-terminally cyclized) product. The study is primarily a biochemical and enzymological characterization conducted in vitro, with no human or animal subjects involved. Limitations include that all work was performed outside of a biological system, and the therapeutic relevance of the resulting macrocyclic GLP-1 analogues in vivo remains to be established. The findings position PapB as a potentially versatile biocatalytic tool for generating conformationally constrained peptide drug candidates.
ACS bio & med chem Au · Oct 2025DOI ↗ In vitro
This study investigated the cellular mechanisms by which larazotide acetate (LA), a synthetic octapeptide in clinical development for celiac disease, protects the intestinal epithelial barrier. Researchers pretreated two intestinal epithelial cell lines — C2BBe1 (human) and IPEC-J2 (a "leaky" porcine line) — with LA before exposing them to anoxia/reoxygenation (A/R) injury, a model of ischemia-reperfusion stress. LA pretreatment significantly increased transepithelial electrical resistance (TEER), a measure of barrier integrity, and preserved the normal localization of tight junction (TJ) proteins. RNA sequencing identified enriched gene sets related to barrier regulation, small GTPase signaling, protein phosphorylation, cell proliferation, and migration. Consistent with transcriptomic findings, LA markedly reduced phosphorylation of myosin light chain-2 (MLC-2), suggesting modulation of the ROCK signaling pathway, which is known to influence TJ dynamics. LA also enhanced epithelial cell proliferation. Limitations include exclusive reliance on in vitro cell culture models with no animal or human data, and the use of a single, fixed LA concentration. The authors conclude that LA stabilizes tight junctions, reduces MLC-2 phosphorylation, and promotes epithelial renewal, supporting its broader potential in gastrointestinal conditions involving mucosal barrier disruption.
Biomedicines · Oct 2025DOI ↗ In vitro
This study developed a dual-mode semi-preparative anion-exchange chromatography (AEX) method to fractionate and characterize charge variants of dulaglutide, a GLP-1 receptor agonist used in type 2 diabetes management. Because dulaglutide is an acidic Fc-fusion protein with complex charge heterogeneity, standard characterization methods are technically challenging. The researchers isolated acidic, main, and basic charge variant fractions and subjected them to comprehensive downstream analyses, including assessments of sialic acid content, post-translational modifications (phosphorylation, sialylation, deamidation, oxidation), size heterogeneity, aggregation, truncation, and biological activity. A key finding was that aggregates in basic variants are primarily held together by non-covalent interactions, while acidic variants contain covalently linked aggregates—a structurally meaningful distinction. Charge variants showed only slight differences in biological activity, potentially linked to aggregate presence. A comparative analysis between the innovator product Trulicity® and a biosimilar candidate revealed minor differences in acidic variants, likely attributable to variations in phosphorylation and sialylation profiles. Limitations include the in vitro nature of the biological activity assessments and the absence of in vivo or clinical data. The study provides a detailed analytical framework for characterizing charge heterogeneity in complex biopharmaceuticals.
International journal of biological macromolecules · Sep 2025DOI ↗ In vitroPreprint
This study investigated whether Tβ4-17, a small bioactive peptide derived from thymosin β4 and identified via iTRAQ technology, could enhance the sensitivity of cisplatin (DDP)-resistant ovarian cancer cells to chemotherapy. Using in vitro models of DDP-resistant ovarian cancer cell lines, the researchers examined the effects of Tβ4-17 alone and in combination with DDP on cell proliferation, migration, and apoptosis. Multiple assays were employed, including CCK8 viability assays, EDU fluorescence proliferation assays, cell scratch (wound healing) assays, qRT-PCR, and Western blot. The study found that Tβ4-17 combined with DDP significantly inhibited proliferation and migration of resistant cells and promoted apoptosis compared to either agent alone. Mechanistically, the researchers reported that NF-κB p65 was highly expressed in DDP-resistant cells, and that Tβ4-17 down-regulated NF-κB p65 protein expression. Use of NF-κB inhibitors and activators further supported this proposed pathway. Key limitations include the exclusive use of cell-line models with no animal or human data, the preprint status of the work, and the absence of in vivo validation. Findings are preliminary and require further study.
Unknown journal · Jul 2025DOI ↗ In vitro
This study investigated the metabolic fate of alexamorelin — a synthetic peptide growth hormone secretagogue (GHS) with potential performance-enhancing properties — to identify biomarkers useful for doping control. Researchers used in silico metabolite prediction software (GLORYx) alongside in vitro incubations with pooled human hepatocytes from 10 donors, analyzing samples via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS/MS). GLORYx predicted 21 possible single-reaction metabolites, with N-acetylation ranked highest in probability (98%), and other transformations such as hydroxylation, N-oxidation, and glucuronidation predicted at lower probabilities. However, after 3 hours of hepatocyte incubation, only one metabolite was experimentally detected: examorelin (hexarelin), produced by carboxypeptidase-mediated cleavage of the C-terminal alanine residue. The parent compound decreased approximately 150-fold over the incubation period, indicating rapid and extensive hepatic metabolism. A key limitation is that examorelin is itself a commercially available GHS compound, meaning it cannot serve as a specific biomarker for alexamorelin use. The authors conclude that direct detection of alexamorelin itself remains the most reliable strategy for confirming its consumption in anti-doping contexts. This study was conducted entirely in vitro and does not involve human subjects or animal models.
Journal of analytical toxicology · Jul 2025DOI ↗ In vitro
This study reports the first-in-class design of unimolecular "tetra-agonists" — single peptide molecules engineered to simultaneously activate four metabolically relevant receptors: GLP-1R, GIPR, GcgR (all class B GPCRs), and Y2R (a class A GPCR). Prior work had yielded dual and triple agonists exploiting sequence homology among class B GPCR ligands, but incorporating Y2R agonism was considered a major challenge due to the structural and sequence divergence between class A and class B GPCRs. The researchers used rational chimeric peptide design to overcome these topological constraints, producing high-potency tetra-agonists. They further showed that lipidation of the scaffold was well tolerated, potentially improving therapeutic viability. The study also explored biased agonism at GLP-1R, demonstrating that cAMP signaling could be selectively amplified while minimizing β-arrestin recruitment — a mechanism that may reduce receptor desensitization. A tunable framework for modulating β-arrestin engagement without compromising cAMP potency is also described. Limitations include that all experiments appear to be conducted in vitro (cell-based receptor activation and signaling assays), with no animal or human efficacy data reported. The work is primarily a molecular pharmacology and peptide chemistry study establishing proof-of-concept at the receptor level.
Journal of the American Chemical Society · Jun 2025DOI ↗ In vitroPreprint
This preprint describes a computational study aimed at designing improved semaglutide analogues — variants of the GLP-1 receptor agonist used in weight-loss and diabetes treatment — in the context of the underperforming CagriSema Phase III trial. The researchers used an automated "natural amino acid scanning" approach, systematically introducing single amino acid mutations across the semaglutide peptide backbone. Using the crystal structure of the GLP-1–GLP-1R complex (PDB: 4ZGM) as a structural template, they performed high-throughput computational modeling with Modeller and estimated binding affinities (Kd) using the Prodigy tool. From this pipeline, the study identified 564 computationally designed semaglutide analogues predicted to show improved binding affinity to the extracellular domain (ECD) of GLP-1R. The authors propose a conceptual "interfacial electrostatic scaffold" consisting of four salt bridges at the peptide–receptor interface as a framework for next-generation GLP-1R agonist development, drawing an analogy to the century-long iterative optimization of insulin. Key limitations include the fully computational nature of the study — no experimental validation (biochemical, cellular, or in vivo) is presented — and reliance on a single structural template and computational binding affinity estimators, which may not fully capture dynamic receptor behavior.
Unknown journal · Apr 2025DOI ↗ In vitro
This study investigated the synthesis and biological activity of novel conjugates combining hyaluronic acid (HA) with the copper-binding tripeptide glycyl-l-histidyl-l-lysine (GHK) at varying loading ratios, forming GHK-HA conjugates. The conjugates were designed to address the individual limitations of HA and GHK, including susceptibility to hydrolytic degradation and oxidative stress. In vitro assays demonstrated that GHK-HA bound copper(II) ions effectively and showed enhanced antioxidant properties compared to the individual components alone. When complexed with copper, the conjugates promoted the expression and release of several growth and trophic factors, including brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and bone morphogenetic protein-2 (BMP-2), suggesting synergistic osteogenic and angiogenic potential. The study also explored a proposed mechanistic pathway, finding that copper's intracellular chaperones — CCS and Atox1 — translocated to the nucleus and appeared to function as transcription factors. Limitations include the exclusively in vitro nature of the experiments, meaning results cannot yet be extrapolated to animal models or human clinical outcomes. No in vivo validation was performed.
Bioconjugate chemistry · Mar 2025DOI ↗ In vitro
This study investigated whether ibutamoren (IBU) — a compound known primarily as a growth hormone secretagogue — might also act as an inhibitor of the MDM2-p53 interaction, a pathway dysregulated in roughly half of human cancers. MDM2 normally tags the tumour suppressor p53 for degradation; blocking this interaction is a recognized anticancer strategy. The researchers first used in silico molecular modelling to predict that IBU binds favorably to the p53-binding pocket of MDM2 and exhibits a low estimated IC50 for MDM2 inhibition. They then tested IBU on immortalized human cancer cell lines in vitro, finding reduced cell viability in lines with an intact, functional MDM2-p53 pathway but not in lines carrying damaging mutations in this pathway. RT-qPCR analysis supported this pattern, showing differential expression of two p53 target genes in wild-type but not mutant cell lines after IBU treatment. Key limitations include exclusive reliance on cell-line models (no animal or human data), use of immortalized rather than primary cells, and the preliminary, exploratory nature of the work. The authors conclude that IBU shows early-stage anticancer activity potentially mediated through the MDM2-p53 axis and warrants further mechanistic investigation.
Basic & clinical pharmacology & toxicology · Jan 2025DOI ↗ In vitro
This study presents a novel analytical application of capillary electrophoresis coupled to inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) to investigate the encapsulation of the copper tripeptide complex GHK-Cu within liposomes — a delivery system commonly used in cosmetic formulations. Using an ethanol injection method to form liposomes, the researchers monitored copper (Cu) and phosphorus (P) signals to confirm liposome formation and quantify the concentration of GHK-Cu encapsulated within them. The study demonstrates that CE-ICP-MS/MS, valued for its high sensitivity and ability to preserve analytes in their intact chemical form under mild, physiologically compatible conditions, can be successfully applied in cosmetic science contexts. The authors propose this methodology could support the development and optimization of diverse liposomal cosmetic formulations. Importantly, this paper is a methodological/analytical chemistry study — it does not test GHK-Cu efficacy in humans or animals, make clinical claims, or evaluate biological outcomes. Its findings are limited to demonstrating an analytical technique for characterizing a cosmetic ingredient's encapsulation efficiency in a laboratory setting.
Electrophoresis · Oct 2024DOI ↗ In vitro
This study explores novel peptide stapling strategies inspired by natural product motifs found in fungal toxins (amatoxins, phallotoxins) and the alkaloid staurosporine. The researchers developed a chemical method using a 5-hydroxypyrroloindoline building block that can react with either a cysteine thiol (forming a tryptathionine staple) or a tryptophan indole (forming a 2,2'-bis-indole staple) to create constrained macrocyclic peptide structures. The authors applied these two stapling approaches to α-melanocyte-stimulating hormone (α-MSH), using the Melanotan-II scaffold as a model, with careful protecting group strategies to achieve chemoselectivity between the two staple types. Both classes of stapled peptides were evaluated for binding affinity at the melanocortin receptor, and the study reports that both series achieved nanomolar inhibition constants (Ki values), with at least one compound reaching sub-nanomolar Ki. Limitations include that all work is conducted in vitro (binding assays and synthetic chemistry), with no cell-based functional data, animal studies, or human data reported. The study is primarily a proof-of-concept for expanding the chemical toolbox of peptide stapling using underexplored natural product-derived cross-links.
Chemistry (Weinheim an der Bergstrasse, Germany) · Feb 2024DOI ↗ In vitro
This study used all-atom molecular dynamics (MD) simulations, complemented by Fourier transform infrared (FTIR) spectroscopy, to investigate how two permeability enhancers (PEs) — sodium caprate and SNAC (sodium N-[8-(2-hydroxybenzoyl)amino]caprylate) — interact with four peptide drugs (octreotide, hexarelin, degarelix, and insulin) in the presence of taurocholate, an intestinal bile salt. The simulations revealed that the two PEs had distinct, peptide-dependent effects: they tended to promote release of more hydrophobic peptides while inhibiting release of more water-soluble ones. At lower peptide concentrations, peptide–peptide interactions decreased while interactions with PEs and taurocholate increased. Introducing all components together produced dynamic mixed aggregates with reduced peptide–peptide hydrogen bonding compared to peptide-only systems. FTIR analysis of insulin showed that SNAC increased β-sheet formation, while sodium caprate favored α-helical and random-coil structures. The authors suggest these molecular-level insights could guide the rational design of PE-based oral peptide formulations. Key limitations include the exclusive use of computational and in vitro/spectroscopic methods, with no cell-based, animal, or human data reported.
Nanoscale · Dec 2023DOI ↗