New and Evolving Techniques for the Characterization of Peptide Therapeutics

D'Addio, Suzanne M.; Bothe, Jameson R.; Neri, Claudia; Walsh, Paul L.; Zhang, Jingtao; Pierson, Elizabeth; Mao, Yun; Gindy, Marian E.; Leone, Anthony; Templeton, Allen C.

doi:10.1016/j.xphs.2016.06.011

Cited by 29 publications

(31 citation statements)

References 131 publications

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“…Amyloid fibrils are formed by the stacking of b-sheets that interact side by side through interdigitation of amino acid side chains to form steric zippers (55). These interactions are mostly noncovalent, mediated through hydrogen bonding, steric or hydrophobic interactions, and/or charge interactions (56). Modifying amino acid side chains through site-specific phosphorylation of hCT interferes with the interactions responsible for steric zipper formation in the core of hCT fibrils.…”

Section: Discussionmentioning

confidence: 99%

Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Renawala

Chandrababu

Topp

2021

Biophysical Journal

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Section: Discussionmentioning

confidence: 99%

Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Renawala

Chandrababu

Topp

2021

Biophysical Journal

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“…The recent blockbuster successes of innovative peptide drugs have rekindled interest in peptide therapeutics, and the number of peptide New Molecular Entity (NME) approvals has grown exponentially, decade by decade, in contrast to the overall approval rate of all NMEs by the US Food and Drug Administration (FDA). 2 This growth is expected to continue, 3 in part due to novel molecular strategies for overcoming the biological barriers to peptide therapy and more robust and economical syntheses. 1,4,5 The increase in the complexity and diversity of peptide molecular design often results in more complex drug product development.…”

Section: Introductionmentioning

confidence: 99%

“…The physical and chemical properties of peptides can vary significantly with molecular weight and sequence, requiring a tailored, molecule-specific drug product development strategy that combines appropriate small molecule and biologics formulation and characterization approaches. 2 While single doses of monoclonal antibodies are typically tens to hundreds of milligrams with dosing frequencies of less than once per week, the high potencies, lower half-lives, and lower molecular weights of peptides have given rise to multiple use injectable dosage forms which deliver doses of a less than 1 mg to a few milligrams of a therapeutic peptide, such as TYMLOS (abaloparatide), VICTOZA (liraglutide), BYETTA (exenatide), SYMLIN (pramlintide acetate), and FORTEO (teriparatide injection). For injectable multidose formulations, preservatives which inhibit microbial growth are essential to ensuring that any inadvertent introduction of a contamination does not compromise the sterility and safety of the drug product.…”

Section: Introductionmentioning

confidence: 99%

Antimicrobial Excipient-Induced Reversible Association of Therapeutic Peptides in Parenteral Formulations

D'Addio

Yin

et al. 2021

Journal of Pharmaceutical Sciences

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New classes of therapeutic peptides are being developed to prosecute biological targets which have been inaccessible to other modalities. Higher potency and longer half-life peptides have given rise to multiuse injectable formulations that enable convenient, low volume, and self-administered dosing; however, inclusion of antimicrobial preservatives to meet bactericidal requirements can impact other attributes of peptide formulations. Peptide-preservative interactions influencing solution-phase self-association of a non-insulin, linear, palmitoylated 31 amino acid peptide and two structurally similar peptides were assessed via turbidity, intrinsic fluorescence shifts and quenching, isothermal titration calorimetry, and 1 H NMR. Meta-cresol and phenol specifically interact with the peptide, result in increased hydrophobicity near the tryptophan residue, and induce conformational changes, while benzyl alcohol does not impact tryptophan fluorescence, demonstrate any interaction enthalpy, or induce conformational changes. These same trends did not hold true for the other palmitoylated peptides evaluated, reinforcing the impacts of unique peptide sequences. Importantly, the presence of benzyl alcohol does increase the physical stability and solubility of the linear, 31 amino acid peptide under salt stress. We report new insights into the physical interactions of peptides with antimicrobial excipients, demonstrating a reversible association phenomenon and highlighting practical implications for formulation design and excipient selection.

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“…The development of molecules for biological or biomedical applications relies on their accurate and precise biophysical/biological characterization (La Gatta et al, 2016;Van Norman, 2016). The necessary data concerning the efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location of such molecules can be gathered using in vitro or in vivo approaches (D'Addio et al, 2016). The collection of this information requires the use of highly sensitive techniques, which usually depend on the use of fluorescent probes (Gonzalez-Vera, 2012;Xu et al, 2018;Gao and Wu, 2019).…”

Section: Introductionmentioning

confidence: 99%

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

Cavaco

Pérez‐Peinado

Valle

et al. 2020

Front. Bioeng. Biotechnol.

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The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood-brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.

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New and Evolving Techniques for the Characterization of Peptide Therapeutics

Cited by 29 publications

References 131 publications

Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Antimicrobial Excipient-Induced Reversible Association of Therapeutic Peptides in Parenteral Formulations

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

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