(T1530-05-27) Analytical Method Development and Characterization of a Novel Antimicrobial Peptide WW504 In Vitro and In Murine Tissues by LC-MS/MS

Purpose: There has been an increase in the emergence of diseases resistant to the traditional antibiotics, leading to a rise in antimicrobial resistance. This has led to greater need for new antimicrobial medications.[1] It has been reported that approximately 90% of antibiotic-resistant infections are primarily due to Gram positive bacteria such as Staphylococcus aureus as well as Gram negative bacteria such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter species. Hence, there is an urgent need to develop a novel antibiotics to curb such infections.[2] Antimicrobial Peptides (AMPs) are small peptides ( < 100 amino acids) found in the innate immune system of all organisms with narrow or broad spectrum antimicrobial activity.[3, 4] They differ from traditional antibiotics by their unique mechanism of action and structure. They frequently target bacterial membranes, making it difficult for bacteria to develop resistance.[1] Both positively charged and hydrophobic amino acids play an important role in bacterial killing.[5] There are few reported studies on the use of LC-MS to evaluate the AMPs in biological matrices due to their amphipathic nature. Hence, our research focuses on developing a robust and sensitive bioanalytical LC-MS/MS method for quantification of a novel AMP, WW504, in biological matrices. WW504, synthesized in D-amino acids, is an improved version of Verine.[2] Additionally, we have conducted in vitro studies to characterize benchtop stability, pH stability, protein binding and in vivo studies to assess plasma and lung concentrations following WW504 administration.

Methods: A LC-MS/MS method for quantification of WW504 was developed. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using positive electrospray ionization mode (ESI). The precursor ion and product ion used were 436.43 >130.10m/z for the target peptide and 425.15 >132.20m/z for the internal standard (IS). An Acquity UPLC Peptide BEH C18 column (1.7 µm, 2.1mm* 50mm) with an Acquity UPLC C18 guard column was used for analyte separation. The mobile phase consisted of 0.1% Formic acid in water and 50:50 acetonitrile: methanol and operated at a gradient flow rate of 0.5 mL/min. The analytical method was validated according to the Bioanalytical Method Validation and Study Samples Analysis ICH M10 Guideline (2022) and was tested for stability under laboratory handling conditions (bench-top), autosampler conditions (4°C)and after three consecutive freeze-thaw cycles. The developed method was used to assess pH stability in different media including simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and plasma stability. The plasma protein binding of the AMP was estimated by Rapid Equilibrium Dialysis Assay (RED). The distribution of WW504 in plasma and lung tissue was determined following an intravenous (IV) injection (5 mg/kg) to healthy male Balb/c mice.

Results: The assay was successfully validated and was linear in mouse plasma and tissues over the concentration range of 1-1000 ng/mL. The percent extraction recovery of the peptide in plasma was found to be 76.9 ± 0.6%. Precision (%RSD) and Accuracy (%Bias) met the acceptable criteria of ± 15% of nominal concentrations and ± 20% at the lower limit of quantitation (LLOQ). WW504 was found stable under the laboratory handling conditions, autosampler conditions and freeze-thaw cycles. It was also stable in SGF ( >90%), SIF ( >80%) and plasma ( >90%) (Figure 1). The plasma protein binding was estimated to be 94%. Following the IV administration of WW504, the C_max was 5002.5 ± 2658.0 ng/mL in plasma. The volume of distribution was found to be 2305.0 ± 627.3 L/kg with an elimination half-life of 4 hours and a systemic clearance of 419.8 ± 63.0 L/h/kg. WW504 exhibited greater exposure in lungs (AUC_0-24h 27344.7 ± 8289.1 hr*ng/g) than in plasma (AUC_0-24hr 11948.5 ± 1666.2 hr*ng/mL).

Conclusion: A robust and sensitive LC-MS/MS method was successfully developed and validated according to the ICH M10 Guideline for the quantification of AMP, WW504 in plasma and lung tissue. The method showed acceptable linearity, precision, accuracy and stability under various conditions. In vitro studies indicated high plasma protein binding and good stability under different pH conditions.
The in vivo pharmacokinetic profile showed high exposure in lungs and systemic distribution, indicating WW504’s potential to act as a novel antibiotic. Ongoing research focuses on the use of polymeric nanoparticles to encapsulate AMPs to control the release and prolong systemic exposure.

References: 1. Talapko, J., et al., Antimicrobial Peptides—Mechanisms of Action, Antimicrobial Effects and Clinical Applications. Antibiotics, 2022. 11(10): p. 1417.
2. Lakshmaiah Narayana, J., et al., Two distinct amphipathic peptide antibiotics with systemic efficacy. Proc Natl Acad Sci U S A, 2020. 117(32): p. 19446-19454.
3. Cesaro, A., et al., Advanced delivery systems for peptide antibiotics. Adv Drug Deliv Rev, 2023. 196: p. 114733.
4. Wang, G., X. Li, and Z. Wang, APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Res, 2016. 44(D1): p. D1087-93.
5. Fillion, M., et al., Membrane Interactions of Synthetic Peptides with Antimicrobial Potential: Effect of Electrostatic Interactions and Amphiphilicity. Probiotics and Antimicrobial Proteins, 2015. 7(1): p. 66-74.


Table 1. PK Parameters Analyzed by Phoenix (Mean ± SD, n=3)


Figure 1. Stability of peptide under plasma and physiological conditions (Mean ± SD, n = 3).


Figure 2. (A) Plasma Concentration and time profile after single intravenous administration. (B) Lung distribution profile of WW504.