Trypsin Digestion

Keywords

1. Daptomycin quantification
2. Trypsin digestion muscle
3. HPLC-UV daptomycin analysis
4.Myopathies side effect
5. Skeletal muscle drug penetration

The urgent need to accurately quantify the concentration of daptomycin (DAP) in skeletal muscles has been highlighted in recent studies, especially to understand its penetration and the myopathies associated as side effects. A pioneering research article published in the Biological & Pharmaceutical Bulletin on September 25, 2019, has made significant strides in addressing this challenge. The study with the DOI 10.1248/bpb.b18-00945, spearheaded by Sakai Yusuke and colleagues from institutions such as The United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, and Gifu Pharmaceutical University, delved into the feasibility of using trypsin digestion as a sample preparation method for the quantification of DAP in murine skeletal muscle tissues in comparison with conventional High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) analysis.

Prior to this study, no substantial efforts were made to measure DAP concentrations in skeletal muscles, despite the recognized consequence of understanding its distribution for therapeutic and safety implications. In their methodological approach, Sakai and the research team employed trypsin, an enzyme, for the digestion of muscle tissues, hypothesizing that it could fully recover DAP from spiked samples. This assumption was rooted in the fact that DAP tends to incorporate into muscle proteins, which might hinder its recovery using standard extraction processes that involve homogenization, centrifugation, and filtration.

Through a series of meticulously executed spike recovery assays, the researchers uncovered that while conventional extraction methods led to less recovery of DAP due to its integration with muscle proteins, the enzymatic digestion with trypsin allowed for a complete recovery of DAP from the spiked skeletal muscle. The success of the trypsin digestion method was validated when HPLC analysis, paired with this innovative sample preparation technique, determined the DAP concentrations in skeletal muscles collected from mice that had been subcutaneously administered with DAP.

The study’s findings are monumental as they propose a robust method of muscle sample preparation that significantly elevates the efficiency of DAP quantification. This could be pivotal for future investigations aimed at understanding drug migration into muscle tissues and unraveling the mechanisms underpinning skeletal muscle injuries, which are observed as adverse reactions in DAP therapy.

The implications of this research are profound. For clinical pharmacology and toxicology, improved quantification methods mean that drug dosages can be tailored more accurately, minimizing the risk of side effects, like myopathies, while still maintaining antimicrobial efficacy. This is particularly important for DAP, a lipopeptide antibiotic commonly used in treating systemic and life-threatening infections caused by Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA).

For clinical practitioners and researchers alike, the study published under the title “Feasibility of Trypsin Digestion as a Sample Preparation for Daptomycin Quantification in Murine Skeletal Muscles” underscores the ongoing quest for precision in the pharmacokinetic profiling of drugs. It encourages the further exploration of enzymatic digestion techniques for the preparation of biological samples as a way to overcome the analytical challenges posed by protein-binding characteristics of certain drugs.

In conclusion, the article by Sakai Yusuke et al. represents a significant advance in the methodology available for the analysis of daptomycin distribution in muscle tissues. The development of the trypsin digestion method represents a leap forward in ensuring the safety and efficacy of daptomycin therapies, by offering a more precise understanding of its pharmacokinetics in skeletal muscle.

References

1. Sakai, Yusuke, et al. “Feasibility of Trypsin Digestion as a Sample Preparation for Daptomycin Quantification in Murine Skeletal Muscles.” Biological & Pharmaceutical Bulletin, vol. 42, no. 5, 2019, pp. 751-757. DOI: 10.1248/bpb.b18-00945.

2. Steenbergen, J. N., et al. “Daptomycin: A Lipopeptide Antibiotic for the Treatment of Serious Gram-Positive Infections.” Journal of Antimicrobial Chemotherapy, vol. 55, no. 3, 2005, pp. 283-288. DOI: 10.1093/jac/dkh546.

3. Arbeit, Robert D., et al. “Safety and Tolerance of Daptomycin in Children.” Pediatric Infectious Disease Journal, vol. 29, no. 12, 2010, pp. 1171-1176. DOI: 10.1097/INF.0b013e3181eb9dbf.

4. Pai, Manjunath P., et al. “Innovations in Antimicrobial Dosing in the Era of Multidrug Resistance: the Role of Pharmacokinetics and Pharmacodynamics.” Clinical Infectious Diseases, vol. 69, no. Supplement_7, 2019, pp. S402-S407. DOI: 10.1093/cid/ciz668.

5. Fowler Jr, Vance G., et al. “Daptomycin versus Standard Therapy for Bacteremia and Endocarditis Caused by Staphylococcus aureus.” New England Journal of Medicine, vol. 355, no. 7, 2006, pp. 653-665. DOI: 10.1056/NEJMoa053783.