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Liposomal drugs, as a major breakthrough in modern medicine, not only significantly enhance drug solubility, stability, and bioavailability but also greatly reduce toxic side effects on healthy tissues. From cancer treatment to vaccine delivery and gene therapy, the applications of liposomal drugs are vast and promising. However, this cutting-edge therapeutic approach also brings unique challenges. The bioanalysis of liposomal drugs is a complex and critical process, involving structural analysis, stability studies, and pharmacokinetics, among other aspects. How can we overcome these challenges? How can we address various difficulties in practice?
The Cloud Lecture Hall has invited a senior scientific research expert in the field of liposomes, Sai Lian, who will provide an in-depth analysis of the practical challenges in the bioanalysis of liposomal drugs, drawing on extensive experience and unique insights
Q1. How to choose the appropriate SPE plate during the method development stage?
Sai Lian: First, we need to assess the polarity of the compound. For weak acids, weak bases, and neutral compounds, it is recommended to choose reversed-phase SPE plates with a silica gel matrix. These are suitable for most small-molecule bioanalyses, such as Waters’ HLB series and Thermo Fisher’s SOLA series plates.
For strong acids or strong alkaline bases, ion-exchange SPE plates should be considered.
You can choose Waters’ multifunctional ion-exchange development plates, such as the Oasis MCX cation exchange plates for basic compounds, the MAX anion exchange plates for acidic compounds, the WCX weak cation exchange plates for strongly basic or quaternary ammonium compounds, and the WAX weak anion exchange plates for strongly acidic compounds.
Additionally, selecting the appropriate plate size is crucial. You can choose based on the sample load, such as 2 mg, 10 mg, or 30 mg.
Micromolecular Drugs Bioanalysis
Q2. In the SPE (Solid Phase Extraction) activation and equilibration stages, why are glucose injection solutions and plasma used for activation and equilibration? What are the underlying considerations?
Sai Lian: When determining the encapsulation rate of free Vincristine, we found that the measured results consistently fall below the values provided by the client. This might indicate that there is liposome fragmentation during the intermediate processing stage or that some protein-bound components have not been completely washed out. Therefore, we initially considered addressing the potential liposome fragmentation issue by adding glucose for pre-treatment before the sample loading operation.
On the other hand, if the silica groups on the stationary phase are not fully eluted from the protein-bound portions, we consider using a blank matrix for pre-occupation. This would prevent subsequent protein-bound components from binding to the stationary phase.
Q3. Why are there so many differences between the overall drug method validation and the free drug method validation in terms of standard curves and quality control preparation?
Sai Lian: All of our samples come from the same animal. Both the total drug concentration and the free drug concentration measurements are based on the same sample. When measuring the total drug concentration, liposomes are typically present in the sample. If the liposomes are not fully fragmented, the measured total drug concentration may be inaccurate. To simulate the actual condition of liposomes in the sample, we use quality control samples containing liposomes for calibration. Only when the liposomes in the standard and the sample are completely matched can we accurately reflect whether the total drug concentration is fully fragmented.
For the measurement of free drug, we derive it by subtracting the results obtained from samples containing liposomes. However, in the final sample, both free drug and liposomes are present simultaneously. Therefore, we need to consider both the fragmentation of liposomes and the stability of free small molecules. To simulate the conditions in real samples, we need to introduce quality control samples that contain both liposomes and free small molecules for study.
Q4. Is the detection of mouse tissues also for measuring both the total drug and the free portion?
Sai Lian: According to liposome guidelines, it is recommended to measure both the total drug and the free drug. However, tissue samples require homogenization, which often makes it challenging to measure the free drug. Therefore, currently, only the total drug is typically measured.
Q5. Do micellar injection drugs and liposomal drugs both require the simultaneous determination of total and free drugs? Can they be considered in the same way? Are there any differences?
Sai Lian: Micellar injection drugs and liposomal injection drugs, as nanomedicines, should be considered equally when measuring drug concentrations in plasma. Both the free and total drug concentrations need to be determined. For more details, please refer to the “Technical Guidelines for Non-clinical Pharmacokinetic Studies of Nanomedicines”. Currently, Medicilon has experience with two micellar injection projects, where the methods for determining free and total drug concentrations are the same. The difference lies in the pre-treatment separation techniques.
Q6. How were the concentration and ratio of the stabilizer glucose optimized and determined?
Sai Lian: Usually, 5% glucose is chosen and added at a ratio of 1:10. If the protection is insufficient, the concentration can be increased, but the addition ratio should generally not be less than 1:10.
Whether you are a senior expert in the pharmaceutical industry or a new learner of cutting-edge medical technology, Medicilon’s Cloud Lecture Hall will open a door to the future of drug development for you. If you have hot topics you want to learn about or are interested in hearing a detailed introduction to preclinical research services in the Cloud Lecture Hall, feel free to leave a comment or ask a question. We look forward to having more discussions with you.
