The brain is a challenging target for therapeutic monoclonal antibodies (mAbs), nanobodies, antibody drug conjugates (ADCs), and other drugs. The blood brain barrier prevents many drugs with otherwise good absorption profiles from crossing into the brain, and also complicates attempts to model how drugs are distributed within the brain.
Prof. Dhaval Shah and PhD student Hsueh-Yuan Chang of the University at Buffalo study the pharmacokinetics/pharmacodynamics (PK/PD) of therapeutic antibodies and ADCs. Their lab recently used the Eicom AtmosLM (large molecule) microdialysis system as a way to quantitate a mAb in multiple brain regions simultaneously, generating data to underpin pharmacokinetic models for the disposition of mAbs in rats (1,2). They found that tissue homogenate and lumbar cerebrospinal fluid samples do not make good proxies for predicting mAb concentrations at their sites of action within the parenchyma of the brain. They also found that the lateral ventricles and the blood-CSF barrier may be an important route for mAb entry.
How Large Molecule microdialysis works
AtmosLM is a push-pull microdialysis system for measuring the levels of large proteins and peptides, as opposed to the catecholamines and other small molecules typically measured by microdialysis. AtmosLM features unique probes that incorporate vents to equalize the pressure inside the membrane of the probe with the outside atmosphere. This prevents ultrafiltration and yields more consistent analyte recovery rates than other push-pull systems. It has been widely used to study levels of Abeta, Tau, synuclein, lipidated ApoE particles, cytokines, and other molecules. Recently, the Derendorf lab
(U of Florida) used AtmosLM to determine tissue interstitial concentrations of mAbs in liver, skin, kidney, and muscle after IV dosing to aid in their development as anticancer drugs.
Microdialysis based PK modeling
Amuza spoke with Hsueh-Yuan (Luke) Chang about how he used AtmosLM in this project, and he also shared several tips for other users of AtmosLM.
Amuza: Could you explain how your PBPK (physiologically-based pharmacokinetic) model can be used by those studying the use of mAb based drugs in the brain?
Hsueh-Yuan: Our current version of the PBKP model was developed to capture nonspecific mAb distribution in the brain and different regions of the brain. Additionally, it can help to quantify the correlation between mAb concentrations in brain CSF and ISF. It may also help to quantify the amount of mAb entering brain parenchyma versus brain CSF compartments.
While the nonspecific mAb PBPK model has not incorporated target binding or receptor-mediated transcytosis yet, both novel delivery mechanisms and target binding kinetics can be mechanistically added into the current basic version of the PBPK model.
The final version of the PBPK model for mAbs may provide an a priori prediction of mAb distribution in the human brain once kon/koff of values of specific mAbs and receptor/target concentrations in the brain have been included. This prediction could be tested in rodents and primates.
Amuza: Central nervous system (CNS) concentrations of mAbs are often determined by taking whole brain homogenate samples or cerebrospinal fluid (CSF) samples from the lumbar region. What are these methods missing?
Hsueh-Yuan: They do not provide direct information of the mAb concentration at the site-of-action as we mentioned in the introduction of the paper. There are many studies suggesting mAb concentrations are different between CSF and ISF.
More importantly, some studies have reported mAb accumulation within brain capillary cells, which may utilize endogenous receptor binding to enhance brain uptake of mAbs.
Shah lab insights for using AtmosLM
Amuza: I’d also like to ask you a few questions about using our AtmosLM system.
You used siliconized sample tubes to prevent adsorption of antibodies in your samples to the plastic. Would blocking the tubes by rinsing with BSA work as well?
Hsueh-Yuan: Yes. However, the storage of low concentration IgG microdialysates requires 0.1-0.15% BSA. BSA is compatible with ELISA. For LC/MS, the BSA method should be replaced.
Amuza: Do you think endogenous IgG could be used similarly to an internal standard to suggest whether or not a microdialysis experiment is working correctly?
Hsueh-Yuan: Yes, ELISA methods can quantify rat, mouse, or human IgG specifically. They can serve as an endogenous IgG reference, a proxy for calculating in vivo recovery of the mAbs themselves. Hemoglobin can be used, too.
Amuza: During in vitro tests, you were very careful when balancing the flow between the syringe pump (push) and the peristaltic pump (pull), adjusting the peristaltic pump until the ratio of fluid pumped in/out of the probe stayed in the range of 97 – 103%.
What happens if fluid recovery is outside of this range?
Hsueh-Yuan: Then convection [bulk flow of solutes and solvent across a membrane due to a pressure imbalance] will happen.
Amuza: This is indeed a problem. Your paper (1) found that recovery rates were strongly changed when the flow was not properly balanced. This data is available in the supplementary material.
How did you measure the amount of fluid recovered in each sample?
Hsueh-Yuan: By measuring their net weight.
Amuza: With AtmosLM probes, this can also be accomplished by visually monitoring the flow exiting the probe during in vivo experiments. If the peristaltic pump is pulling fluid out of the probe faster than the syringe pump is pushing fluid in, air will enter the system through the vent in the probe and be visible as bubbles in the tubing. If instead the syringe pump is pushing more fluid than the peristaltic pump is removing, the excess fluid will exit the probe through the vent hole. The vent hole is downstream from the membrane, and does not interfere with microdialysis.
What suggestions do you have for others using AtmosLM to study antibody concentrations?
- Endogenous IgG or another internal reference should be measured to validate that the BBB is intact.
- Fresh CSF perfusion buffer should be used. BSA may precipitate if the CSF is left sitting at room temperature.
- Due to the instability of low concentrations of antibodies in microdialysate, samples should be analyzed ASAP. Alternatively, standards should be diluted to their final concentrations and stored together with the samples until analysis.
- Always check the inlet and outlet of the probe before connecting the probe to the push-pull system.
Amuza: Do you have future projects in mind for large molecule microdialysis?
Hsueh-Yuan: Yes. We have been working on several projects using AtmosLM microdialysis.
- Chang, H. Y., Morrow, K., Bonacquisti, E., Zhang, W., & Shah, D. K. (2018, August). Antibody pharmacokinetics in rat brain determined using microdialysis. In MAbs (Vol. 10, No. 6, pp. 843-853). Taylor & Francis.https://doi.org/10.1080/19420862.2018.1473910
- Chang, H. Y., Wu, S., Meno-Tetang, G., & Shah, D. K. (2019). A translational platform PBPK model for antibody disposition in the brain. Journal of pharmacokinetics and pharmacodynamics, 1-20.https://doi.org/10.1007/s10928-019-09641-8
- Jadhav, S. B., Khaowroongrueng, V., Fueth, M., Otteneder, M. B., Richter, W., & Derendorf, H. (2017). Tissue distribution of a therapeutic monoclonal antibody determined by large pore microdialysis. Journal of pharmaceutical sciences, 106(9), 2853-2859. https://doi.org/10.1016/j.xphs.2017.03.033
The Amuza FC-90 is a game changer for any microdialysis experiment:
4 channel operation: collect from up to 4 animals simultaneously.
Ideal for large and small molecule microdialysis.
Refrigerated storage in 96 well plates.
Small footprint: 7″ wide.