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.
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 is developed to capture nonspecific mAb distribution in the brain and different regions of the brain. It can help to quantify the correlation between mAb in CSF and mAb in brain ISF. It may 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 current basic version of PBPK model.
The final version of PBPK model for mAb may provide an a priori prediction of mAb distribution in the human brain by inputting kon/koff of values of mAb, receptor/target concentration in the brain. The validation of 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 important, mAb accumulation within brain capillary cells has been reported in some studies that work with endogenous receptor binding to enhance brain uptake of mAb.
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: Positive. 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 for calculating in vivo recovery. 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.
Amuza: 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 when sitting at room temperature.
Samples should be analyzed ASAP due to low concentration (or prepare a standard curve on the same day and store them with samples together).
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
For approximately 200,000 US veterans, the 1991 Persian Gulf War marked the beginning of their experience with Gulf War Illness (GWI). GWI encompasses a cluster of chronic symptoms including memory and cognitive problems, fatigue, and fibromyalgia.
GWI has long been associated with a combination of several possible contributory factors: the stress of deployment, altered immune function, and exposure to acetylcholinesterase inhibitors (AChEI), but the exact cause or causes have remained elusive. The AChEI pyridostigmine bromide (PB) was administered to soldiers as a prophylactic against the risk of nerve agent weapons, but many veterans were also exposed to AChEI based pesticides, further complicating the etiology of this illness.
To elucidate the relationship between these factors, Dr. Victoria Macht, her advisor Prof. Lawrence Reagan, and colleagues at the University of South Carolina School of Medicine studied rats exposed to pyridostigmine bromide and repeated restraint stress. The rats were then given either an immune challenge or an acute immobilization stress challenge during in vivo microdialysis. It is the first study to use an in vivo method (microdialysis) to show that PB changes the response of the central cholinergic system to both stress and immune challenges, and does so in a brain region specific manner.
By measuring acetylcholine levels via microdialysis and subsequent HPLC-ECD, they found that cholinergic responses were attenuated in the PFC and hippocampus after immobilization stress. Lipopolysaccharide (LPS) was administered as an immune challenge, after which cholinergic responses were attenuated in the hippocampus but not the PFC. These results indicate that PB and stress interact to shift the cholinergic response to future psychological and immunological stressors, providing a potential mechanism for the persistent and exacerbated cognitive symptoms evidenced in soldiers with GWI.
Mike Churchill: What story do the different responses to the immune challenge and the immobilization challenge tell?
Victoria Macht: By using two different types of challenges, we were able to test both the diversity and consistency of effects of PB and stress on the cholinergic system. LPS is a novel challenge which specifically elicits a response from the innate immune system. The immobilization challenge is more of a psychological stressor, and as it shares some similar qualities with the prior restraint stress, this allowed us to test if rats with PB and restraint stress had impaired neurochemical adaptations to recurrent stressors.
MC: How might these results relate to changes in fear memory and cognitive function?
VM: ACh is an important regulator for a variety of factors in fear memory including coordination of local circuits to help with sensory and cortical processing of stimuli as well as the consolidation process. Interestingly, regional differences in the cholinergic response of the PFC and hippocampus to immobilization stress suggested that PB impairs cortical processing of novel stressful stimuli and impairs the neurochemical adaptation to recurrent stressful stimuli. In our fear conditioning studies, we similarly found impairments in the way PB and stress interacted to impair context and cue related retrieval. This suggested to us that impairments in the function of the cholinergic systems impacts a variety of psychological stressful stimuli, indicating that this is a global deficit in cognitive function rather than a specific deficit to only one type of stressor.
MC: How do the microdialysis results relate to the tests for inflammation you ran?
VM: ACh is really fascinating because while it is not only central in learning and memory, it is also an important negative regulator for the inflammatory response via α7 nicotinic ACh receptors. We found that PB blunted the central cholinergic response to an innate immune challenge, which could suggest an exacerbated chronic inflammatory response in the brain. Interestingly, these microdialysis results for acetylcholine parallel some of our findings with peripheral inflammatory markers. Peripheral levels of c-reactive protein were elevated after the LPS challenge in rats which had received PB, suggesting a dysregulated inflammatory response. While we need to confirm these results with cytokine levels in the brain, our results suggest that impaired cholinergic feedback to inflammatory stimuli could underlie some of the changes in the sensitivity of the immune system which are evident in clinical populations with GWI.
MC: Does PB have to cross the BBB to cause these effects?
VM: It does not. There has been a big debate on this topic. One suggestion was that stress caused a leaky barrier, allowing PB to get through. However, tests on this have been inconsistent on this. What our studies demonstrate is that PB changes the function of the central cholinergic system regardless of whether it is able to get through the BBB.
MC: What will be the next steps for this project?
VM: Prof. Reagan will continue the project: measuring cytokine responses in the brain to see if they match peripheral cytokine responses. There is also an opportunity to see if aging exacerbates the decline of the cholinergic responses and cognitive deficits in our model of GWI. The goal would be to see if animal models of GWI can predict further changes in veterans as they age, and plan treatment accordingly. We have a unique opportunity with this population for the preclinical research on treatments to get ahead of the patient population as they age.
MC: How did you like using the Eicom HTEC HPLC-ECDs in Prof. Jim Fadel’s lab?
VM: It is amazing! I can’t imagine having done these projects without it, and I miss using it. We used the system daily for two years to measure acetylcholine without any real problems. It made my dissertation a much more pleasant experience!
MC: Had you used HPLCs before using the HTEC?
VM: We used a different system before but it was not reliable, so when it was working people felt they had to immediately run all of their samples before it went down again, and watch it all of the time when it was running.
MC: How many samples do you think you ran over the course of this project?
VM: That makes my head spin! We looked at both ACh and glutamate, in two brain regions, each rat underwent microdialysis 2 separate days, there were approximately 8 animals per group, and 4 groups. So at least 3500 samples – plus the pilot study! Plus there were other studies going on during this time which were also using the HTEC.
MC: Where is your career taking you next?
VM: I am now doing a postdoc at UNC Chapel Hill, working with Prof. Fulton Crews, studying the long term effects of binge drinking in adolescents. Interestingly, while this is a different clinical population, changes in the cholinergic system and innate immune system are also common features here.
The article appears in the April issue of Brain, Behavior, and Immunity:
Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness V.A. Macht, J.L. Woodruff, E.S. Maissy, C.A. Grillo, M.A. Wilson, J.R. Fadel, L.P. Reagan doi: 10.1016/j.bbi.2019.04.015
After being conditioned to drink alcohol, 15% of rats continue to seek alcohol after being given the choice of switching to sweetened water. This group of rats shows a high motivation to pursue alcohol even when it is paired with a negative stimulus such as an electric shock, mimicking the compulsion as well as the frequency of alcohol-related problems in humans. Further examination of this group revealed the GAT-3 γ-aminobutyric acid transporter protein was significantly downregulated in the amygdalas of these rats, leading to decreased clearance of γ-aminobutyric acid (GABA) from synapses in this region.
To confirm this model, the Heilig group of the Linköping University in Sweden then downregulated GAT-3 in the amygdala in rats which until that point showed no preference for alcohol. As the injected vector began to downregulate GAT-3, the rats showed a stronger and stronger preference for alcohol over sweetened water.
This model also seems to hold true for humans: postmortem examination of human subjects showed lower GAT-3 expression in the amygdalas of those with alcohol dependence.
Monitoring GABA levels in research animals is what we do: please visit the Amuza Neuroscience website to learn more about microdialysis and HPLC-ECD analysis for GABA and other analytes.
The study is being lauded not only for proceeding from basic research to translational significance in humans, but also for its addition of choice to the basic experimental design. Allowing the research animals to choose between self-administering alcohol or switching to another reward after the conditioning period required a more lengthy and larger scale project (over 600 rats), but it revealed how the animals should be grouped together for further analysis.
While this study indicates a causal relationship between a GABA transporter and alcohol dependence, it may not point directly to the best drug target for addiction therapy. Increasing GABA transport back into neurons in the amygdala may prove more difficult than other ways of controlling GABA levels in the region.
The Heilig group of the Linköping University in Sweden presented the results at the Research Society on Alcoholism meeting in San Diego and subsequently in the journal Science:
A molecular mechanism for choosing alcohol over an alternative reward Science 22 Jun 2018: Vol. 360, Issue 6395, pp. 1321-1326, DOI: 10.1126/science.aao1157
We are excited to announce that Amuza Inc have been awarded an R41 Technology Transfer grant by the National Institute of Neurological Disorders (NINDS/NIH, USA) to develop “a unified system of wireless optogenetics and brain microdialysis”. The grant proposal was based on research which combined Amuza’s HPLC-ECD, microdialysis, and optogenetics instruments.
The funding is for Phase I (prototype development and validation) of a Small Business Technology Transfer (STTR) project in collaboration with Drs. Nobuyoshi Suto (The Scripps Research Institute), Thomas Jhou (Medical University of South Carolina) and Matthew Buczynski (Virginia Tech).
After research and commercialization phases have been completed the final opto-dialysis system will be available from Amuza as well. We are currently undertaking the pilot experiments in freely moving animals (both rats and mice) using our opto-dialysis prototypes. We will post the results as they become available. Please stay tuned!
We are pleased to announce the launch of a new Microdialysis Fraction Collector, Model FC-90, designed by Amuza.
The newFC-90 Microdialysis Fraction Collector saves lab space and your time when collecting microdialysis samples from multiple animals, up to 4 at once. The FC-90’s quiet operation and narrow footprint are ideal for small spaces. Programmed and controlled by Android™ devices via Bluetooth. Robustly designed for microdialysis.
The Android robot is reproduced or modified from work created and shared by Google and used according to terms described in the Creative Commons 3.0 Attribution License.
Eicom can increase the throughput, sensitivity, and temporal resolution of your next microdialysis experiment. Fast microdialysis allows basal level monitoring of dopamine and serotonin in mice with 3 minute online HPLC analyses and 6 minute sampling times. These conditions allow a detection limit for serotonin of 0.8 fMol with an Eicom HTEC-500 HPLC.
Developed by neuroscientists Anne Andrews and Hongyan Yang at UCLA in conjunction with Eicom, this application makes full use of the short analysis time by staggering injections from two different animals onto one Eicom HTEC 500 HPLC-ECD equipped with a PP-ODS II separation column.
The 6 minute sample time has already proven useful in unmasking physiologically relevant changes in extracellular serotonin levels too fast to examine with standard 20 minute samples. Furthermore, online microdialysis allowed them to troubleshoot microdialysis sampling at the beginning of their experiments instead of after hours of collection.
Two Eicom EAS-20s autoinjectors are programmed to collect 6 minute samples from two animals and alternate making injections on the HPLC. This allows 2 complete runs, one from each animal, in each 6 minute chromatogram.