PBPK model for antibody disposition in mouse brain: validation using large-pore microdialysis data.

The objective of this manuscript was to validate a physiologically-based pharmacokinetic (PBPK) model developed to characterize brain pharmacokinetics (PK) of monoclonal antibodies (mAbs) using novel large-pore microdialysis data generated in mice. To support this objective, brain, CSF, and ISF PK of a human anti-tetanus toxin (TeTx) antibody was measured in mice following intraperitoneal (IP) administration. This antibody has no binding in mice. In addition, our recently published mouse brain PK data generated following intravenous (IV) and IP administration of trastuzumab in mice, and other published PK data for brain disposition of antibody in mice, were used to evaluate the PBPK model. All the model parameters were obtained from literature or kept the same as in our previously published manuscript. The revised PBPK model was able to characterize the PK of antibodies in mice brain, CSF, and ISF reasonably well (i.e., within a three-fold error). However, a priori selected parameters led to underprediction of ISF PK during the initial phase of the profile. A local sensitivity analysis suggested that minor changes in several brain-related parameters can help overcome this discrepancy, where an increase in the convective flow of antibodies across BBB was found to be the most parsimonious way to capture all the PK profiles well. However, the presence of this pathway needs further validation. As such, here we have presented an improved PBPK model to characterize and predict the PK of mAbs in different regions of the mouse brain following systemic administration. This model can serve as a quantitative tool to facilitate the discovery, preclinical evaluation, and preclinical-to-clinical translation of novel antibodies targeted against CNS disorders.

Adaptive Mechanisms of Somatostatin-Positive Interneurons after Traumatic Brain Injury through a Switch of α Subunits in L-Type Voltage-Gated Calcium Channels.

Unilateral traumatic brain injury (TBI) causes cortical dysfunctions spreading to the primarily undamaged hemisphere. This phenomenon, called transhemispheric diaschisis, is mediated by an imbalance of glutamatergic versus GABAergic neurotransmission. This study investigated the role of GABAergic, somatostatin-positive (SST) interneurons in the contralateral hemisphere 72 h after unilateral TBI. The brain injury was induced to the primary motor/somatosensory cortex of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice at postnatal days 19-21 under anesthesia in vivo. Single GFP+ interneurons of the undamaged, contralateral cortex were isolated by fluorescence-activated cell sorting and analyzed by mass spectrometry. TBI caused a switch of 2 α subunits of pore-forming L-type voltage-gated calcium channels (VGCC) in GABAergic interneurons, an increased expression of CaV1.3, and simultaneous ablation of CaV1.2. This switch was associated with 1) increased excitability of single SST interneurons in patch-clamp recordings and (2) a recovery from early network hyperactivity in the contralateral hemisphere in microelectrode array recordings of acute slices. The electrophysiological changes were sensitive to pharmacological blockade of CaV1.3 (isradipine, 100 nM). These data identify a switch of 2 α subunits of VGCCs in SST interneurons early after TBI as a mechanism to counterbalance post-traumatic hyperexcitability.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Proteasome and Autophagy-Mediated Impairment of Late Long-Term Potentiation (l-LTP) after Traumatic Brain Injury in the Somatosensory Cortex of Mice.

Traumatic brain injury (TBI) can lead to impaired cognition and memory consolidation. The acute phase (24-48 h) after TBI is often characterized by neural dysfunction in the vicinity of the lesion, but also in remote areas like the contralateral hemisphere. Protein homeostasis is crucial for synaptic long-term plasticity including the protein degradation systems, proteasome and autophagy. Still, little is known about the acute effects of TBI on synaptic long-term plasticity and protein degradation. Thus, we investigated TBI in a controlled cortical impact (CCI) model in the motor and somatosensory cortex of mice ex vivo-in vitro. Late long-term potentiation (l-LTP) was induced by theta-burst stimulation in acute brain slices after survival times of 1-2 days. Protein levels for the plasticity related protein calcium/calmodulin-dependent protein kinase II (CaMKII) was quantified by Western blots, and the protein degradation activity by enzymatical assays. We observed missing maintenance of l-LTP in the ipsilateral hemisphere, however not in the contralateral hemisphere after TBI. Protein levels of CaMKII were not changed but, interestingly, the protein degradation revealed bidirectional changes with a reduced proteasome activity and an increased autophagic flux in the ipsilateral hemisphere. Finally, LTP recordings in the presence of pharmacologically modified protein degradation systems also led to an impaired synaptic plasticity: bath-applied MG132, a proteasome inhibitor, or rapamycin, an activator of autophagy, both administered during theta burst stimulation, blocked the induction of LTP. These data indicate that alterations in protein degradation pathways likely contribute to cognitive deficits in the acute phase after TBI, which could be interesting for future approaches towards neuroprotective treatments early after traumatic brain injury.

Acute Cortical Transhemispheric Diaschisis after Unilateral Traumatic Brain Injury.

Focal neocortical brain injuries lead to functional alterations, which can spread beyond lesion-neighboring brain areas. The undamaged hemisphere and its associated disturbances after a unilateral lesion, so-called transhemispheric diaschisis, have been progressively disclosed over the last decades; they are strongly involved in the pathophysiology and, potentially, recovery of brain injuries. Understanding the temporal dynamics of these transhemispheric functional changes is crucial to decipher the role of the undamaged cortex in the processes of functional reorganization at different stages post-lesion. In this regard, little is known about the acute-subacute processes after 24-48 h in the brain hemisphere contralateral to injury. In the present study, we performed a controlled cortical impact to produce a unilateral traumatic brain injury (TBI) in the motor and somatosensory cortex of mice. In vitro extracellular multi-unit recordings from large neuronal populations, together with single-cell patch-clamp recordings in the cortical network contralateral to the lesion, revealed a strong, but transient, neuronal hyperactivity as early as 24-48 h post-TBI. This abnormal excitable state in the intact hemisphere was not accompanied by alterations in neuronal intrinsic properties, but it was associated with an impairment of the phasic gamma aminobutyric acid (GABA)ergic transmission and an increased expression of GABAA receptor subunits related to tonic inhibition exclusively in the contralateral hemisphere. These data unravel a series of early transhemispheric functional alterations after diffuse unilateral cortical injury, which may compensate and stabilize the disrupted brain functions. Therefore, our findings support the hypothesis that the undamaged hemisphere could play a significant role in early functional reorganization processes after a TBI.

In vitro activities of synthetic host defense propeptides processed by neutrophil elastase against cystic fibrosis pathogens.

The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.