Eric Brunette

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-d-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol‐rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen–glucose deprivation (OGD), N‐methyl‐d‐aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only β‐, methylated β‐ and sulfated β‐CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD‐95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X‐100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated β‐CD to protect against NMDA toxicity, and reversed NR2B, PSD‐95 and nNOS localization to Triton X‐100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor‐mediated increases in intracellular Ca2+ concentration ([Ca2+]i), but did suppress OGD‐induced increases in [Ca2+]i. β‐CD, but not Mβ‐CD, also caused a slight block of NMDA‐induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent‐resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol‐rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca2+ levels.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics

The blood‐brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic “arm” is combined with a BBB‐transcytosing arm can significantly enhance their brain delivery. The BBB‐permeable single‐domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single‐domain antibody phage display library. In this study, FC5 was engineered as a mono‐ and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30‐fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5‐compared with control domain antibody‐Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB‐impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5‐Fc fusion proteins. Improved serum pharmacokinetics of Fc‐fused FC5 contributed to a 60‐fold increase in pharmacological potency compared with the single‐domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB‐carrier arm in bispecific antibodies or antibody‐drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.—Farrington, G. K., Caram‐Salas, N., Haqqani, A. S., Brunette, E., Eldredge, J., Pepinsky, B., Antognetti, G., Baumann, E., Ding, W., Garber, E., Jiang, S., Delaney, C., Boileau, E., Sisk, W. P., Stanimirovic, D. B., A novel platform for engineering blood‐brain barrier‐crossing bispecific biologics. FASEB J. 28, 4764–4778 (2014). www.fasebj.org

Chlortetracycline and Demeclocycline Inhibit Calpains and Protect Mouse Neurons against Glutamate Toxicity and Cerebral Ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-d-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.

Dynamic Analysis of the Blood-Brain Barrier Disruption in Experimental Stroke Using Time Domain In Vivo Fluorescence Imaging

The blood-brain barrier (BBB) disruption following cerebral ischemia can be exploited to deliver imaging agents and therapeutics into the brain. The aim of this study was (a) to establish novel in vivo optical imaging methods for longitudinal assessment of the BBB disruption and (b) to assess size selectivity and temporal patterns of the BBB disruption after a transient focal ischemia. The BBB permeability was assessed using in vivo time domain near-infrared optical imaging after contrast enhancement with either free Cy5.5 (1 kDa) or Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa) in mice subjected to either 60- or 20-minute transient middle cerebral artery occlusion (MCAO) and various times of reperfusion (up to 14 days). In vivo imaging observations were corroborated by ex vivo brain imaging and microscopic analyses of fluorescent tracer extravasation. The in vivo optical contrast enhancement with Cy5.5 was spatially larger than that observed with BSA-Cy5.5. Longitudinal studies after a transient 20-minute MCAO suggested a bilateral BBB disruption, more pronounced in the ipsilateral hemisphere, peaking at day 7 and resolving at day 14 after ischemia. The area differential between the BBB disruption for small and large molecules could potentially be useful as a surrogate imaging marker for assessing perinfarct tissues to which neuroprotective therapies of appropriate sizes could be delivered.

ELEVATED SYNAPTIC ACTIVITY PRECONDITIONS NEURONS AGAINST AN IN VITRO MODEL OF ISCHEMIA

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-d-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the “brink of death.” We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K+-channel blocker, 4-aminopyridine (20–2500 μm), and a GABAA receptor antagonist, bicuculline (50 μm) (4-AP/bic), for 1–2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca2+ spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or l-type voltage-gated Ca2+ channels (L-VGCCs) applied during preconditioning decreased Ca2+ spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca2+/calmodulin-dependent protein kinases (CaMKs) also reduced Ca2+ spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.

In vivo time domain optical imaging of renal ischemia-reperfusion injury: discrimination based on fluorescence lifetime.

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.

An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD‐active MnIIIporphyrins meso‐substituted with N, N′‐dimethylimidazolium or N‐alkylpyridinium groups did not protect neurons against oxygen‐glucose deprivation (OGD), although lower SOD‐active and ‐inactive para isomers protected against N‐methyl‐d‐aspartate (NMDA) exposure. MnIIImeso‐tetrakis(4‐benzoic acid)porphyrin (MnIIITBAP), as well as SOD‐inactive metalloTBAPs and other phenyl ring‐ or β‐substituted metalloporphyrins that contained redox‐insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD‐ or NMDA‐induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor‐mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD‐inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.

Brain penetration, target engagement, and disposition of the blood–brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood‐brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB‐crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high‐affinity, selective antibody antagonist of the metabotropic glutamate receptor‐1 (BBB‐mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB‐NiP). Both BBB‐NiP and BBB‐mGluR1 demonstrated a similar 20‐fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo. Comparative assessment of BBB‐NiP and BBB‐mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target‐mediated removal of the mGluR1‐engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose‐dependent inhibition of mGluR‐1‐mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved inmGluR1‐mediated pain processing. We demonstrate the feasibility of targeting central G‐protein‐coupled receptors using a BBB‐crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.—Webster, C. I., Caram‐Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood‐brain barrier‐crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1 FASEB J. 30, 1927–1940 (2016). www.fasebj.org

Multiplexed Evaluation of Serum and CSF Pharmacokinetics of Brain- Targeting Single-Domain Antibodies Using a NanoLC−SRM-ILIS Method

FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p < 0.01) enhanced transport (50-100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.

Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery

It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain–CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally‐infused antibodies exhibited size‐dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full‐length IgG could be enhanced by intrathecal co‐infusion of hyperosmolar mannitol. Pores or stomata present on CSF‐facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain–CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies.