Nadia L. Caram-Salas

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

Role of opioid receptors in the reduction of formalin-induced secondary allodynia and hyperalgesia in rats

This study assesses the effects of peripheral or intrathecal pre-treatment or post-treatment with micro, delta, kappa and nociceptin/orphanin FQ (NOP) opioid receptor agonists (morphine, U-50488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride], DADLE [D-Ala2-Leu5-enkephalin] and nociceptin, respectively) on formalin-induced secondary mechanical allodynia and hyperalgesia in rats. 1% Formalin injection produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term tactile secondary allodynia and hyperalgesia. Neither peripheral (into the formalin-injected paw) nor intrathecal morphine post-treatment reversed formalin-induced secondary allodynia and hyperalgesia. In contrast, morphine pre-treatment prevented the development of these pain behaviors. Intrathecal and peripheral post- but not pre-treatment with U-50488 or DADLE significantly reduced secondary allodynia and hyperalgesia. Interestingly, nociceptin reduced both pain behaviors regardless of the administration site or treatment time. Local antinociceptive effects of morphine, DADLE, U-50488 or nociceptin were blocked by naltrexone, naltrindole, 5-guanidinonaltrindole and [Nphe(1)]nociceptin(1-13)NH(2), respectively. These results suggest that the long-term nociceptive behaviors induced by formalin are differentially modulated by selective opioid receptor agonists. In addition, data suggest that peripheral and spinal delta and kappa opioid receptors are important when nociceptive behaviors are established. In contrast, micro opioid receptors are more important at the beginning of the injury when the sensory system has not changed. NOP receptors participate diminishing both the development and maintenance of nociceptive behaviors. Results suggest that a barrage of afferent input induced by formalin injection initiates a long-term differential change in peripheral and spinal processing that affect the efficacy of opioid receptor agonists.

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.

Thiamine and Cyanocobalamin Relieve Neuropathic Pain in Rats: Synergy with Dexamethasone

Treatment of neuropathic pain is an area of largely unmet medical need. Therefore, this pain may require the development of novel drug entities. In the search for alternatives, B vitamins have been found to be a clinically useful pharmacological tool for patients with neuropathic pain. However, preclinical studies supporting this use are lacking. In this study, we assessed the possible antiallodynic effects of thiamine, pyridoxine, and cyanocobalamin as well as dexamethasone and their combination on spinal nerve ligation induced allodynia. Sub cutaneous administration of thiamine (75–600 mg/kg), pyridoxine (75–600 mg/kg), cyanocobalamin(0.75–6 mg/kg), and dexamethasone (4–32 mg/kg) significantly reduced tactile allodynia in rats. Maximal antiallodynic effects were reached with 600 mg/kg of thiamine (∼58%), 600 mg/kg of pyridoxine (∼22%), 6 mg/kg of cyanocobalamin (∼73%), and 32 mg/kg of dexamethasone (∼68%). Since a small antiallodynic effect was observed with pyridoxine, this drug was not further analyzed in the combinations. Coadministration of thiamine or cyanocobalamin and dexamethasone remarkably reduced spinal nerve ligation induced allodynia (∼90%), showing a synergistic interaction between either thiamine or cyanocobalamin and dexamethasone. Our data indicate that thiamine and pyridoxine as well as the combination of B vitamins and dexamethasone are able to reduce tactile allodynia in rats and suggest the possible clinical use of these drugs in the treatment of neuropathic pain in human beings.

Isobolographic Analyses of the Gabapentin-Metamizol Combination after Local Peripheral, Intrathecal and Oral Administration in the Rat

This study was designed to evaluate the possible antinociceptive interaction between gabapentin and metamizol on formalin-induced nociception. Gabapentin, metamizol or a fixed dose-ratio combination of both drugs were assessed after local peripheral, intrathecal and oral administration in rats. Isobolographic analyses were employed to define the nature of the interaction between drugs. Gabapentin, metamizol and gabapentin-metamizol combinations yielded a dose-dependent antinociceptive effect when administered by the three different routes. ED30 values were estimated for the individual drugs and isobolograms were constructed. Theoretical ED30 values for the combination estimated from the isobolograms were 21.11 ± 1.17 µg/paw, 104.6 ± 5.5 µg/rat and 78.8 ± 5.5 mg/kg for the local peripheral, intrathecal and oral administration routes, respectively. These values were significantly higher than the experimentally obtained ED30 values which were 11.3 ± 1.5 µg/paw, 36.8 ± 3.1 µg/rat and 15 ± 1.2 mg/kg indicating a synergistic interaction. Systemic administration resulted in the highest synergism. Data confirm that low doses of the gabapentin and metamizol can interact synergistically to reduce formalin-induced nociceptive behavior suggesting that this combination could be useful to treat inflammatory pain in humans.

In Vitro and In Vivo Methods for Assessing FcRn-Mediated Reverse Transcytosis Across the Blood–Brain Barrier

The neonatal Fc receptor, FcRn, mediates endocytic recycling pathway that prevents degradation of IgG and is expressed in most endothelial cells. The blood-brain barrier (BBB), formed by brain endothelial cells sealed with tight junctions, restricts transport of IgG from the blood to the brain. In contrast, it has been suggested that IgG undergoes efflux from the brain parenchyma via reverse transcytosis across the BBB mediated by FcRn. The fast elimination of therapeutic antibodies from the brain via this route may limit their therapeutic potency. In vitro and in vivo methods described in this chapter were developed to facilitate research into mechanisms and dynamics of brain efflux of compounds, including FcRn-mediated reverse transcytosis across the BBB. The in vitro model uses immortalized adult rat brain endothelial cells which express high levels of FcRn. In vivo models use Prospective optical imaging to measure the clearance rate of intracerebrally injected FcRn-transported molecules tagged with near-infrared fluorescent probes.

Sildenafil and glyceryl trinitrate reduce tactile allodynia in streptozotocin-injected rats

The possible antiallodynic effect of phosphodiesterase 5 inhibitor sildenafil and nitric oxide donor glyceryl trinitrate as well as the changes in phosphodiesterase 5A2 mRNA expression in dorsal root ganglion and spinal cord of allodynic diabetic rats was assessed. Diabetes was induced by streptozotocin (50mg/kg, i.p.) in male Wistar rats. Streptozotocin injection produced hyperlglycemia, polydipsia, polyphagia and polyuria as well as long-term tactile allodynia (12 weeks) and a reduction of phosphodiesterase 5A2 mRNA expression in spinal cord of diabetic rats. Systemic administration of sildenafil (1-5.6 mg/kg, i.p.) reduced tactile allodynia in a dose-dependent manner in diabetic rats. Likewise, glyceryl trinitrate patches (0.2mg/h) also reduced tactile allodynia in diabetic rats. Moreover, both drugs reversed streptozotocin-induced phosphodiesterase 5A2 mRNA expression reduction. Our results indicate that glyceryl trinitrate and sildenafil reduce tactile allodynia in diabetic rats suggesting that nitric oxide and cyclic GMP supply is an important step in their mechanism of action of these drugs in diabetic animals. Data suggest that nitric oxide donors (as glyceryl trinitrate) and drugs which increase cyclic GMP levels (as sildenafil) could have a role in the pharmacotherapy of tactile allodynia in diabetic patients.

Ultra-Low Doses of Naltrexone Enhance the Antiallodynic Effect of Pregabalin or Gabapentin in Neuropathic Rats: SYNERGY OF GABAPENTINOIDS AND NALTREXONE

Preclinical Research