Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.

Neuroprognostication in the Post Cardiac Arrest Patient: A Canadian Cardiovascular Society Position Statement.

Cardiac arrest (CA) is associated with a low rate of survival with favourable neurologic recovery. The most common mechanism of death after successful resuscitation from CA is withdrawal of life-sustaining measures on the basis of perceived poor neurologic prognosis due to underlying hypoxic-ischemic brain injury. Neuroprognostication is an important component of the care pathway for CA patients admitted to hospital but is complex, challenging, and often guided by limited evidence. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system to evaluate the evidence underlying factors or diagnostic modalities available to determine prognosis, recommendations were generated in the following domains: (1) circumstances immediately after CA; (2) focused neurologic exam; (3) myoclonus and seizures; (4) serum biomarkers; (5) neuroimaging; (6) neurophysiologic testing; and (7) multimodal neuroprognostication. This position statement aims to serve as a practical guide to enhance in-hospital care of CA patients and emphasizes the adoption of a systematic, multimodal approach to neuroprognostication. It also highlights evidence gaps.

Development of a multigram synthesis of the bradykinin receptor 2 agonist FR-190997 and analogs thereof.

Using Fujisawa's B2R agonist FR-190997, we recently demonstrated for the first time that agonism at the bradykinin receptor type 2 (B2R) produces substantial antiproliferative effects. FR-190997 elicited an EC50 of 80 nM in the triple-negative breast cancer cell line MDA-MB-231, a much superior performance to that exhibited by most approved breast cancer drugs. Consequently, we initiated a program aiming primarily at synthesizing adequate quantities of FR-190997 to support further in vitro and in vivo studies toward its repurposing for various cancers and, in parallel, enable the generation of novel FR-190997 analogs for an SAR study. Prerequisite for this endeavor was to address the synthetic challenges associated with the FR-190997 scaffold, which the Fujisawa chemists had constructed in 20 steps, 13 of which required chromatographic purification. We succeeded in developing a 17-step synthesis amenable to late-stage diversification that eliminated all chromatography and enabled access to multigram quantities of FR-190997 and novel derivatives thereof, supporting further anticancer research based on B2R agonists.

Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene.

Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3-protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and ß-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.

Pharmacological Modulation of Blood-Brain Barrier Permeability by Kinin Analogs in Normal and Pathologic Conditions.

The blood-brain barrier (BBB) is a major obstacle to the development of effective diagnostics and therapeutics for brain cancers and other central nervous system diseases. Peptide agonist analogs of kinin B1 and B2 receptors, acting as BBB permeabilizers, have been utilized to overcome this barrier. The purpose of the study was to provide new insights for the potential utility of kinin analogs as brain drug delivery adjuvants. In vivo imaging studies were conducted in various animal models (primary/secondary brain cancers, late radiation-induced brain injury) to quantify BBB permeability in response to kinin agonist administrations. Results showed that kinin B1 (B1R) and B2 receptors (B2R) agonists increase the BBB penetration of chemotherapeutic doxorubicin to glioma sites, with additive effects when applied in combination. B2R agonist also enabled extravasation of high-molecular-weight fluorescent dextrans (155 kDa and 2 MDa) in brains of normal mice. Moreover, a systemic single dose of B2R agonist did not increase the incidence of metastatic brain tumors originating from circulating breast cancer cells. Lastly, B2R agonist promoted the selective delivery of co-injected diagnostic MRI agent Magnevist in irradiated brain areas, depicting increased vascular B2R expression. Altogether, our findings suggest additional evidence for using kinin analogs to facilitate specific access of drugs to the brain.

Haemorrhagic Transformation of a MELAS Stroke-Like Lesion.

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a rare progressive maternally inherited mitochondrial disease that clinically harbours various neurological and systemic manifestations.

Efficacy of Combining PRP and MMP Inhibitors in Treating Moderately Damaged Tendons Ex Vivo.

Platelet-rich plasma (PRP) and broad-spectrum matrix metalloproteinase inhibitors (MMPIs) have been used as therapeutic options for tendinopathy. However, mixed results have been reported regarding their efficacy. We posited that the combination of these two treatment strategies would be more beneficial for healing tendons than each treatment alone. Rat tail tendons were harvested and cultured without mechanical stress for 0, 4, or 10 days. Single and combination treatment with PRP and MMPIs with either broad- or narrow-spectrum (MMP-13 selective), was administered to 4-day stress-deprived (SD) tendons, an ex vivo model for moderate tendinopathy. This treatment was applied to the damaged tendons over 6 days. At the end of their culture time, the tendons were subjected to traction testing and pathohistology, immunohistochemistry, and viability assays. The results showed better histological features for the PRP + narrow-spectrum MMPI group compared with all individual treatment modalities. Moreover, higher fiber density, more elongated nucleus shape, smaller space between fibers, and a trend toward higher mechanical strength were noted for PRP + narrow-spectrum MMPI group compared with 10-day SD tendons. This study shows that the combination of PRP + narrow-spectrum MMPI is a potentially effective treatment approach for tendinopathy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1838-1847, 2019.

Characterization of moderate tendinopathy in ex vivo stress-deprived rat tail tendons.

BACKGROUND: Stress deprivation (SD) has frequently been used as a model to study tendinopathy. Most of these studies have investigated either short-term (early tendinopathy) or long-term SD (advanced tendinopathy), while the transient mid-term SD has been given less attention. Therefore, the main objective of this study was to characterize mid-term SD. METHODS: To this end, live, healthy rat tail tendons (RTTs) were harvested and cultured without mechanical stress and then were divided into five groups based on their culture time (fresh, 2-day SD, 4-day SD, 6-day SD, and 10-day SD). For each group, the tendons were subjected to traction testing and pathohistology, immunohistochemistry, and viability assays. RESULTS: Our results showed that 4 days of SD resulted in moderate pathological changes in RTTs. These changes included increases in the space area between fibers, cell density, and fiber tortuosity as well as a decrease in collagen density and elongation of cell nuclei. No changes in the stress at failure of tendons were observed at this time point. CONCLUSIONS: This simple ex vivo model is expected to be useful for studying the progression of tendinopathy as well as for testing potential mechanobiological or pharmacological therapy strategies to stop or reverse the progression of the pathology.

Antitumor activity of cell-penetrant kinin B1 receptor antagonists in human triple-negative breast cancer cells.

High nuclear expression of G protein-coupled receptors, including kinin B1 receptors (B1R), has been observed in several human cancers, but the clinical significance of this is unknown. We put forward the hypothesis that these "nuclearized" kinin B1R contribute to tumorigenicity and can be a new target in anticancer strategies. Our initial immunostaining and ultrastructural electron microscopy analyses demonstrated high B1R expression predominantly located at internal/nuclear compartments in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line as well as in clinical samples of patients with TNBC. On the basis of these findings, in the present study, we evaluated the anticancer therapeutic potential of newly identified, cell-permeable B1R antagonists in MDA-MB-231 cells (ligand-receptor binding/activity assays and LC-MS/MS analyses). We found that these compounds (SSR240612, NG67, and N2000) were more toxic to MDA-MB-231 cells in comparison with low- or non-B1R expressing MCF-10A normal human mammary epithelial cells and COS-1 cells, respectively (clonogenic, MTT proliferative/cytocidal assays, and fluorescence-activated cell-sorting (FACS)-based apoptosis analyses). By comparison, the peptide B1R antagonist R954 unable to cross cell membrane failed to produce anticancer effects. Furthermore, the putative mechanisms underlying the anticancer activities of cell-penetrant B1R antagonists were assessed by analyzing cell cycle regulation and signaling molecules related to cell survival and apoptosis (FACS and western blot). Finally, drug combination experiments showed that cell-penetrant B1R antagonists can cooperate with suboptimal doses of chemotherapeutic agents (doxorubicin and paclitaxel) to promote TNBC death. This study provides evidence on the potential value of internally acting kinin B1R antagonists in averting growth of breast cancer.

Jamestown Canyon encephalitis with NORSE and electrographic response to ribavirin: a case report.

New-onset refractory status epilepticus (NORSE) is a rare condition where refractory seizures occur with no obvious cause in an otherwise healthy individual. We report here the first case of a patient with NORSE caused by Jamestown Canyon virus, an emerging virus throughout North America. This is the first patient with Jamestown Canyon virus encephalitis treated by a specific antiviral agent, in this case intravenous ribavirin, with electroencephalography (EEG) evidence of improvement. This is finally the first reported death caused by this virus, here secondary to intensive care unit complications. Therefore, Jamestown Canyon virus should be considered as a NORSE etiology, and ribavirin may be an effective treatment of that condition, especially if used early.

Targeting intracellular B2 receptors using novel cell-penetrating antagonists to arrest growth and induce apoptosis in human triple-negative breast cancer.

G protein-coupled receptors (GPCRs) are integral cell-surface proteins having a central role in tumor growth and metastasis. However, several GPCRs retain an atypical intracellular/nuclear location in various types of cancer. The pathological significance of this is currently unknown. Here we extend this observation by showing that the bradykinin B2R (BK-B2R) is nuclearly expressed in the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and in human clinical specimens of TNBC. We posited that these "nuclearized" receptors could be involved in oncogenic signaling linked to aberrant growth and survival maintenance of TNBC. We used cell-penetrating BK-B2R antagonists, including FR173657 and novel transducible, cell-permeable forms of the peptide B2R antagonist HOE 140 (NG68, NG134) to demonstrate their superior efficacy over impermeable ones (HOE 140), in blocking proliferation and promoting apoptosis of MDA-MB-231 cells. Some showed an even greater antineoplastic activity over conventional chemotherapeutic drugs in vitro. The cell-permeable B2R antagonists had less to no anticancer effects on B2R shRNA-knockdown or non-B2R expressing (COS-1) cells, indicating specificity in their action. Possible mechanisms of their anticancer effects may involve activation of p38kinase/p27Kip1 pathways. Together, our data support the existence of a possible intracrine signaling pathway via internal/nuclear B2R, critical for the growth of TNBC cells, and identify new chemical entities that enable to target the corresponding intracellular GPCRs.

Severe cerebellar hemorrhage following transverse sinus stenting for idiopathic intracranial hypertension.

We report a severe adverse event occurring in the course of a cohort study (ISRCTN13784335) aimed at measuring the efficacy and safety of venous stenting in the treatment of patients with medically refractory idiopathic intracranial hypertension (IIH). The patient was a 41-year-old woman who was not overweight, who presented with severe headache, grade 1 bilateral papilledema and transient tinnitus, refractory to medical treatment. Right transverse sinus stenting was successfully performed. Following surgery, the patient's state of consciousness decreased acutely with rapid and progressive loss of brainstem reflex. CT scan revealed acute cerebellar and intraventricular hemorrhage with obstructive hydrocephalus. Angioscan revealed normal venous sinus patency and cerebral MRI showed acute mesencephalic ischemia. Mechanical impairment of cerebellar venous drainage by the stent or venous perforation with the large guidewire used in this technique are two logical ways to explain the cerebellar hemorrhage seen in our patient. The risk of such a complication could probably be reduced using alternative tools and technique. However, given the low level of evidence around the safety of transverse sinus stenting in IIH, its formal assessment in clinical trials is required.

Prognostication in critically ill patients with severe traumatic brain injury: the TBI-Prognosis multicentre feasibility study.

OBJECTIVE: Severe traumatic brain injury is a significant cause of morbidity and mortality in young adults. Assessing long-term neurological outcome after such injury is difficult and often characterised by uncertainty. The objective of this feasibility study was to establish the feasibility of conducting a large, multicentre prospective study to develop a prognostic model of long-term neurological outcome in critically ill patients with severe traumatic brain injury. DESIGN: A prospective cohort study. SETTING: 9 Canadian intensive care units enrolled patients suffering from acute severe traumatic brain injury. Clinical, biological, radiological and electrophysiological data were systematically collected during the first week in the intensive care unit. Mortality and functional outcome (Glasgow Outcome Scale extended) were assessed on hospital discharge, and then 3, 6 and 12 months following injury. OUTCOMES: The compliance to protocolised test procedures was the primary outcome. Secondary outcomes were enrolment rate and compliance to follow-up. RESULTS: We successfully enrolled 50 patients over a 12-month period. Most patients were male (80%), with a median age of 45 years (IQR 29.0-60.0), a median Injury Severity Score of 38 (IQR 25-50) and a Glasgow Coma Scale of 6 (IQR 3-7). Mortality was 38% (19/50) and most deaths occurred following a decision to withdraw life-sustaining therapies (18/19). The main reasons for non-enrolment were the time window for inclusion being after regular working hours (35%, n=23) and oversight (24%, n=16). Compliance with protocolised test procedures ranged from 92% to 100% and enrolment rate was 43%. No patients were lost to follow-up at 6 months and 2 were at 12 months. CONCLUSIONS: In this multicentre prospective feasibility study, we achieved feasibility objectives pertaining to compliance to test, enrolment and follow-up. We conclude that the TBI-Prognosis prospective multicentre study in severe traumatic brain injury patients in Canada is feasible.

Pulvinar sign in a case of anti-HU paraneoplastic encephalitis.

This article reports the case of a 68-year-old patient with anti-HU antibodies paraneoplastic encephalitis. The clinical manifestations were atypical and the paraclinical work-up, notably the magnetic resonance imaging (MRI) showing bilateral posterior thalamic hyperintensities (pulvinar sign), misleadingly pointed towards a variant Creutzfeld-Jakob disease. After presenting the case, the differential diagnosis of the pulvinar sign is discussed along with other important diagnostic considerations.

Limbic Encephalitis Associated With GAD65 Antibodies: Brief Review of the Relevant literature.

Recently, many cases of autoimmune limbic encephalitis with positive GAD65 (glutamic acid decarboxylase) antibodies have been described in the scientific literature. However, it remains an understudied topic of great relevance to practicing neurologists. Thus, we report here a review of published cases, in English, of autoimmune limbic encephalitis with this type of antibodies, focusing on presenting symptoms and signs, associated conditions, and findings upon investigation. We also report treatment responses. We aim to offer a better description of the clinical spectrum of autoimmune limbic encephalitis associated with GAD65 antibodies as well as to expose its paraclinical features and outcome.

Associated Factors and Prognostic Implications of Stimulus-Induced Rhythmic, Periodic, or Ictal Discharges.

IMPORTANCE: The implications of stimulus-induced rhythmic, periodic, or ictal discharges (SIRPIDs) sometimes found on prolonged electroencephalographic (EEG) recordings are uncertain. OBJECTIVE: To evaluate the incidence of SIRPIDs and their clinical implications in critically ill patients. DESIGN, SETTING, AND PARTICIPANTS: A multicenter, international retrospective study was performed from October 1, 2012, through September 30, 2014, of consecutive adult patients hospitalized in intensive care units with alteration of consciousness who underwent EEG recordings at 3 separate centers. Demographic data, including admission diagnosis, age, sex, history of epilepsy, and EEG findings, were noted. Characteristics of SIRPIDs were documented. Data were evaluated for predictors of SIRPIDs and in-hospital mortality. Data analysis was performed from January 16, 2015, to June 15, 2015. MAIN OUTCOMES AND MEASURES: Incidence of SIRPIDs, association of SIRPIDs with mortality and other EEG characteristics, and EEG and clinical predictors of mortality. RESULTS: A total of 416 patients were studied. The median age of patients was 60 years (interquartile range, 46-71 years), and 252 (60.6%) were male. A total of 104 patients (25.0%) did not survive to hospital discharge. SIRPIDs were identified in 43 patients (10.3%). The proportion of patients with SIRPIDs was not significantly different across the 3 sites (P = .34). Anoxic brain injury (odds ratio [OR], 3.80; 95% CI, 1.73-8.33; P < .001), the use of antiepileptic medications (OR, 3.24; 95% CI, 1.31-8.00; P = .01), electrographic seizures (OR, 2.85; 95% CI, 1.13-7.19; P = .03), generalized periodic discharges with triphasic morphologic features (OR, 3.66; 95% CI, 1.67-8.02; P = .001), and sporadic sharp waves and periodic discharges (OR, 2.59; 95% CI, 1.13-5.92; P = .02) were independently associated with the presence of SIRPIDs. Older age (OR, 1.02; 95% CI, 1.01-1.04; P = .005), anoxic brain injury (OR, 3.49; 95% CI, 1.96-6.21; P ≤ .001), and absence of EEG reactivity (OR, 8.14; 95% CI, 4.20-15.79; P < .001) but not SIRPIDs (OR, 1.73; 95% CI, 0.79-3.78; P = .17) were independently associated with in-hospital mortality. CONCLUSIONS AND RELEVANCE: In critically ill patients undergoing EEG recordings, SIRPIDs occurred in 43 (10.3%) and were associated with other electrographic abnormalities previously reported to indicate poor prognosis. However, SIRPIDs were not independently associated with in-hospital mortality.