Joseph K. Eibl

Structural, biological, and pharmacological strategies for the inhibition of nerve growth factor

Nerve growth factor (NGF) is critical for the development and maintenance of sympathetic and sensory neurons in the developing nervous system, including nociceptors. In the adult nervous system, NGF is known to produce significant pain signals by binding to the TrkA and p75NTR receptors. Several pathological pain disorders are associated with nerve growth factor dysregulation, including neuropathic pain, osteoarthritic pain, and hyperalgesia. Currently, clinical management of these pathologies has relied on the use of opioid and non-steroidal anti-inflammatory drugs (NSAID). However, several chronic pain conditions demonstrate insensitivity to NSAID treatment or the development of detrimental opioid-related side effects, including addiction. As NGF plays an important role in pain generation; antibodies, small molecules and peptides have been designed to antagonize NGF. In this review, we discuss the structural biology of NGF ligand/receptor interaction, and we review current biological and pharmacological strategies to modulate NGF-related pathologies.

Multipotent neurotrophin antagonist targets brain-derived neurotrophic factor and nerve growth factor.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are members of the neurotrophin family that normally play a role in the development and maintenance of the nervous system. However, neurotrophin dysregulation has been implicated in several neurodegenerative diseases and psychiatric disorders including Alzheimers disease, Parkinsons disease, neuropathic pain, depression, and substance abuse. Despite their central role in the nervous system, neurotrophins have proved to be an elusive pharmacological target. Here, we describe a novel multipotent neurotrophin antagonist, 3-[(5E)-4-oxo-5-[[5-(4-sulfamoylphenyl)-2-furyl]methylene]-2-thioxo-thiazolidin-3-yl]propanoic acid (Y1036). Y1036 binds BDNF (KD = 3.5 ± 0.3 μM) and NGF (KD = 3.0 ± 0.4 μM) preventing either BDNF or NGF from interacting with their obligate receptor(s). Y1036 prevents both BDNF- and NGF-mediated trk activation, downstream activation of the p44/42 mitogen-activated protein kinase pathway, and neurotrophin-mediated differentiation of dorsal-root ganglion sensory neurons. Identification of a BDNF- and NGF-specific antagonist is of considerable interest in the study and treatment of diseases where dysregulation of multiple neurotrophins has been implicated.

Zinc induces motor neuron death via a selective inhibition of brain-derived neurotrophic factor activity

Amyotrophic lateral sclerosis is a debilitating disease that results from the deterioration and loss of motor neurons. The neurotoxic potential of Zn(2+), both in vitro and in vivo, has been well established; however, the mechanism(s) of zincs toxicity remain unclear. Our laboratory has demonstrated that Zn(2+)-mediated inhibition of neurotrophins can induce cell death. The present study investigates the neurotoxic mechanism(s) of this metal ion by assessing zincs selectivity in altering the neurotrophin BDNF, but not the neural cytokine CNTF, with respect to motor neuron survival. Embryonic day 15 rat spinal motor neuron cultures were maintained in either BDNF or CNTF. Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining showed that exposure to 100microM Zn(2+) significantly increased the number of pro-apoptotic neurons in cultures maintained with BDNF, while these conditions had no effect on cultures maintained with CNTF. We also demonstrate that BDNF protomer cross-linking efficiency and TrkB receptor cross-linking to BDNF are significantly inhibited by Zn(2+), suggesting that a Zn(2+)-induced change in BDNF conformation inhibits receptor-binding activity. This study reveals a mechanism by which zinc toxicity is mediated via a selective loss in neurotrophin activity resulting in motor neuron death.Amyotrophic lateral sclerosis is a debilitating disease that results from the deterioration and loss of motor neurons. The neurotoxic potential of Zn2+, both in vitro and in vivo, has been well established; however, the mechanism(s) of zincs toxicity remain unclear. Our laboratory has demonstrated that Zn2+-mediated inhibition of neurotrophins can induce cell death. The present study investigates the neurotoxic mechanism(s) of this metal ion by assessing zincs selectivity in altering the neurotrophin BDNF, but not the neural cytokine CNTF, with respect to motor neuron survival. Embryonic day 15 rat spinal motor neuron cultures were maintained in either BDNF or CNTF. Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining showed that exposure to 100µM Zn2+ significantly increased the number of pro-apoptotic neurons in cultures maintained with BDNF, while these conditions had no effect on cultures maintained with CNTF. We also demonstrate that BDNF protomer cross-linking efficiency and TrkB receptor cross-linking to BDNF are significantly inhibited by Zn2+, suggesting that a Zn2+-induced change in BDNF conformation inhibits receptor-binding activity. This study reveals a mechanism by which zinc toxicity is mediated via a selective loss in neurotrophin activity resulting in motor neuron death.

Zinc-metallothionein: a potential mediator of antioxidant defence mechanisms in response to dopamine-induced stress.

Clinically, Parkinsons disease (PD) is a neurodegenerative disorder characterized by the development of tremors and rigidity that is found primarily in patients over the age of 50. At the cellular level, it is clear that the pathology of PD results from the progressive loss of dopaminergic neurons in the substantia nigra. Several lines of evidence have implicated oxidative stress as a contributing factor to the depletion of dopaminergic neurons in PD. Under conditions of oxidative stress, the neurotransmitter dopamine can be oxidized to form neurotoxic quinone and semiquinone products. While dopaquinones are known to be extremely reactive towards sulfhydryl groups of many cellular substrates, mounting evidence suggests that their toxic effects can be quenched by intrinsic antioxidant mechanisms (e.g., glutathione). However, to respond appropriately to differing levels of oxidative stress, cells require a mechanism to regulate an appropriate response. This manuscript proposes metallothionein as a major cellular sensor of oxidized dopamine stress and metallothionein-mediated Zn2+ mobilization as an effecter signal that is used by the cell to manage oxidized dopamine as an intrinsic neurotoxin.

Evaluating the Effectiveness of First-Time Methadone Maintenance Therapy Across Northern, Rural, and Urban Regions of Ontario, Canada

Objectives:Our objective was to determine the impact that a patients geographic status has on the efficacy of first-time methadone maintenance therapy (MMT) retention. Methods:We conducted an observational cohort study using administrative health care databases for patients who commenced methadone therapy between 2003 and 2012. Patients were stratified on the basis of their location of residence into 1 of 4 groups—Southern Urban, Southern Rural, Northern Urban, or Northern Rural. The primary outcome was continuous retention in treatment, defined as 1 year of uninterrupted therapy on the basis of prescription refill data. Mortality was measured as a secondary outcome. Results:We identified 17,211 patients initiating first-time MMT during this 10-year period. Nearly half of patients initiating therapy in northern regions completed 1 year of treatment (48.9%; N = 258 and 47.0%; N = 761 in Northern Rural and Urban regions, respectively), whereas lower rates of 40.6% (N = 410) and 39.3% (N = 5,518) occurred in Southern Rural and Urban regions, respectively. Patients residing in Northern Rural and Northern Urban regions were 31% (adjusted odds ratio = 1.31; 95% confidence interval [CI], 1.09%–1.58%] and 14% (adjusted odds ratio = 1.14; 95% CI, 1.02%–1.27%] more likely to be retained in treatment compared with those residing in Southern Urban regions. There was no significant difference in treatment retention between those residing in Southern Rural and Southern Urban regions. A mortality rate of 3% was observed within 1 year of patients initiating treatment, with patients in the Southern Rural region having the highest rate (4.85%). Conclusions:Our study identified regional differences in retention rates and mortality of first-time MMT. These findings may relate to geographic isolation and limited methadone program availability experienced in northern regions. We interpret the data to suggest that patients who have reduced access to treatment experience higher retention rates when they are able to access therapy.

Identification of novel pyrazoloquinazolinecarboxilate analogues to inhibit nerve growth factor in vitro.

Nerve growth factor (NGF) is known to regulate the development and survival of select populations of neurons via its binding/activation of the TrkA and p75(NTR) receptors. However, in some physiological circumstances NGF dysregulation can result in debilitating pathologies, including diabetic neuropathies, interstitial cystitis and fibromyalgia. Thus, the identification of small molecules which inhibit NGF signalling have significant therapeutic potential. PD 90780, Ro 08-2750, and ALE 0540 are small molecules that have been reported to bind and inhibit NGF activity. Importantly, the docking site of these compounds is hypothesised to occur at the loop I/IV cleft of NGF-a region which is required for efficient and selective binding of this neurotrophin to its receptor(s). Molecular modelling predicts a number of previously reported NGF antagonists (PD 90780, ALE 0540, and Ro 08-2750) share conserved molecular features, and these drug-like small molecules have the ability to bind and modify the molecular topology of NGF. In order to understand the putative mechanism of binding, we synthesised a pyrazoloquinazolinecarboxilate analogue series and tested each compound in an NGF-dependent PC12 cell differentiation assay. In vitro data confirms that the pyrazoloquinazolinecarboxilate analogues functionally inhibit NGFs effects on PC12 cell differentiation. The results of this study provide evidence to refine the docking mode of pyrazoloquinazolinecarboxilate based compounds for the purposes of inhibiting NGF in vitro. In addition, we identified series analogue PQC 083 (IC50=7.0 µM; CI=5.4-10.1 µM) which displays markedly higher potency than previously described NGF antagonists.

Covalent arylation of metallothionein by oxidized dopamine products: A possible mechanism for zinc-mediated enhancement of dopaminergic neuron survival

Metallothioneins are a group of low molecular weight proteins which can be induced upon exposure to metal ions, including Zn2+. These cysteine-rich proteins are believed to have anti-oxidant-like properties due to their ability to scavenge free radicals with their multiple sulfhy-dryl groups. Dopamine is a neurotransmitter that can form toxic quinone and semi-quinone products in an oxidative environment. While Zn2+ is known to be toxic to some neuron subtypes, here we report a beneficial effect of Zn2+ on dopaminergic neurons and identify a mechanism through which metallothionein may scavenge toxic dopamine oxidation products. Cultured embryonic neurons were treated with Zn2+, and the number of dopaminergic neurons surviving after two or three weeks in culture was determined. We demonstrate that under these conditions metallothionein is upregulated and is able to form covalent arylation products with dopamine and 6-hydroxydopamine bothin vitro and in culture. These experiments suggest that Zn2+ enhances the survival of dopaminergic neurons, and we propose that as a mechanism, upregulated metallothioneins form covalent adducts with both dopamine and 6-hydroxydopamine, resulting in the observed neuroprotective effect of Zn2+ on these cells. As Zn2+ homeostasis and modulation of metallothionein expression are often markers of neurodegeneration, these studies may have significant implications for understanding the underlying basis of degenerative diseases involving dopaminergic neurons, including Parkinson’s disease.

Up-regulation of integrin expression in lung adenocarcinoma cells caused by bacterial infection: in vitro study.

Integrins are a large family of adhesion receptors that are known to be key signaling molecules in both physiological and pathological processes. Previous studies have demonstrated that the expression of integrin receptors in the pulmonary epithelium can change under various pathological conditions, such as injury, inflammation, or malignant transformation. We hypothesize that integrin expression can be altered by stimulation of lung epithelial cells with an opportunistic bacterial pathogen Pseudomonas aeruginosa. Using the A549 adenocarcinoma cell line that expressed a low level of several integrin subunits we have demonstrated that P. aeruginosa infection in vitro caused a rapid up-regulation of α5, αv, β1, and β4 integrins at both the mRNA and protein level. Neither heat-killed P. aeruginosa strain PAK nor its live isogenic mutants lacking pili or lipopolysaccharide (LPS) core oligosaccharide showed any effect on integrin expression in A549 cells as compared to the use of the wild-type PAK strain. These results establish that up-regulation of integrin expression is dependent on the internalization of live bacteria possessing intact pili and LPS. Gene silencing of integrin-linked kinase in A549 cells caused a significant decrease in the release of proinflammatory cytokines in response to P. aeruginosa stimulation. Although further studies are warranted towards understanding the precise role of integrin receptors in prominent inflammation caused by P. aeruginosa, our findings suggest a possibility of using specific integrin inhibitors for therapy of pulmonary inflammatory conditions caused by pathogenic micro-organisms.

The State of Opioid Agonist Therapy in Canada 20 Years after Federal Oversight

Opioid agonist therapy was introduced in Canada in 1959 with the use of methadone for the treatment of opioid dependence. The regulation of methadone was the responsibility of Health Canada until 1995, when oversight was transferred to the provincial health systems. During the more than 20 years since the federal health authority transferred oversight of methadone to the provincial level, methadone programming has evolved differently in every province. The landscape of opioid dependence treatment is varied across the country, with generally increasing treatment capacity in all provinces and dramatic increases in some. Each province has an independent methadone program with differing policies, contingency management strategies, laboratory monitoring policies, and delivery methods. Treatment options have increased, with buprenorphine- and heroin-assisted treatment becoming available to limited degrees. Despite this, access remains a challenge in many parts of the country (particularly rural and remote areas) because the demand for treatment has increased even more rapidly than the capacity. Although treatment access remains a priority in many jurisdictions, there is also a need to attend to treatment quality as treatment access expands, including integration with addiction counselling, primary care, and mental health care. As well, coordinated monitoring and reporting of treatment need, quality, and delivery are required; implementing a national policy to promote planning would have tremendous value.

The effectiveness of telemedicine-delivered opioid agonist therapy in a supervised clinical setting

OBJECTIVE Opioid use disorder has been declared a public health crisis across North America and opioid agonist therapy (OAT) is the standard of care for these patients. Despite the increasing adoption of telemedicine as a delivery method for OAT, its effectiveness has not yet been evaluated against traditional in-person treatment. This study compared treatment outcomes for in-person versus telemedicine-delivered OAT. METHODS We conducted a non-randomized cohort comparison study using an administrative database for patients who commenced OAT between 2011 and 2012 across 58 clinic sites in the province of Ontario, Canada. Patients were stratified by primary treatment modality as being: in-person (<25% appointments by telemedicine), mixed (25-75% by telemedicine), or via telemedicine (>75% appointments by telemedicine). The primary outcome was continuous retention in treatment as defined by one year of uninterrupted therapy, based on pharmacy dosing records. RESULTS A total of 3733 OAT initiating patients were identified. Patients treated via telemedicine were more likely to be retained in therapy than patients treated in-person (n=1590; aOR=1.27; 95% CI 1.14-1.41; p<0.001). Telemedicine patients demonstrated a retention rate of 50% at one year whereas in-person patients were retained at a rate of 39%. The mixed group also had higher likelihood of retention than the in-person group (n=418; aOR=1.26; 95% CI 1.08-1.47; p=0.001) and had a retention rate of 47% at one year. CONCLUSION Telemedicine may be an effective alternative to delivering in person OAT, and it has the potential to expand access to care in rural, remote, and urban regions.