Enhancing Pharmacovigilance from the US Experience: Current Practices and Future Opportunities.

This review is intended to present perspectives from the US experience in enhancing pharmacovigilance on current practices and future opportunities. Best practices concepts could be applied worldwide through the presentation of how three pillars of pharmacovigilance: (1) medical and scientific excellence, (2) operational and compliance excellence, and (3) knowledge sharing and experts development in the field could serve as a framework for the establishment of an efficient and successful global pharmacovigilance system.

Hormesis pervasiveness and its potential implications for pharmaceutical research and development.

This mini-review illustrates that hormesis is not only confined to the areas of biochemistry, radiation biology and toxicology, where it is traditionally known, but illustrates, by citing published scientific literature, that it is found across a wide range of biomedical science and clinical medicine such as neuroscience, cardiology and oncology. The use of techniques and technology, including high through-put screening, micro-dosing or phase 0 studies, pharmacometrics and adaptive trial design in the clinic, are proposed to illustrate how acknowledging the potential impact of hormesis throughout different stages of drug discovery and development, including hurdles related to efficacy and safety, could help the pharmaceutical industry address some of its major and frequently mentioned challenges.

Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework.

Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.

Delayed treatment with nicotinamide inhibits brain energy depletion, improves cerebral microperfusion, reduces brain infarct volume, but does not alter neurobehavioral outcome following permanent focal cerebral ischemia in Sprague Dawley rats.

Delayed treatment with nicotinamide (NAm) reduces infarction induced by middle cerebral artery occlusion (MCAO) in rats. This study explored some potential mechanisms by which delayed NAm treatment may confer protection in the brain of Sprague-Dawley rats following permanent MCAO (pMCAO). NAm (500 mg/kg) or vehicle was given 2 h after the onset of pMCAO. Cortical microperfusion, brain and rectal temperature were serially measured. Neurobehavioral examinations were performed at 24 h post-ischemia followed by sacrifice for histologic assessment. Some rats were also sacrificed at 4 h post-ischemia for analyses of ATP, ADP, AMP, and adenosine. Permanent MCAO induced spontaneous hyperthermia and a sharp decrease in cortical microperfusion, ATP concentration, and the sum of adenine nucleotides (p < 0.05). At 4 h post-ischemia, NAm improved ATP recovery, the sum of adenine nucleotides (p < 0.05) and attenuated the ischemia-induced systemic hyperthermia (p < 0.05) without affecting brain temperature or cortical microperfusion. At 24 h, NAm improved cortical microperfusion in the ischemic hemisphere and reduced total infarct volume (p < 0.05), but did not affect behavioral scores. The data suggest that NAm attenuated brain damage following pMCAo initially by improving cerebral bioenergetic metabolism during the sub-acute phase of ischemia, followed by a delayed improvement in microvascular perfusion.

Nicotinamide modulates energy utilization and improves functional recovery from ischemia in the in vitro rabbit retina.

The central nervous system depends critically on a regular supply of oxygen and glucose for the formation of adenosine triphosphate (ATP) and the sustenance of its energy metabolism. Consequently, a significant reduction in the supply of oxygen and glucose to neuronal tissue causes an imbalance between the energy supply and demand, inducing the onset of neuronal ischemia and triggering many metabolic cascades leading to irreversible injury and cell death. Nicotinamide (NAm), an essential precursor to nicotinamide adenine dinucleotide (NAD+), which raises brain ATP levels, may improve cerebral blood flow and is neuroprotective against ischemia-induced injury. We therefore chose to examine the metabolic and electrophysiologic/functional effects of NAm (0.1 mM, 1.0 mM, 10.0 mM) under normal, control, and ischemic conditions, as well as following the early stages of reperfusion ("return-to-control" conditions) using an in vitro rabbit retina model where blood flow effects are excluded. Under nonischemic, control conditions, the protective concentration of NAm (10.0 mM) increased glucose utilization (34%, P < 0.01) and decreased lactate production (44%, P < 0.01), but had no significant effect on electrophysiologic function. After 2 h of ischemia, glucose utilization was significantly decreased (41%, P < 0.01) and lactate production was unaffected by NAm (10 mM). Following 3 h of "reperfusion", NAm (10 mM) significantly improved glucose utilization (217%, P < 0.01), lactate production (40%, P < 0.01), and electrophysiologic function (264%, P < 0.01) relative to controls. Thus, the functional neuroprotective effects of NAm may be independent of blood flow effects, but related, at least in part, to its improvement of tissue glucose utilization and lactate production.

The acoustic startle reflex in Sprague-Dawley rats is altered by permanent middle cerebral artery occlusion.

The startle reflex is an unconditioned, quantifiable behavior used to study sensory modalities. We examined whether the acoustic startle reflex (ASR) was sensitive to lesions induced by focal cerebral ischemia. Sprague-Dawley rats were pre-screened for startle reflex responses 3-6 days prior to surgery and there were no differences in mean startle amplitude across groups. Animals were subjected to permanent middle cerebral artery occlusion (pMCAo) or a sham surgical procedure. Twenty-four hours later rats were evaluated for ASR prior to sacrifice. Infarct volumes were subsequently determined by quantitative image analysis of 2,3,5-triphenyltetrazolium chloride-stained brain sections. Infarct volumes of rats undergoing pMCAO ranged from 0 to 48%. Data were divided into three groups based upon percent infarction: mild (0-20%), moderate (21-35%), and severe (>35%). A within-subject analysis revealed a significant decrease in mean startle amplitude of only severely infarcted rats relative to their pre-surgery startle responses. Furthermore, the lesioned brain areas observed in these animals provide an anatomical basis for these results. Our findings demonstrate that ASR is affected in a model of stroke. Further work is needed to characterize this behavioral test and to determine whether it may have application as a surrogate endpoint for clinical stroke studies.

Acute studies of a new primate model of reversible middle cerebral artery occlusion.

The recent failure of many clinical trials of neuroprotective compounds may be due in part to poor animal models of human stroke. We have developed an endovascular stroke model in nonhuman primates that is compatible with serial magnetic resonance imaging (MRI) monitoring. Using cynomologous macaques (n = 4), a microcatheter was navigated transarterially (under fluoroscopic guidance) from the femoral artery to the middle cerebral artery (MCA). The microcatheter was wedged in a branch of the MCA for 3 hours to cause focal cerebral ischemia, as verified angiographically. During occlusion and/or reperfusion, animals were scanned with MRI, and imaging findings were compared with the stained brain sections. All animals demonstrated small stroke lesions in the expected vascular territory, as seen on diffusion-weighted MRI and confirmed by postmortem examination. Reperfusion after 3 hours was confirmed angiographically (n = 2) and also by MRI (n = 4). The mean initial lesion volume, measured on the postreperfusion MRI scans, was 2.3 +/- 1.3 mL (n = 4). There was good agreement between anatomic location of the lesion on MRI and postmortem histological staining (n = 3). A "minimally invasive" primate model of focal cerebral ischemia was developed that is ideally suited to MRI studies of both acute and chronic stroke. By using serial MRI scans to measure changes in lesion size over time, we will be able to control for variability in lesion size/location. This model should prove useful as a test bed for new stroke therapies, in which noninvasive imaging findings are readily comparable to human stroke.

ACEA 1021: flip or flop?

Inasmuch as glutamate is the main excitatory neurotransmitter in the central nervous system, strategies aimed at counteracting glutamate excitotoxicity, which is at least partially involved in many acute neurologic, chronic neurodegenerative and psychiatric diseases, are challenging. Blockade of the NMDA receptor was identified as one way of achieving selective antagonism and overcoming glutamate neurotoxicity, yet not without liabilities. Glycine site antagonism of the NMDA receptor in 1987 offered a significant advance in blocking this receptor because such drugs were shown to lack most of the side effects, such as memory impairment, ataxia, lack of motor coordination and psychotomimetic effects, which accompanied competitive and non-competitive NMDA receptor antagonists. To date, much has been done to improve the structure-activity relationship (SAR) of compounds resulting in the synthesis of ACEA 1021. It is unclear, however, whether further chemical substitutions will lead to an improved compound. Many studies have been performed with ACEA 1021 and although there are much in vitro and in vivo data to support its neuroprotective effects and improved safety profile, there is very little published information regarding its clinical pharmacology. In order to properly evaluate the true potential for ACEA 1021 in acute and chronic CNS disorders additional longer term safety and efficacy data in humans are needed.

Delayed treatment with 5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione, a glycine site N-methyl-D-aspartate antagonist, protects against permanent middle cerebral artery occlusion in male rats.

The glycine site, N-methyl-D-aspartate antagonist 5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione (ACEA1021) was previously tested only in models of transient stroke with pre-treatment paradigms. We therefore tested whether it would protect in two models of permanent stroke in two rat strains with delayed treatment. Intravenous ACEA1021 reduced cerebral infarction by 62% (15 min treatment delay) and 42% (2 h treatment delay), relative to vehicle-injected rats, when subjected to a modified Tamura and permanent intraluminal filament model of stroke, respectively. In comparison, intravenous nicotinamide (500 mg/kg), which was tested in separate animal cohorts, had no significant effect on infarction. These data show that ACEA1021 protects against permanent focal cerebral ischemia, even with a 2 h post-treatment delay. Characterization of the therapeutic window with longer outcome times including infarction and neurobehavioral endpoints is needed.

Metabolic effects of hypothermia and its neuroprotective effects on the recovery of metabolic and electrophysiological function in the ischemic retina in vitro.

OBJECTIVE: Reduction in energy usage has been investigated as the mechanism by which hypothermia provides protection during ischemia. We describe experiments using hypothermia in the rabbit retina in vitro that show a correlation between hypothermia-induced reductions in energy usage and neuroprotection. METHODS: We examined energy metabolism and electrophysiological function under control/nonischemic conditions during 1 or 2 hours of "ischemia" (induced by decreasing glucose from 6 to 1 mmol/L and oxygen from 95 to 15%) and during 3 to 4 hours of "return-to-control" conditions. Glucose utilization and lactate production were measured as indices of energy metabolism, and light-evoked compound action potentials were monitored to assess functional recovery. RESULTS: Nonischemic retinas subjected to both mild (33 +/- 0.5 degrees C) and moderate (30 +/- 0.5 degrees C) hypothermia exhibited a decrease of 38% in the rate of glucose utilization and lactate production compared with normothermic retinas (36 +/- 0.5 degrees C) (analysis of variance, P < 0.001). In retinas that were made ischemic, mild or moderate hypothermia further reduced the rates of glucose utilization (18 and 39%, respectively) and lactate production (21 and 28%, respectively) (P < 0.001 for glucose, P < 0.01 for lactate). Retinas that had been mildly or moderately hypothermic during ischemia exhibited improved recovery of glucose utilization (65 and 57%, respectively) and lactate production (72 and 74%, respectively) compared with normothermic retinas (18% for glucose and 54% for lactate; repeated-measures analysis of variance, P < 0.001). Recovery of compound action potentials for retinas kept at 36, 33, and 30 degrees C was 15, 36, and 53%, respectively (repeated-measures analysis of variance, P < 0.001). CONCLUSION: Our studies in an avascular neuronal model of ischemia demonstrate that hypothermia protects against ischemic injury. We interpret the smaller reductions in energy generation and usage caused by ischemia when the retinas were hypothermic as evidence that hypothermia had reduced energy requirements more than energy production, and we propose that this at least in part explains its protection.

Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats.

We examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves (EGb 761) in permanent and transient middle cerebral artery (MCA) occlusion models in Sprague-Dawley rats. Forty-four animals were given either EGb 761 (50-200 mg/kg) or vehicle intraperitoneally, 1 hr before permanent MCA occlusion, to evaluate the dose-response effects. An additional 58 animals received EGb 761 (200 mg/kg) or vehicle, 0.5- 4 hr after permanent MCA occlusion, for establishing the therapeutic window. Delayed treatment was also employed in 110 animals treated with either EGb 761 (100-200 mg/kg) or vehicle at 2-3 hr following transient focal cerebral ischemia induced by MCA occlusion for 2 hr. Neurobehavioral scores were determined 22-24 hr after permanent MCA occlusion and either 3 or 7 days after transient MCA occlusion, and brain infarction volumes were measured upon sacrifice. Local cortical blood flow (LCBF) was serially measured in a subset of animals receiving EGb 761 (100-200 mg/kg) or vehicle, 0.5 hr and 2 hr after permanent and transient MCA occlusion, respectively. Relative to vehicle-treated controls, rats pretreated with EGb761 (100 and 200 mg/kg) had significantly reduced infarct volumes, by 36% and 49%, respectively, and improved sensory behavior (P < 0.05). Delayed treatment with EGb 761 also significantly reduced brain infarction, by 20-29% and 31%, when given up to 2 and 3 hr following transient and permanent MCA occlusion, respectively, whereas improved neurobehavioral scores were noted up to 2 hr after the onset of MCA occlusion (P < 0.05). LCBF was significantly improved in the ipsilateral cortex following the EGb 761 treatment, whereas a higher dose showed a more sustained effect. In conclusion, EGb 761 protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hr. This neuroprotection may be at least partially attributed to the beneficial effects of selectively improved LCBF in the area at risk of infarction.

Functional CT perfusion imaging in predicting the extent of cerebral infarction from a 3-hour middle cerebral arterial occlusion in a primate stroke model.

BACKGROUND AND PURPOSE: Our purpose was to determine whether cerebral perfusion functional CT (fCT), performed after endovascular middle cerebral artery (MCA) occlusion, can be used to predict final cerebral infarction extent in a primate model. METHODS: fCT with bolus tracking was performed before and 30 and 150 minutes after 3-hour digital subtraction angiography (DSA)-guided endovascular MCA occlusion in five baboons. Parametric cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) maps were constructed by voxel-by-voxel gamma variate fitting and used to determine lesion sizes. Animals were sacrificed 48 hours after the occlusion, and ex vivo MR imaging was performed. Lesion sizes on fCT and MR images were compared. RESULTS: Hypoperfusion was clearly identified on all images obtained after MCA occlusion. Thirty and 150 minutes after occlusion onset, respectively, mean lesion sizes were 737 mm(2) +/- 33 and 737 mm(2) +/- 44 for CBF, 722 mm(2) +/- 32 and 730 mm(2) +/- 43 for CBV, and 819 mm(2) +/- 14 and 847 mm(2) +/- 11 for MTT. Mean outcome infarct size on MR images was 733 mm(2) +/- 30. Measurements based on CBV and CBF (R(2) = 0.97 and 0.96, P <.001), but not MTT (R(2) = 0.40, P >.5), were highly correlated with final lesion size. CONCLUSION: An endovascular approach to MCA occlusion provides a minimally invasive, reproducible animal model for controlled studies of cerebral ischemia and infarction. Derived cerebral perfusion maps closely predict the 48-hour infarct size after 3-hour MCA occlusion.

Therapeutic window for nicotinamide following transient focal cerebral ischemia.

The therapeutic window with the neuroprotectant nicotinamide (NAm) was tested in a model of stroke. Either 2, 4 or 6 h after the onset of transient (2 h) focal cerebral ischemia, Wistar rats received either saline or NAm (500 mg/kg). Sensory and motor behavioral scores and weight of the animals were obtained before surgery, and 2 h, 3 and 7 days after stroke onset. Cerebral infarct volumes were measured on day 7 after sacrifice. NAm given 4 or 6 h after stroke onset significantly (p<0.05) reduced the cerebral infarction and improved the behavioral scores, respectively, compared to saline-injected animals. There was a non-significant improvement in weight gained by NAm-treated rats at 3 and 7 days following stroke compared to the saline-injected controls.

Delayed treatment with nicotinamide (vitamin B3) reduces the infarct volume following focal cerebral ischemia in spontaneously hypertensive rats, diabetic and non-diabetic Fischer 344 rats.

Since hypertension and/or hyperglycemia are risk factors for stroke, we examined whether the putative neuroprotectant, nicotinamide (NAm), could protect spontaneously hypertensive rats (SHR) or diabetic Fischer 344 rats against focal cerebral ischemia using a model of permanent middle cerebral artery occlusion (MCAo). Intravenous NAm given 2 h after MCAo significantly reduced the infarct volume of SHR (750 mg/kg, 31%, P<0.01) and diabetic (500 mg/kg, 56%, P<0.01) as well as non-diabetic (500 mg/kg, 73%, P<0.01) Fischer 344 rats when compared with saline-injected controls. Thus delayed treatment with NAm protected hypertensive and hyperglycemic rats against a robust model of stroke.