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Background: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. Methods: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. Results: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1β, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. Conclusions: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

Multiple sclerosis (MS) is a chronic, inflammatory demyelinating disease of the CNS. In addition to motor, sensory and visual deficits, MS is also characterized by hippocampal demyelination and memory impairment. We recently demonstrated that a recombinant human-derived monoclonal IgM antibody, which is designated rHIgM22 and currently in clinical development for people with MS, accelerates remyelination of the corpus callosum in the brains of cuprizone-treated mice. Here, we investigated the effects of rHIgM22 in the hippocampus and on hippocampal-dependent learning and memory in the same mouse model of cuprizone-induced demyelination and spontaneous remyelination. The degree of hippocampal myelination of cuprizone-fed mice treated with a single dose of rHIgM22 (10 mg/kg of body weight) was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. Four or five mice per treatment group were examined for each time point. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated hippocampus. Using two additional cohorts of mice and eight animals per treatment group for each cohort, we also demonstrate that the enhancing effects of rHIgM22 on hippocampal remyelination were accompanied by improved performance in the Morris water maze and amelioration of the memory deficits induced by cuprizone. These results further confirm the remyelination-promoting capabilities of rHIgM22 and support additional investigation of its therapeutic potential in MS.

GGF2 is a recombinant human neuregulin-1β in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.

Failure of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is thought to be a major cause of the limited ability of the central nervous system to repair plaques of immune-mediated demyelination in multiple sclerosis (MS). Current therapies for MS aim to lessen the immune response in order to reduce the frequency and severity of attacks, but these existing therapies do not target remyelination or stimulate repair of the damaged tissue. Thus, the promotion of OPC differentiation and remyelination is potentially an important therapeutic goal. Previous studies have shown that a recombinant human-derived monoclonal IgM antibody, designated rHIgM22, promotes remyelination, particularly of the spinal cord in rodent models of demyelination. Here, we examined the effects of rHIgM22 in remyelination in the brain using the mouse model of cuprizone-induced demyelination, which is characterized by spontaneous remyelination. The myelination state of the corpus callosum of cuprizone-fed mice treated with rHIgM22 was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated corpus callosum. The remyelination-enhancing effects of rHIgM22 were found across different, anatomically distinct regions of the corpus callosum, and followed a spatiotemporal pattern that was similar to that of the spontaneous remyelination process. These enhancing effects were also accompanied by increased differentiation of OPCs into mature oligodendrocytes. Our data indicate strong remyelination-promoting capabilities of rHIgM22 and further support its therapeutic potential in MS.

Neuregulins are important growth factors involved in cardiac development and response to stress. Certain isoforms and fragments of neuregulin have been found to be cardioprotective. The effects of a full-length neuregulin-1β isoform, glial growth factor 2 (GGF2; USAN/INN; also called cimaglermin) were investigated in vitro. Various dosing regimens were then evaluated for their effects on left ventricular (LV) function in rats with surgically-induced myocardial infarction. In vitro, GGF2 bound with high affinity to erythroblastic leukemia viral oncogene (ErbB) 4 receptors, potently promoted Akt phosphorylation, as well as reduced cell death following doxorubicin exposure in HL1 cells. Daily GGF2 treatment beginning 7-14 days after left anterior descending coronary artery ligation produced improvements in LV ejection fraction and other measures of LV function and morphology. The improvements in LV function (e.g. 10% point increase in absolute LV ejection fraction) with GGF2 were dose-dependent. LV performance was substantially improved when GGF2 treatment was delivered infrequently, despite a serum half-life of less than 2h and could be maintained for more than 10 months with treatment once weekly or once every 2 weeks. These studies confirm previous findings that GGF2 may improve contractile performance in the failing rat heart and that infrequent exposure to GGF2 may improve LV function and impact remodeling in the failing myocardium. GGF2 is now being developed for the treatment of heart failure in humans.

Background. 4-Aminopyridine (4-AP) is a Food and Drug Administration–approved drug to improve motor function in people with multiple sclerosis. Preliminary results suggest the drug may act on intact neural circuits and not just on demyelinated ones. Objective. To determine if 4-AP at clinically relevant levels alters the excitability of intact motor circuits. Methods. In anesthetized rats, electrodes were placed over motor cortex and the dorsal cervical spinal cord for electrical stimulation, and electromyogram electrodes were inserted into biceps muscle to measure responses. The motor responses to brain and spinal cord stimulation were measured before and for 5 hours after 4-AP administration both in uninjured rats and rats with a cut lesion of the pyramidal tract. Blood was collected at the same time as electrophysiology to determine drug plasma concentration with a goal of 20 to 100 ng/mL. Results. We first determined that a bolus infusion of 0.32 mg/kg 4-AP was optimal: it produced on average 61.5 ± 1.8 ng/mL over the 5 hours after infusion. This dose of 4-AP increased responses to spinal cord stimulation by 1.3-fold in uninjured rats and 3-fold in rats with pyramidal tract lesion. Responses to cortical stimulation also increased by 2-fold in uninjured rats and up to 4-fold in the injured. Conclusion. Clinically relevant levels of 4-AP strongly augment physiological responses in intact circuits, an effect that was more robust after partial injury, demonstrating its broad potential in treating central nervous system injuries.

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb- and hindlimb-placing scores (proprioceptive behavioral tests), body-swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin-mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose-frequency study used the dose-ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose- and frequency-dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth-associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment.

Cimaglermin alfa (GGF2) is a recombinant human protein growth factor in development for heart failure. Phase I trials were suspended when two cimaglermin alfa-treated subjects experienced concomitant elevations in serum aminotransferases and total bilirubin, meeting current US Food and Drug Administration criteria for a serious liver safety signal (i.e., "Hy's Law"). We assayed mechanistic biomarkers in archived clinical trial serum samples which confirmed the hepatic origin of the aminotransferase elevations in these two subjects and identified apoptosis as the major mode of hepatocyte death. Using a mathematical model of drug-induced liver injury (DILIsym) and a simulated population, we estimated that the maximum hepatocyte loss in these two subjects was

Background and Purpose— Stroke survivors often have permanent deficits that are only partially addressed by physical therapy. This study evaluated the effects of dalfampridine, a potassium channel blocker, on persistent sensorimotor deficits in rats with treatment initiated 4 or 8 weeks after stroke. Methods— Rats underwent permanent middle cerebral artery occlusion. Sensorimotor function was measured using limb-placing and body-swing symmetry tests, which normally show a partial recovery from initial deficits that plateaus ≈4 weeks after permanent middle cerebral artery occlusion. Dalfampridine was administered starting at 4 or 8 weeks after permanent middle cerebral artery occlusion in 2 blinded, vehicle-controlled studies. Plasma samples were collected and brain tissue was processed for histologic assessment. Results— Dalfampridine treatment (0.5–2.0 mg/kg) improved forelimb- and hindlimb-placing responses and body-swing symmetry in a reversible and dose-dependent manner. Plasma dalfampridine concentrations correlated with dose. Brain infarct volumes showed no differences between treatment groups. Conclusions— Dalfampridine improves sensorimotor function in the rat permanent middle cerebral artery occlusion model. Dalfampridine extended-release tablets (prolonged release fampridine outside the United States) are used to improve walking in patients with multiple sclerosis, and these preclinical data provide a strong rationale for examining the potential of dalfampridine to treat chronic stable deficits in stroke patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT01605825

Background Dalfampridine (4-aminopyridine; 4-AP) is a potassium channel blocker that has been available in the United States as a treatment to improve walking in patients with multiple sclerosis. 4-AP is well-characterized in vitro with regard to inhibition of neuronal potassium channels, but the potential contribution of its metabolites to clinical activity has not been determined. This study evaluated the concentration–response of 4-AP and its two primary metabolites, 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate, for inhibition of the potassium channels Kv 1.1, Kv 1.2, and Kv 1.4, which are considered candidates for mediating effects of 4-AP on action potential conduction because of their presence in axonal membranes. Methods Stable transfection of cDNA for Kv 1.1, Kv 1.2, and Kv 1.4 was performed into HEK293 cells, and colonies of cells containing each channel were selected and maintained under appropriate cell culture conditions. Electrophysiological measurements were performed using a patch-clamp technique in at least three cells for each concentration (50, 500, 5000, and 50,000 μM) of 4-AP and the two metabolites, with each cell acting as its own control. Concentration–response curves were constructed for 4-AP and each metabolite. Data were analyzed using nonlinear least-squares fit, and concentrations inhibiting the channels by 50% (IC50) were estimated. Results 4-AP induced similar concentration-dependent inhibition profiles of all three potassium channels, resulting in a narrow range of IC50 values across channels (242 µM to 399 µM). Across the three channels, the IC50 values of 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate were 1–2 orders of magnitude higher (less potent) than those of 4-AP. Conclusions 3-Hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate demonstrated low in vitro potency for Kv 1.1, Kv 1.2, and Kv 1.4 inhibition, suggesting that these metabolites are unlikely to contribute to the positive pharmacodynamic effects of 4-AP. A limitation of this study is that while the metabolites were substantially less active at these representative potassium channels in vitro, the untested possibility exists that they may be active at one or more of the many other channel types that occur in vivo.

Magnesium (Mg2+) homeostasis is impaired following spinal cord injury (SCI) and the loss of extracellular Mg2+ contributes to secondary injury by various mechanisms, including glutamate neurotoxicity. The neuroprotective effects of high dose Mg2+ supplementation have been reported in many animal models. Recent studies found that lower Mg2+ doses also improved neurologic outcomes when Mg2+ was formulated with polyethylene glycol (PEG), suggesting that a PEG/ Mg2+ formulation might increase Mg2+ delivery to the injured spinal cord, compared with that of MgSO4 alone. Here, we assessed spinal extracellular Mg2+ and glutamate levels following SCI in rats using microdialysis. Basal levels of extracellular Mg2+ (∼0.5 mM) were significantly reduced to 0.15 mM in the core and 0.12 mM in the rostral peri-lesion area after SCI. A single intravenous infusion of saline or of MgSO4 at 192 μmoL/kg did not significantly change extracellular Mg2+ concentrations. However, a single infusion of AC105 (a MgCl2 in PEG...

Background Peripheral nerve injury (PNI) can result in neurodegenerative changes leading to motor, sensory and autonomic dysfunction. Injury to the rat sciatic nerve is used to model pathophysiologic processes following PNI and assess the efficacy of therapeutic interventions. Frequently, temporal changes in the sciatic functional index (SFI), a measure of sensorimotor integration are measured in rats to assess functional recovery following sciatic nerve injury. However, multiple rat strains and behavioral endpoints have been employed to investigate pathophysiology of PNI and impact of therapeutic intervention on recovery, raising the possibility that rat strain may influence the outcome of such studies. New method The temporal course of recovery from sham, sciatic nerve crush or transection injury was assessed using SFI determined by two methods (footprint and DigiGait), and proprioceptive hind limb placement (a measure of proprioceptive integrity) of the sciatic nerve innervation, in male Sprague Dawley, Lewis, Fischer, Wistar and Long Evans rats. Results The SFI profile, as assessed by both inked footprint analysis and DigiGait, following sciatic nerve injury was remarkably conserved across strains. Dramatic strain-related differences were observed in the latency to place the crush- or transection-injured hind limb following proprioceptive hind limb stimulation. Comparison with existing method The novelty of this study is the parallel comparison of multiple strains using existing and novel tests. Conclusion These results suggest that some sensorimotor function tests may be sensitive to the choice of strain, as evidenced by the differences between SFI and proprioceptive function outcomes.

In this communication we show that the same principle that underlies the use of the isobolograph for assessing agonist interactions also leads to a method for analyzing the opposing effects of a single agonist. This is the principle of dose equivalence whose application is illustrated here and applied to the endothelium-dependent relaxing component of two putative vasoconstrictor peptides. These studies, employing angiotensin II and endothelin-1, were conducted with isolated preparations of rat aorta that were measured for agonist-induced isometric tension development in both endothelial-denuded and -intact vessels. The dose–effect relation of the relaxing component of each agonist, which should not be calculated from simple effect subtraction, was derived by the method described here.

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb‐ and hindlimb‐placing scores (proprioceptive behavioral tests), body‐swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin‐mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose‐frequency study used the dose‐ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose‐ and frequency‐dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth‐associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Background Neuregulin‐1β ( NRG ‐1β) is a growth factor critical for cardiac development and repair with therapeutic potential for heart failure. We previously showed that the glial growth factor 2 ( GGF 2) isoform of NRG ‐1β improves cardiac function in rodents after myocardial infarction ( MI ), but its efficacy in a large animal model of cardiac injury has not been examined. We therefore sought to examine the effects of GGF 2 on ventricular remodeling, cardiac function, and global transcription in post‐ MI swine, as well as potential mechanisms for anti‐remodeling effects. Methods and Results MI was induced in anesthetized swine (n=23) by intracoronary balloon occlusion. At 1 week post‐ MI , survivors (n=13) received GGF 2 treatment (intravenous, biweekly for 4 weeks; n=8) or were untreated (n=5). At 5 weeks post‐ MI , fractional shortening was higher (32.8% versus 25.3%, P =0.019), and left ventricular ( LV ) end‐diastolic dimension lower (4.5 versus 5.3 cm, P =0.003) in GGF 2‐treated animals. Treatment altered expression of 528 genes, as measured by microarrays, including collagens, basal lamina components, and matricellular proteins. GGF 2‐treated pigs exhibited improvements in LV cardiomyocyte mitochondria and intercalated disk structures and showed less fibrosis, altered matrix structure, and fewer myofibroblasts (myoFbs), based on trichrome staining, electron microscopy, and immunostaining. In vitro experiments with isolated murine and rat cardiac fibroblasts demonstrate that NRG ‐1β reduces myoFbs, and suppresses TGF β‐induced phospho‐ SMAD 3 as well as α SMA expression. Conclusions These results suggest that GGF 2/ NRG ‐1β prevents adverse remodeling after injury in part via anti‐fibrotic effects in the heart.

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5′ untranslated region, the coding region, and the 3′ untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.

The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.

Background Dalfampridine (4-aminopyridine; 4-AP) is a potassium channel blocker available in the United States to improve walking in patients with multiple sclerosis as demonstrated by an increase in walking speed. Its pharmacokinetics have been evaluated in human studies but its metabolites are not well characterized. This study characterizes the metabolic profile of dalfampridine in two animal species that were used to support nonclinical toxicology evaluation. Methods Metabolic profiling of single oral 14C-4-AP doses was performed in 12 adult male Sprague–Dawley rats. Similarly, metabolic profiling was performed in beagle dogs in two studies that administered 14C-4-AP by gastric intubation; the first study included six animals (three males, three females), and the second study included two animals (one male, one female). Blood and urine samples were evaluated using high performance liquid chromatography, thin layer chromatography, and radioanalysis (liquid scintillation counting), with further identification of components by gas chromatography/mass spectrometry. Results Five radioactive components, M1–M5, were detected in rat plasma, although most of the radioactivity corresponded with unchanged 4-AP. Based on Rf values, M1 and M2 coseparated with reference standards of 3-hydroxy-4-AP and 4-AP, respectively. Additionally, components M1, M2, and M3 coseparated with the same components isolated from the urine of a dog dosed with 14C-4-AP and identified as 3-hydroxy-4-AP, 4-AP, and 3-hydroxy-4-AP sulfate, respectively; M4 and M5 could not be identified because of low concentrations. In dogs, most of the radioactivity was excreted within the first 24 hours as unchanged compound. Conclusions Following oral dosing, 4-AP was rapidly absorbed in rats and dogs, with rapid excretion and almost complete urinary recovery in dogs. The primary metabolites in both animal models were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate. Systemic clearance not accounted for by renal excretion of 4-AP may occur by liver metabolism by hydroxylation of 4-AP to 3-hydroxy-4-AP followed by sulfate conjugation to 3-hydroxy-4-AP sulfate.

Kainic acid was microinjected or microdialyzed into the rostral medial aspect of the fastigial nucleus to determine its effect on mean arterial pressure and heart rate. This was carried out in both the awake and the anesthetized (α-chloralose) rat. In awake animals, kainic acid elicited an initial phasic pressor response which was followed by a long-term elevation of mean arterial pressure that lasted for the duration of the experiment (2 h). Rats anesthetized with α-chloralose exhibited only a tonic depressor response. This converted to a pressor response as the rats began to emerge from anesthesia after 2 h. Both the awake and the anesthetized rats exhibited regular phasic changes in mean arterial pressure that was superimposed on the longer term changes in the mean arterial pressure. Similar results were obtained in both the microinjected and the microdialyzed animals. Thus, stimulation of the intrinsic fastigial neurons by kainic acid evokes an elevation of the mean arterial pressure in the awake rat. This is manifested as a decrease in pressure in the anesthetized animal. Thus, stimulation of the cardiovascular region of the fastigial nucleus can increase or decrease mean arterial pressure. It is possible that the direction of the change in mean arterial pressure is dependent on the level of afferent or intrinsic fastigial neural activity.

Neuregulin-1β is essential for fetal cardiac development and adult cardiac function. Previous reports indicate that neuregulins improve left ventricular function in heart failure models, however the duration of the functional improvements with early or late initiation of neuregulin treatment has not been characterized. The present studies examine the effects of early and delayed initiation of intravenous GGF2 treatment on left ventricular (LV) function in rats with myocardial infarction (MI). Rats underwent surgically-induced MI by left anterior coronary artery ligation. Treatment with vehicle or GGF2 (2.6 mg/kg) was initiated at 2 or 16 w post-MI and continued once or twice weekly or once every two weeks for the in-life duration of the study (approximately 40 weeks). LV function was assessed echocardiographically up to once weekly for the duration of the study. Early and delayed initiation of GGF2 treatment caused sustained and significant improvement (p < 0.05) in both ejection fraction (EF) and fractional shortening (FS) in all regimens tested. The greatest improvements were seen with the once weekly dosing paradigm after early initiation (average EF (%) at 40 weeks post initiation of dosing: vehicle = 44.4 ± 6.0, n = 8 rats, vs. GGF2 = 64.7±6.1. n = 9 rats) and twice weekly dosing paradigm after delayed initiation (average EF (%) at 4 weeks post initiation of dosing: vehicle = 34.18±1.6, n = 7 rats, vs. GGF2 = 50.69±4.68, n = 7 rats). In addition, LV function improved when rats were re-challenged with GGF2 following an extended wash out period. This observation indicates potential efficacy for treatment paradigms that utilize intermittent dosing. These findings suggest that GGF2 produces sustained improvement in LV function after early or delayed initiation of treatment following MI in rats.

Neuregulins are a family of growth factors essential for normal cardiac and nervous system development. The EGF-like domain of neuregulins contains the active site which binds and activates signaling cascades through ErbB receptors. A neuregulin-1 gene EGF-like fragment demonstrated neuroprotection in the transient middle cerebral artery occlusion (MCAO) stroke model and drastically reduced infarct volume (Xu et al., 2004). Here we use a permanent MCAO rat model to initially compare two products of the neuregulin-1 gene and also assess levels of recovery with acute versus delayed time to treatment. In the initial study full-length glial growth factor 2 (GGF2) and an EGF-like domain fragment were compared with acute intravenous delivery. In a second study GGF2 only was delivered starting at 24h, 3 days or 7 days after permanent ischemia was induced. In both studies daily intravenous administration continued for 10 days. Recovery of neurological function was assessed using limb placing and body swing tests. GGF2 had similar functional improvements compared to the EGF-like domain fragment at equimolar doses, and a higher dose of GGF2 demonstrated more robust functional improvements compared to a lower dose. GGF2 improved sensorimotor recovery with all treatment paradigms, even enhancing recovery of function with a delay of 7 days to treatment. Histological assessments did not show any associated reduction in infarct volume at either 48 h or 21 days post-ischemic event. Neurorestorative effects of this kind are of great potential clinical importance, given the difficulty of delivering neuroprotective therapies within a short time after an ischemic event in human patients. If confirmed by additional work including additional data on mechanism(s) of improved outcome with verification in other stroke models, one can make a compelling case to bring GGF2 to clinical trials as a neurorestorative approach to improving outcome following stroke injury.

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 μg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-μg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P.05, ANOVA) or control infusion (P> .05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P

Repifermin (truncated, recombinant human keratinocyte growth factor-2, KGF-2) was evaluated in cynomolgus monkeys and healthy humans during a phase 1 trial. Monkeys received vehicle or repifermin at 20, 75, or 200 μg/kg IV or 750 μg/kg subcutaneous (SC) daily for 29 days. Clinical observations were made during the entire dosing period. Gross and microscopic changes were assessed at necropsy. Pharmacokinetic parameters and immunogenicity were evaluated in these monkeys and in humans, following a single or 7 daily IV bolus injections of 1, 5, 25, or 50 μg/kg repifermin. In monkeys, repifermin was well tolerated, and histologic evaluation demonstrated dose-dependent, reversible thickening of the mucosa throughout the alimentary tract, except for the stomach. In the alimentary tract tissues, nonepithelial tissues were not affected, indicating a specificity of repifermin for epithelial cells. Pharmacokinetics in both monkeys and humans were dose proportional, showed lack of drug accumulation with repeated daily dosing, and were characterized by high volumes of distribution and clearance rates, indicating substantial tissue binding and metabolism. Repifermin was not markedly immunogenic following multiple daily IV injections in either species. Serum repifermin concentrations in humans were comparable to those attained in monkeys that produced significant pharmacological effects on epithelial cells in the alimentary tract. These findings provide additional support for the ongoing clinical development of repifermin for diseases involving epithelial injury.

Vascular endothelial growth factor (VEGF) is a growth factor that contributes to the angiogenesis of developing tumors. To interfere with the action of VEGF, a nuclease-stabilized ribozyme, ANGIOZYME, has been developed against VEGF receptor subtype Flt-1 mRNA. To determine which routes of administration would be useful for systemic delivery of this ribozyme, a dose of 30 mg/kg [32P]ANGIOZYME was administered as an i.v., i.p., or s.c. bolus. Concentrations of ANGIOZYME in plasma, femur, kidney, liver, and lung were examined. ANGIOZYME was well absorbed after i.p. (90%) or s.c. administration (77%), with peak plasma concentrations occurring 30 minutes after dosing. Total body clearance after a single dose of 30 mg/kg ANGIOZYME was 20 ml/min/kg, and the elimination half-life was 33 minutes. The apparent volume of distribution at steady-state ranged from 0.5 to 1.3 L/kg. ANGIOZYME was detected in the four tissues examined through the 3 hour sampling period after i.v. or i.p. administration. After s.c. administration, ANGIOZYME was detected in femur, kidney, and lung but not in the liver. The highest concentrations of ANGIOZYME were found in kidney and femur with all three routes. Because of the rapid and extensive absorption after extravascular injections, either i.p. or s.c. administration could be considered for use in pharmacodynamic studies examining the effects of ANGIOZYME or other ribozymes with similar chemical modifications.

The effects of dopaminergic stimulation of the subthalamic nucleus (STh) on motor behavior were examined in conscious rats. Unilateral infusion of apomorphine (0.1 to 3.2 micrograms) into the STh induced a dose-dependent increase in abnormal, nondirected orofacial movements without altering turning, sniffing, grooming, or rearing behaviors. Orofacial movements elicited by local infusion of apomorphine (1.0 microgram) into the STh were blocked by peripheral administration of the D1 antagonist, SCH 23390 (0.1 mg/kg, sc), but not by the D2 antagonists haloperidol (1.0 mg/kg, sc) or sulpiride (50 mg/kg, sc). Furthermore, coinfusion of SCH 23390 (1.0 microgram), but not sulpiride (5.0 micrograms), with apomorphine (1.0 microgram) into the STh blocked oral dyskinesia. Oral movements could not be reelicited by an infusion of apomorphine into the STh after a kainic acid lesion of the STh. In addition, infusion of apomorphine (1.0 microgram) into sites proximal to but deliberately outside of the STh failed to elicit nondirected oral movements above baseline levels. The results indicate that stimulation of D1 dopaminergic receptors within the STh induces abnormal orofacial movements. This highlights the importance of the dopaminergic input to the STh in the regulation of motor function and suggests that D1 receptor antagonists could prove useful in the treatment of orofacial dyskinesia in humans.

A method for restraining awake rats using head implant immobilization is described. In order to reduce stress to the individual animal, rats were restrained side by side in pairs during the adaptation and experimental periods. This technique was used in studies of central regulation of cardiovascular function. In particular, microdialysis probes were placed stereotaxically in deep nuclei of the cerebellum while mean arterial pressure and heart rate were monitored in the awake rat. After initial habituation, normal levels of heart rate and blood pressure obtained during restraint indicated that this is essentially a nonstressful procedure. This technique could also be used for a variety of other experimental conditions where head movement is not desirable, such as during oculomotor or vestibular experiments.

The object of the present study was to examine the effects of temperature, oxidation, and pH on in vitro relative recovery of catecholamine and indoleamine neurotransmitters and their metabolites using microdialysis probes. Relative recovery of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), homovanillic acid (HVA), and 5-hydroxytryptamine (5HT) increased with temperature from 0 to 46°C. For each compound, the increase in the amount recovered with increasing temperature was different. The stability of norepinephrine and dopamine was not affected at any temperature using deoxygenated calibration standard solutions containing ascorbic acid but was greatly reduced when exposed to ambient air without antioxidant treatment; catecholamine metabolites and the indole compounds were less affected. No change for in vitro relative recovery was observed by varying the pH of the perfusing solution from 6 to 8. Thus, temperature control in probe calibration as well as analyte stability using antioxidant treatment are important in reducing the error when estimating extracellular concentrations of neurotransmitter and metabolites.