The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia.

In previous work we evaluated an opioid glycopeptide with mixed µ/δ-opioid receptor agonism that was a congener of leu-enkephalin, MMP-2200. The glycopeptide analogue showed penetration of the blood-brain barrier (BBB) after systemic administration to rats, as well as profound central effects in models of Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesia (LID). In the present study, we tested the glycopeptide BBI-11008 with selective δ-opioid receptor agonism, an analogue of deltorphin, a peptide secreted from the skin of frogs (genus Phyllomedusa). We tested BBI-11008 for BBB-penetration after intraperitoneal (i.p.) injection and evaluated effects in LID rats. BBI-11008 (10 mg/kg) demonstrated good CNS-penetrance as shown by microdialysis and mass spectrometric analysis, with peak concentration levels of 150 pM in the striatum. While BBI-11008 at both 10 and 20 mg/kg produced no effect on levodopa-induced limb, axial and oral (LAO) abnormal involuntary movements (AIMs), it reduced the levodopa-induced locomotor AIMs by 50% after systemic injection. The N-methyl-D-aspartate receptor antagonist MK-801 reduced levodopa-induced LAO AIMs, but worsened PD symptoms in this model. Co-administration of MMP-2200 had been shown prior to block the MK-801-induced pro-Parkinsonian activity. Interestingly, BBI-11008 was not able to block the pro-Parkinsonian effect of MK-801 in the LID model, further indicating that a balance of mu- and delta-opioid agonism is required for this modulation. In summary, this study illustrates another example of meaningful BBB-penetration of a glycopeptide analogue of a peptide to achieve a central behavioral effect, providing additional evidence for the glycosylation technique as a method to harness therapeutic potential of peptides.

Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.

Highly-selective µ-opioid receptor antagonism does not block L-DOPA-induced dyskinesia in a rodent model.

OBJECTIVES: Dopamine-replacement utilizing L-DOPA is still the mainstay treatment for Parkinson's disease (PD), but often leads to development of L-DOPA-induced dyskinesia (LID), which can be as debilitating as the motor deficits. There is currently no satisfactory pharmacological adjunct therapy. The endogenous opioid peptides enkephalin and dynorphin are important co-transmitters in the direct and indirect striatofugal pathways and have been implicated in genesis and expression of LID. Opioid receptor antagonists and agonists with different selectivity profiles have been investigated for anti-dyskinetic potential in preclinical models. In this study we investigated effects of the highly-selective µ-opioid receptor antagonist CTAP (> 1200-fold selectivity for µ- over δ-opioid receptors) and a novel glycopeptide congener (gCTAP5) that was glycosylated to increase stability, in the standard rat LID model. RESULTS: Intraperitoneal administration (i.p.) of either 0.5 mg/kg or 1 mg/kg CTAP and gCTAP5 completely blocked morphine's antinociceptive effect (10 mg/kg; i.p.) in the warm water tail-flick test, showing in vivo activity in rats after systemic injection. Neither treatment with CTAP (10 mg/kg; i.p.), nor gCTAP5 (5 mg/kg; i.p.) had any effect on L-DOPA-induced limb, axial, orolingual, or locomotor abnormal involuntary movements. The data indicate that highly-selective µ-opioid receptor antagonism alone might not be sufficient to be anti-dyskinetic.

The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced l-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia.

Dopamine (DA)-replacement therapy utilizing l-DOPA is the gold standard symptomatic treatment for Parkinson's disease (PD). A critical complication of this therapy is the development of l-DOPA-induced dyskinesia (LID). The endogenous opioid peptides, including enkephalins and dynorphin, are co-transmitters of dopaminergic, GABAergic, and glutamatergic transmission in the direct and indirect striatal output pathways disrupted in PD, and alterations in expression levels of these peptides and their precursors have been implicated in LID genesis and expression. We have previously shown that the opioid glycopeptide drug MMP-2200 (a.k.a. Lactomorphin), a glycosylated derivative of Leu-enkephalin mediates potent behavioral effects in two rodent models of striatal DA depletion. In this study, the mixed mu-delta agonist MMP-2200 was investigated in standard preclinical rodent models of PD and of LID to evaluate its effects on abnormal involuntary movements (AIMs). MMP-2200 showed antiparkinsonian activity, while increasing l-DOPA-induced limb, axial, and oral (LAO) AIMs by ∼10%, and had no effect on dopamine receptor 1 (D1R)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (D2R)-like-induced LAO AIMs. The locomotor AIMs were reduced by MMP-2200 in all three conditions. The N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 has previously been shown to be anti-dyskinetic, but only at doses that induce parkinsonism. When MMP-2200 was co-administered with MK-801, MK-801-induced pro-parkinsonian activity was suppressed, while a robust anti-dyskinetic effect remained. In summary, the opioid glycopeptide MMP-2200 reduced AIMs induced by a D2R-like agonist, and MMP-2200 modified the effect of MK-801 to result in a potent reduction of l-DOPA-induced AIMs without induction of parkinsonism.

Differential release of dopamine in the nucleus accumbens evoked by low-versus high-frequency medial prefrontal cortex stimulation.

The medial prefrontal cortex (mPFC) coordinates goal-directed behaviors, which may be mediated through mPFC regulation of dopamine release in the nucleus accumbens (NAc). Furthermore, frequency-specific oscillatory activity between the frontal cortex and downstream structures may facilitate inter-region communication. Although high-frequency (e.g., 60 Hz) mPFC stimulation is known to increase basal dopamine levels in the NAc, little is known about how phasic dopamine release is affected by mPFC stimulation. Understanding the frequency-specific control of phasic dopamine release by mPFC stimulation could elucidate mechanisms by which the mPFC modulates other regions. It could also inform optimization of deep brain stimulation for treatment of neurological disorders. OBJECTIVE: The goal of this work was to characterize the frequency response of NAc dopamine release resultant from mPFC stimulation. We hypothesized that the magnitude of dopamine release in the NAc would increase with increasing stimulation frequency. METHODS: Electrical stimulation of the mPFC of anesthetized rats was delivered at 4-60 Hz and at varying durations while measuring NAc dopamine release with fast-scan cyclic voltammetry. RESULTS: mPFC stimulation resulted in phasic dopamine release in the NAc. Furthermore, 20 Hz stimulation evoked the largest peak response for stimulation intervals >5 s when compared to higher or lower frequencies. CONCLUSIONS: Activation of the mPFC drives dopamine release in the NAc in a complex frequency- and duration-dependent manner. This has implications for the use of deep brain stimulation treatment of disorders marked by dopaminergic dysregulation, and suggest that mPFC may exert more specialized control over neuromodulator release than previously understood.

Platform to Enable Combined Measurement of Dopamine and Neural Activity.

Complex behaviors depend on the coordination of the activities of ensembles of neurons and the release of neuromodulators such as dopamine. The mechanisms underlying such coordination are not well-understood due to a lack of instrumentation for combined and real-time monitoring of neuromodulator release and the activities of large ensembles of neurons. Here we describe a measurement platform that allows for the combined monitoring of electrophysiology from a high-density electrode array and dopamine dynamics from a carbon-fiber microelectrode. Integration of these two measurement systems was achieved through modification of the existing instrumentation. A shared grounded reference electrode was used in both systems to minimize electrical interference. Further, an optional solid-state-relay array positioned between the electrophysiological electrode array and amplifiers was added to provide additional electrical isolation. The capacity of the integrated measurement platform, termed DANA (Dopamine And Neural Activity), to measure action potentials (high frequency) and local-field oscillations (low frequency) was characterized in vitro using an artificial cerebral spinal fluid gelatin. In vivo recordings from the DANA platform in anesthetized rats demonstrated the ability of the system for near-simultaneous measurement of dopamine release and activity from multiple neurons both in distant brain regions (striatum and hippocampus) and within the same brain region (striatum). Furthermore, this system was shown to be sufficiently compact to measure activity in freely moving animals through recording of single-neuron activity, high-frequency local-field oscillations, and dopamine release.

Long-term effect of sub-anesthetic ketamine in reducing L-DOPA-induced dyskinesias in a preclinical model.

Low-dose sub-anesthetic ketamine infusion treatment has led to a long-term reduction of treatment-resistant depression and posttraumatic stress disorder (PTSD) symptom severity, as well as reduction of chronic pain states, including migraine headaches. Ketamine also is known to change oscillatory electric brain activity. One commonality between migraine headaches, depression, PTSD, Parkinson's disease (PD) and l-DOPA-induced dyskinesias (LID) is hypersynchrony of electric activity in the brain, including the basal ganglia. Therefore, we investigated the use of low-dose sub-anesthetic ketamine in the treatment of LID. In a preclinical rodent model of LID, ketamine (5-20mg/kg) led to long-term dose-dependent reduction of abnormal involuntary movements, only when low-dose ketamine was given for 10h continuously (5× i.p. injections two hours apart) and not after a single acute low-dose ketamine i.p. injection. Pharmacokinetic analysis of plasma levels showed ketamine and its major metabolites were not detectable any more at time points when a lasting anti-dyskinetic effect was seen, indicating a plastic change in the brain. This novel use of low-dose sub-anesthetic ketamine infusion could lead to fast clinical translation, and since depression and comorbid pain states are critical problems for many PD patients could open up the road to a new dual therapy for patients with LID.

The coaction of tonic and phasic dopamine dynamics.

Tonic neurochemical dopamine activity underlies many brain functions; however a consensus on this important concentration has not yet been reached. In this work, we introduce in vivo fast-scan controlled-adsorption voltammetry to report tonic dopamine concentrations (90 ± 9 nM) and the dopamine diffusion coefficient (1.05 ± 0.09 × 10(-6) cm(2) s(-1)) in the mouse brain.

Fast voltammetry of metals at carbon-fiber microelectrodes: copper adsorption onto activated carbon aids rapid electrochemical analysis.

Rapid, in situ trace metal analysis is essential for understanding many biological and environmental processes. For example, trace metals are thought to act as chemical messengers in the brain. In the environment, some of the most damaging pollution occurs when metals are rapidly mobilized and transported during hydrologic events (storms). Electrochemistry is attractive for in situ analysis, primarily because electrodes are compact, cheap and portable. Electrochemical techniques, however, do not traditionally report trace metals in real-time. In this work, we investigated the fundamental mechanisms of a novel method, based on fast-scan cyclic voltammetry (FSCV), that reports trace metals with sub-second temporal resolution at carbon-fiber microelectrodes (CFMs). Electrochemical methods and geochemical models were employed to find that activated CFMs rapidly adsorb copper, a phenomenon that greatly advances the temporal capabilities of electrochemistry. We established the thermodynamics of surface copper adsorption and the electrochemical nature of copper deposition onto CFMs and hence identified a unique adsorption-controlled electrochemical mechanism for ultra-fast trace metal analysis. This knowledge can be exploited in the future to increase the sensitivity and selectivity of CFMs for fast voltammetry of trace metals in a variety of biological and environmental models.

Differential effects of the NMDA receptor antagonist MK-801 on dopamine receptor D1- and D2-induced abnormal involuntary movements in a preclinical model.

Dopamine-replacement therapy with l-DOPA is still the gold standard treatment for Parkinson's disease (PD). One drawback is the common development of l-DOPA-induced dyskinesia (LID) in patients, which can be as disabling as the disease itself. There is no satisfactory adjunct therapy available. Glutamatergic transmission in the basal ganglia circuitry has been shown to be an important player in the development of LID. The N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 has previously been shown to reduce l-DOPA-induced abnormal involuntary movements (AIMs) in a rat preclinical model but only at concentrations that worsen parkinsonism. We investigated the contribution of the direct and indirect striatofugal pathways to these effects. In the direct pathway, dopamine D1 receptors (D1R) are expressed, whereas in the indirect pathway, dopamine D2 receptors (D2R) are expressed. We used the 6-hydroxydopamine-lesioned hemi-parkinsonian rat model initially primed with l-DOPA to induce dyskinesia. When the rats were then primed and probed with the D1R agonist SKF81297, co-injection of MK-801 worsened the D1R-induced limb, axial, and orolingual (LAO) AIMs by 18% (predominantly dystonic axial AIMs) but did not aggravate parkinsonian hypokinesia as reflected by a surrogate measure of ipsiversive rotations in this model. In contrast, when the rats were then primed and probed with the D2R agonist quinpirole, co-injection of MK-801 reduced D2R-induced LAO AIMs by 89% while inducing ipsiversive rotations. The data show that only inhibition of the indirect striatopallidal pathway is sufficient for the full anti-dyskinetic/pro-parkinsonian effects of the NMDA receptor antagonist MK-801, and that MK-801 modestly worsens dyskinesias that are due to activation of the direct striatonigral pathway alone. This differential activation of the glutamatergic systems in D1R- and D2R-mediated responses is relevant to current therapy for PD which generally includes a mixture of dopamine agonists and l-DOPA.

Fast-scan controlled-adsorption voltammetry for the quantification of absolute concentrations and adsorption dynamics.

Fast-scan cyclic voltammetry has depended on background subtraction to quantify small changes in neurotransmitter concentration. Because of this requirement, measurements of absolute concentrations using fast-scan cyclic voltammetry have been limited. Here we develop and characterize fast-scan controlled-adsorption voltammetry (FSCAV), which enables direct measurements of absolute concentrations in vitro without the use of flow injection to change the concentration. This enables probing the diffusion-controlled adsorption dynamics of biogenic amines and other adsorbing species. An implicit finite-difference model of mass-transport-limited adsorption was developed and is in agreement with experimental results. Optimization of FSCAV yielded a sensitivity of 81 ± 11 nA/µM for dopamine, corresponding to a limit of detection of 3.7 ± 0.5 nM. Through the combination of novel instrumentation and validated computer simulations, we show that FSCAV is an important measurement tool that can be used to determine absolute concentrations and study mass-transport-limited adsorption.

Electrochemical solid-state phase transformations of silver nanoparticles.

Adenosine triphosphate (ATP)-capped silver nanoparticles (ATP-Ag NPs) were synthesized by reduction of AgNO(3) with borohydride in water with ATP as a capping ligand. The NPs obtained were characterized using transmission electron microscopy (TEM), UV-vis absorption spectroscopy, X-ray diffraction, and energy-dispersive X-ray analysis. A typical preparation produced ATP-Ag NPs with diameters of 4.5 ± 1.1 nm containing ~2800 Ag atoms and capped with 250 ATP capping ligands. The negatively charged ATP caps allow NP incorporation into layer-by-layer (LbL) films with poly(diallyldimethylammonium) chloride at thiol-modified Au electrode surfaces. Cyclic voltammetry in a single-layer LbL film of NPs showed a chemically reversible oxidation of Ag NPs to silver halide NPs in aqueous halide solutions and to Ag(2)O NPs in aqueous hydroxide solutions. TEM confirmed that this takes place via a redox-driven solid-state phase transformation. The charge for these nontopotactic phase transformations corresponded to a one-electron redox process per Ag atom in the NP, indicating complete oxidation and reduction of all Ag atoms in each NP during the electrochemical phase transformation.