TAT-Modified ω-Conotoxin MVIIA for Crossing the Blood-Brain Barrier.

As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.

Intrathecal ziconotide: a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics.

Ziconotide (Prialt(®)) is a synthetic conopeptide analgesic that acts by selectively antagonizing N-type voltage-gated calcium channels. Intrathecal ziconotide is the only non-opioid intrathecal analgesic that is FDA-approved for use in patients with treatment-refractory, chronic pain. The efficacy of intrathecal ziconotide was demonstrated in randomized, double-blind, placebo-controlled trials in patients with treatment-refractory noncancer-related pain or cancer- or AIDS-related pain. Across trials, ziconotide recipients had significantly greater reductions in pain intensity during ziconotide treatment than those receiving placebo (primary endpoint). At the end of the titration period, approximately one-sixth to one-third of patients with noncancer chronic pain and one-half with cancer- or AIDS-related pain who received ziconotide reached a pain response threshold (≥30 % reduction in the pain intensity score). In ziconotide responders, analgesic effects were enduring, with some patients continuing treatment over extended periods. Across trials, the chief tolerability concerns in ziconotide recipients during the titration phase and during extended treatment were related to CNS adverse events. These were mostly of mild to moderate intensity, although serious adverse events were commonly attributed to ziconotide treatment, especially in trials with rapid ziconotide titration and that permitted higher dosages. In general, clinically important non-CNS adverse events were infrequent, and during the ziconotide titration phase, relatively few patients discontinued treatment because of adverse events. Ongoing research will assess various strategies for selecting patients for ziconotide treatment and for enhancing its efficacy and tolerability. At the present time, intrathecal ziconotide provides a treatment option for patients with severe, unremitting pain who have failed to respond to other intensive analgesic regimens.

Pharmacokinetic analysis of ziconotide (SNX-111), an intrathecal N-type calcium channel blocking analgesic, delivered by bolus and infusion in the dog.

BACKGROUND AND PURPOSE: Ziconotide is a peptide that blocks N-type calcium channels and is antihyperalgesic after intrathecal (IT) delivery. We here characterize the spinal kinetics of IT bolus and infused ziconotide in dog. EXPERIMENTAL APPROACH: Male beagle dogs (N= 5) were prepared with chronic IT lumbar injection and cerebrospinal fluid (LCSF) sampling catheters connected to vest-mounted pumps. Each dog received the following: 1) IT bolus ziconotide (10 µg + 1 µCi (3) H-inulin); 2) IT infusion for 48 hours of ziconotide (1 µg/100 µL/hour); 3) IT infusion for 48 hours of ziconotide (5 µg/100 µL/hour); and 4) intravenous injection of ziconotide (0.1 mg/kg). After IT bolus, LCSF ziconotide and inulin showed an initial peak and biphasic (distribution/elimination) clearance (ziconotide T(1/2-α/ß) = 0.14 and 1.77 hours, and inulin T(1/2-α/ß) = 0.16 and 3.88 hours, respectively). The LCSF : plasma ziconotide concentration ratio was 20,000:1 at 30 min and 30:1 at eight hours. IT infusion of 1 and then 5 µg/hour resulted in LCSF concentrations that peaked by eight hours and remained stable at 343 and 1380 ng/mL, respectively, to the end of the 48-hour infusions. Terminal elimination T(1/2) after termination of continuous infusion was 2.47 hours. Ziconotide LCSF : cisternal CSF : plasma concentration ratios after infusion of 1 and 5 µg/hour were 1:0.017:0.001 and 1:0.015:0.003, respectively. IT infusion of ziconotide at 1 µg/hour inhibited thermal skin twitch by 24 hours and produced modest trembling, ataxia, and decreased arousal. Effects continued through the 48-hour infusion period, increased in magnitude during the subsequent 5 µg/hour infusion periods, and disappeared after drug clearance. CONCLUSIONS AND IMPLICATIONS: After IT bolus or infusion, ziconotide displays linear kinetics that are consistent with a hydrophilic molecule of approximately 2500 Da that is cleared slightly more rapidly than inulin from the LCSF. Behavioral effects were dose dependent and reversible.

Ziconotide: an update and review.

BACKGROUND: Ziconotide is the only N-type calcium channel blocker approved by the US FDA for the treatment of chronic pain. The approved indication is for the management of severe chronic pain in patients for whom intrathecal therapy is warranted and who are intolerant of or refractory to other treatments such as systemic analgesics, adjunctive therapies or intrathecal morphine. OBJECTIVE: The purpose of this article was to review the available safety, efficacy and dosing information for ziconotide. METHODS: The sources searched for literature from 1980 to January 2008 included Pub Med, MEDLINE and PREMEDLINE using the words ziconotide, conotoxins and pain. RESULTS/CONCLUSION: Ziconotide is administered intrathecally by infusion pump to block nociceptive signal transmission in the spinal cord. It is a synthetic neuroactive peptide equivalent to the omega conotoxin MVIIA, a constituent of the venom of the fish-hunting marine snail Conus magus. It is highly potent, has a steep dose-response curve, a slow onset of action and a narrow margin of safety and responses to dose adjustments are slow. Patients receiving ziconotide should be under the care of physicians experienced in the management of intrathecal infusion therapy for pain control and should have convenient access to medical facilities.

Intrathecal ziconotide for refractory chronic pain.

OBJECTIVE: To describe the pharmacology, efficacy, and safety of ziconotide for treatment of severe chronic pain in patients who are candidates for intrathecal therapy. DATA SOURCES: A PubMed/MEDLINE search (1966-June 2006) was conducted using the terms ziconotide, Prialt, and SNX-111. Manufacturer-provided data, the Food and Drug Administration medical review of ziconotide, and abstracts presented at American Pain Society meetings (2001-2006) were also reviewed. STUDY SELECTION AND DATA EXTRACTION: Human studies evaluating the efficacy and safety of ziconotide for the treatment of chronic pain were considered. Animal data were excluded. DATA SYNTHESIS: Ziconotide is the first and only neuronal-type (N-type) calcium-channel blocker. Ziconotide must be administered intrathecally via continuous infusion. A programmable implanted variable-rate microinfusion device, or an external microinfusion device and catheter must be utilized. In double-blind, placebo-controlled studies, ziconotide significantly improved patient perception of pain from baseline to the end of the study periods, which ranged from 11 to 21 days. Patients enrolled in clinical trials were intolerant of or refractory to other treatment modalities. There have been no studies that directly compared ziconotide with other intrathecal or systemic analgesics. Key ziconotide-related adverse events are neuropsychiatric, including depression, cognitive impairment, and hallucinations; depressed levels of consciousness; and elevation of creatine kinase levels. Ziconotide is also associated with a risk of meningitis due to possible contamination of the microinfusion device. CONCLUSIONS: Ziconotide is a therapeutic option for treatment of severe chronic pain in patients who have exhausted all other agents, including intrathecal morphine, and for whom the potential benefit outweighs the risks of serious neuropsychiatric adverse effects and of having an implanted device. Further studies are needed to determine the comparative efficacy of ziconotide and other pain therapies.

Ziconotide, an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain.

Ziconotide is a novel peptide that blocks the entry of calcium into neuronal N-type voltage-sensitive calcium channels, preventing the conduction of nerve signals. N-type calcium channels are present in the superficial laminae of the dorsal horn of the spinal cord. In various animal models of pain, intrathecal administration of ziconotide blocked nerve transmission and nociception. The United States Food and Drug Administration recently approved ziconotide intrathecal infusion for the management of severe chronic pain in patients who require intrathecal therapy and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. The drug has a narrow therapeutic window and a lag time for the onset and offset of analgesia and adverse events. In early clinical trials, frequent and severe psychiatric and central nervous system adverse effects were associated with rapid intrathecal infusion (0.4 microg/hr) and frequent up-titration (every 12 hrs). Therefore, patients with psychiatric symptoms are not candidates for this drug. Drug trials of external intrathecal catheters and microinfusion devices demonstrated a 3% risk of meningitis. A low initial infusion rate of 0.1 microg/hour and limiting infusion rate increases to 2-3 times/week are now recommended. Patients responsive to intrathecal ziconotide require an implanted infusion system to receive long-term therapy.

Pharmacokinetics and pharmacodynamics of intrathecal ziconotide in chronic pain patients.

The pharmacokinetics and pharmacodynamics of ziconotide were assessed over a 48-hour period following intrathecal (i.t.) administration (1, 5, 7.5, or 10 micrograms) to 22 patients with chronic, nonmalignant pain. Plasma and cerebrospinal fluid (CSF) samples were obtained over a 24-hour period. Analgesic efficacy was monitored using Visual Analog Scale of Pain Intensity (VASPI) and Category Pain Relief Scores (CPRS) measurements. Pharmacokinetic (PK) parameters were calculated by noncompartmental methods. Plasma ziconotide data were insufficient for PK calculations. In CSF, the median half-life of ziconotide was 4.5 hours. The median CSF clearance and volume of distribution were 0.26 mL/min and 99 mL, respectively. CSF pharmacokinetics of ziconotide were linear, based on cumulative exposure and peak CSF concentrations. A dose-related analgesia was observed. Pharmacokinetic-pharmacodynamic efficacy and safety analyses showed that higher CSF ziconotide concentrations were generally associated with analgesia and increased incidence of nervous system adverse events following a 1-hour i.t. infusion.

A confirmation of 125I-omega-conotoxin labeled sites in a crude membrane fraction from chick brain as the alpha1 subunit of N-type calcium channels.

Omega-conotoxin GVIA (omega-CTX), as a selective blocker for an N-type Ca2+ channel, has been conveniently used in many molecular biochemical and pharmacological experiments. There has been little elucidation of 125I-omega-CTX binding sites (mainly the 135-kDa band) in the crude membranes from chick brain, although the characteristics of specific 125I-omega-CTX binding and labeling sites in chick brain membranes have been investigated in our previous research. In this work, our goal is to further identify 125I-omega-CTX labeling sites in chick brain membranes by using anti-B1Nt antibodies (against the N-terminal segment B1Nt of N- or P-type Ca2+ channel alpha1-subunits). The 25I-omega-CTX-labeled sites in chick brain membranes could be solubilized and immunoprecipitated by using an anti B1Nt antibody. The molecular weight of the immunoprecipitated protein was determined as 135 kDa, which is inconsistent with that of the specific 125I-omega-CTX binding protein reported previously. Moreover, the 125-omega-CTX-labeled protein could be purified by the method of preparative SDS-PAGE and recognized by anti-B1Nt antibodies in Western blotting analysis. These results indicated that anti-B1Nt antibodies could truly recognize 125I-omega-CTX-labeled sites as the main band of 135 kDa from chick brain membranes, and the omega-CTX-labeled site (mainly the 135-kDa band) should be N-type Ca2+ channel alpha1-subunits.

Age-related changes in the subtypes of voltage-dependent calcium channels in rat brain cortical synapses.

Age-related changes in the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to depolarization-induced Ca(2+) influx and in the density of VDCC subtypes in cortical synapses were investigated using synaptosomes and their membrane preparations from brain cortices of Wistar rats. The relative contribution of VDCC subtypes to Ca(2+) influx was determined by measuring the inhibition of depolarization-induced Ca(2+) influx with four VDCC subtype-specific peptide blockers. In adult rat synaptosomes, L-, N-, P- and Q-type channels accounted for 24, 32, 27 and 12% of the total Ca(2+) influx, respectively. Brain aging significantly reduced the relative contributions of N- and P-type channels and increased the contribution of the channels resistant to the four blockers used. The densities of VDCC subtypes, determined by binding experiments using radiolabeled PN200 -110, omega-conotoxin GVIA and omega-conotoxin MVIIC, were found to be significantly decreased in aged synaptic plasma membranes. On the contrary, the dissociation constants of the blockers were not changed except for PN200-110-sensitive L-type channels. These results suggest that aging alters the relative contributions of each VDCC subtype to depolarization-induced Ca(2+) influx and decreases the number of VDCCs in rat brain cortical synapses. These changes in VDCCs may lead to age-related hypofunction of synaptic neurotransmission in brain cortices.

Bioavailability of Ziconotide in brain: influx from blood, stability, and diffusion.

Ziconotide is a selective peptide antagonist of the N-type calcium channel currently in clinical trials for analgesia. Ziconotide reached a maximal brain concentration of between 0.003 and 0.006% of the injected material per gram of tissue at 3-20 min after i.v. injection, and this decayed to below 0.001%/g after 2 h. The structurally distinct conopeptide SNX-185 (synthetic TVIA) was considerably more persistent in brain after i.v. administration, with 0.0035% of the injected material present at 2-4 h after i.v. injection, and 0.0015% present at 24 h. Similar results (i.e. greater persistence of SNX-185) were obtained when the peptides were perfused through in vivo dialysis probes implanted into the hippocampus. Image analysis and serial sectioning showed that diffusion of Ziconotide in the extracellular fluid around the dialysis probe was minimal, with the peptide located within 1 mm of the probe after 2 h. In vitro diffusion through cultured bovine brain microvessel endothelial cells (BBMEC) verified that a close structural analog of Ziconotide (SNX-194) passed through this blood-brain barrier (BBB) model as expected for peptides of similar physical properties (permeability coefficient of 6.5 x 10(-4) cm/g). Passage from blood to brain was also verified by in situ perfusion through the carotid artery. A statistically greater amount of radioactivity was found to cross the BBB after perfusion of radioiodinated Ziconotide compared to [14C]inulin. Capillary depletion experiments and HPLC analysis defined the brain location and stability.