Physicochemical Stability Study of the Morphine-Bupivacaine-Ziconotide Association.

OBJECTIVE: The aim of this study was to investigate the physicochemical stability of morphine-bupivacaine-ziconotide mixtures used in intrathecal analgesia in polypropylene syringes and intrathecal pumps. MATERIALS AND METHODS: The stability study method was conceived according to International Council for Harmonisation guidelines. For propylene syringes, six different mixtures of morphine-bupivacaine and ziconotide were assessed over seven days. Two storage temperatures were tested (5 °C ± 3 °C and 25 °C ± 2 °C). For implantable pumps, nine different mixtures were assessed over 60 days and stored at 37 °C. Assays were performed using ultrahigh-pressure liquid chromatography. Turbidity and pH also were measured throughout the study. RESULTS: Results confirmed excellent physicochemical stability for morphine and bupivacaine in the study for all conditions investigated (pumps at 37 °C, polypropylene syringes at 5 °C ± 3 °C and 25 °C ± 2 °C). Concerning ziconotide, after seven days, our study showed that every 95% confidence interval calculated had lower bounds >90% for all mixtures stored in polypropylene syringes. In implantable pumps, a decrease of the concentration was observed in all the mixtures studied. Moreover, the appearance of a degradation product confirmed the ziconotide degradation. CONCLUSION: All results are in favor with a physicochemical stable preparation for six mixture profiles when stored in polypropylene syringes at 5 °C ± 3 °C and 25 °C ± 2 °C. For mixtures stored in implantable pumps, the efficacy should decrease over time owing to the degradation of ziconotide. A trade-off between high morphine concentration and increased refill interval will need to be found by clinicians.

Study of Physicochemical Stability of Ziconotide in Medication Cassette Reservoir.

OBJECTIVE: To determine the physicochemical stability of ziconotide solutions for intrathecal administration in the Medication Cassette Reservoir (MCR). MATERIALS AND METHODS: A stability indicating UPLC-DAD method was developed and validated according to the ICH guidelines. Two mixtures of ziconotide (0.40 µg/mL and 0.60 µg/mL) stored in MCR stored at 25 ± 2°C were evaluated for 14 days and compared to the initial observed concentrations. RESULTS: The physicochemical stability of the two solutions was demonstrated for two days thanks to relative concentrations, pH measurement, visual inspections, and turbidity assays. A degradation product was observed and increased during the study. CONCLUSION: This study showed a very low physicochemical stability of diluted ziconotide stored at 25 ± 2°C in the MCR. The intrathecal administration of ziconotide does not seem appropriate with this device for outpatients.

Development of an ic-ELISA and immunochromatographic strip based on IgG antibody for detection of ω-conotoxin MVIIA.

ω-conotoxin MVIIA(ω-CTX MVIIA) is a peptide consisting of 25 amino acid residues secreted mainly by Conus magus. In view of the toxin threat to humans and animals and defined application in analgesic therapy, it is necessary to develop a rapid, effective and accuracy method for the quantification and analysis of ω-CTX MVIIA in real samples. In the present study, a hybridoma cell named 2E5 stable secreting IgG antibody against ω-CTX MVIIA was selected successfully, and the subtype of Mab 2E5 was IgG1. The purified monoclonal antibody(Mab) 2E5 has high affinity (about 2.79 × 109 L/mol), and shows high specificity to ω-CTX MVIIA antigen. The linear range of ic-ELISA to detect ω-CTX MVIIA was 0.20˜7.22 µg/mL, with a lower detection limit (LOD) of 0.14 ng/mL. The average recovery of intra- and inter-assay were (85.45 ±â€¯2.28)% and (88.03 ±â€¯4.80)% respectively, with a coefficient of variation from 2.59% to 5.42%. The LOD of colloidal strip by naked eye was 1 µg/mL, and the detection time was less than 10 min without any equipment. The developed ELISA and colloidal test strips based on this IgG antibody could be used to detect ω-CTX MVIIA residue in real Conus samples.

Building upon Nature's Framework: Overview of Key Strategies Toward Increasing Drug-Like Properties of Natural Product Cyclopeptides and Macrocycles.

The pharmaceutical industry has focused mainly in the development of small-molecule entities intended for oral administration for the past decades. As a result, the majority of existing drugs address only a narrow range of biological targets. In the era of post-genomics, transcriptomics, and proteomics, there is an increasing interest on larger modulators of proteins that can span larger surfaces, access new therapeutic mechanisms of action, and provide greater target specificity. Traditional drug-like molecules developed using "rule-of-five" (Ro5) guidelines have been proven ineffective against a variety of challenging targets, such as protein-protein interactions, nucleic acid complexes, and antibacterial modalities. However, natural products are known to be effective at modulating such targets, leading to a renewed focus by medicinal chemists on investigating underrepresented chemical scaffolds associated with natural products. Here we describe recent efforts toward identification of novel natural cyclopeptides and macrocycles as well as selected medicinal chemistry strategies to increase drug-like properties or further exploration of their activity.

Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides.

Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Cav2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Cav2.2 in fluorimetric assays and rat Cav2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA (125I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC50 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Cav2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.

Chemical Stability of Morphine, Ropivacaine, and Ziconotide in Combination for Intrathecal Analgesia.

Pain is the most feared symptom amongst individuals living with cancer. In 15% to 20% of patients, conventional analgesic therapy either fails to relieve pain or induces adverse effects. Intrathecal drug delivery systems may present an effective alternative for pain management. The Cancerology Center Paul Papin protocol includes an admixture of morphine, ropivacaine, and ziconotide in intrathecal preparations. These drugs are administered by a fully implantable or an external pump. Syringes or polyolefin infusion bags are prepared for refill just before use. Few centers in France use the method of intrathecal analgesia. Therefore, for those patients receiving intrathecal preparations, each filling requires that the patients be transported from their local hospital (or their home) to a referral center where the patients are monitored. They sometimes must travel up to a hundred kilometers to have a pump filled. The preparation and the analytical control of the mixture are carried out only by those centers meeting the proper criteria, which includes the proper equipment. To spare the patient this travel, a peripheral center may be subcontracted to manage the patient's pump refill. No data are available concerning the chemical stability of admixtures in syringes or polyolefin infusion bags. The aim of this study was to evaluate, with a new analytical method using ultra high-performance liquid chromatography, the chemical stability of these admixtures in syringes or in polyolefin infusion bags. Ziconotide 1 µg/mL was combined with ropivacaine (7.5 mg/mL) and morphine (3.5 mg/mL) in syringes at 5°C, 21°C, and 31°C, and in polyolefin infusion bags at 21°C. Assays were performed using ultra high-pressure liquid chromatography. In syringes stored at 21°C and 31°C, concentrations after 6 hours were not in the acceptable criterion of 10% variability. When syringes were stored at 5°C, the residual concentration of ziconotide after 3 days was 100.5% +/- 2.6% [92.7% to 108.4%]. In polyolefin infusion bags, the residual concentration of ziconotide after 14 days was 96.9% +/- 2.2% [90.1% to 103.6%]. This study demonstrates the chemical stability of this admixture in syringes stored at 5°C for 3 days and in polyolefin plastibags stored at 21°C for 14 days.

Molecular basis of toxicity of N-type calcium channel inhibitor MVIIA.

MVIIA (ziconotide) is a specific inhibitor of N-type calcium channel, Cav2.2. It is derived from Cone snail and currently used for the treatment of severe chronic pains in patients unresponsive to opioid therapy. However, MVIIA produces severe side-effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, that limit its wider application. We previously identified a novel inhibitor of Cav2.2, ω-conopeptide SO-3, which possesses similar structure and analgesic activity to MVIIA's. To investigate the key residues for MVIIA toxicity, MVIIA/SO-3 hybrids and MVIIA variants carrying mutations in its loop 2 were synthesized. The substitution of MVIIA's loop 1 with the loop 1 of SO-3 resulted in significantly reduced Cav2.2 binding activity in vitro; the replacement of MVIIA loop 2 by the loop 2 of SO-3 not only enhanced the peptide/Cav2.2 binding but also decreased its toxicity on goldfish, attenuated mouse tremor symptom, spontaneous locomotor activity, and coordinated locomotion function. Further mutation analysis and molecular calculation revealed that the toxicity of MVIIA mainly arose from Met(12) in the loop 2, and this residue inserts into a hydrophobic hole (Ile(300), Phe(302) and Leu(305)) located between repeats II and III of Cav2.2. The combinative mutations of the loop 2 of MVIIA or other ω-conopeptides may be used for future development of more effective Cav2.2 inhibitors with lower side effects.

A New, High Yield, Rapid, and Cost-Effective Protocol to Deprotection of Cysteine-Rich Conopeptide, Omega-Conotoxin MVIIA.

Almost all conopeptides purified from Conus venoms are cysteine-rich peptides. Among them, omega-conotoxin MVIIA, FDA approved peptide drug (Prialt(®)), selected as a cysteine-rich model that its protection from oxidation is critical during solid phase synthesis. Deprotection of cysteines is a crucial step after peptide synthesis. The current study aimed to set up a new highly efficient deprotection protocol for omega-conotoxin MVIIA. Deprotection was performed based on mercury acetate with significant major modification. The protocol accomplished based on the best molar ratio of peptide/mercury/2-ME that adjusted to 0.2 mm/3 mm/10 mm (50 µg/1 mg/10 µL). The yield and purity of omega-conotoxin MVIIA obtained at 93 and 95%, respectively. The total time of protocol shortened to 90 min instead of 6-20 h in routine methods. In this study, omega-conotoxin MVIIA was recovered in high yield and in the shortest time. Despite of other known protocols, molar ratio adjusted to minimum amount. In conclusion, this protocol would be suggested to cost-effective deprotection of thiol groups for similar cysteine-rich peptides.

Bioactive Mimetics of Conotoxins and other Venom Peptides.

Ziconotide (Prialt®), a synthetic version of the peptide ω-conotoxin MVIIA found in the venom of a fish-hunting marine cone snail Conus magnus, is one of very few drugs effective in the treatment of intractable chronic pain. However, its intrathecal mode of delivery and narrow therapeutic window cause complications for patients. This review will summarize progress in the development of small molecule, non-peptidic mimics of Conotoxins and a small number of other venom peptides. This will include a description of how some of the initially designed mimics have been modified to improve their drug-like properties.

Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers.

The blood brain barrier (BBB) is often an insurmountable obstacle for a large number of candidate drugs, including peptides, antibiotics, and chemotherapeutic agents. Devising an adroit delivery method to cross the BBB is essential to unlocking widespread application of peptide therapeutics. Presented here is an engineered nanocontainer for delivering peptidic drugs across the BBB encapsulating the analgesic marine snail peptide ziconotide (Prialt®). We developed a bi-functional viral nanocontainer based on the Salmonella typhimurium bacteriophage P22 capsid, genetically incorporating ziconotide in the interior cavity, and chemically attaching cell penetrating HIV-Tat peptide on the exterior of the capsid. Virus like particles (VLPs) of P22 containing ziconotide were successfully transported in several BBB models of rat and human brain microvascular endothelial cells (BMVEC) using a recyclable noncytotoxic endocytic pathway. This work demonstrates proof in principle for developing a possible alternative to intrathecal injection of ziconotide using a tunable VLP drug delivery nanocontainer to cross the BBB.

Rationale for Prospective Assays of Intrathecal Mixtures Including Morphine, Ropivacaine and Ziconotide: Prevention of Adverse Events and Feasibility in Clinical Practice.

BACKGROUND: Use of intrathecal admixtures is widespread, but compounding these is sometimes challenging and may result in errors and complications causing super-potency or sub potency adverse events in patients or malfunctions in the pump itself. OBJECTIVE: The purpose of this study is to evaluate the accuracy of compounding of intrathecal admixtures through a prospective, systematic quantitative analysis of each component of the mixture before delivery to patients. STUDY DESIGN: Observational follow up prospective study of intrathecal mixtures components concentrations before refills. SETTINGS: Assays were performed on all intrathecal admixtures produced by the ICO-Paul Papin compounding pharmacy between January 2013 and October 2014 using Ultra High Performance Liquid Chromatography (U.H.P.L.C.). In addition, pH levels of admixtures have been measured since June 2014. When measured concentrations were 15% above or below the required concentrations, the mixture was excluded and compounded again. RESULTS: 1729 mixtures were analyzed. Mean deviation from theoretical values was -1.17% ± 0.28% for morphine, -0.95% ± 1.07% for ropivacaine, and 4.82% ± 0.6% for ziconotide. Exclusion rates were 8.33% overall, but fell from 11.67% in 2013 to 4.97% in 2014. Most exclusions were caused by inaccuracy in the dose of ziconotide. Average mixture pH of the 603 tested admixtures was 4.83 ± 0.6%. LIMITATIONS: This study is monocentric and limitations include also its non-randomized nature with no clinical comparison of the rate of adverse events with a refill process without control of each component concentrations. CONCLUSION: Prospective assays provide benefits in ensuring accuracy of intrathecal mixture compounding and in preventing overdosing or sub dosing, most notably concerning Ziconotide.

Influence of pH and temperature on ziconotide stability in intrathecal analgesic admixtures in implantable pumps and syringes.

PURPOSE: The aim of our study was to investigate the influence of pH and temperature on the stability of ziconotide in analgesic admixtures containing morphine and ropivacaine. METHODS: All admixtures were combined using a wide range of concentrations, in implantable pumps and syringes, using temperatures from 4°C to 37°C. Quantification was made thanks to a specific chromatographic technique. pH has also been measured throughout the study. RESULTS: Admixtures confirm excellent stability for morphine and ropivacaine. Concerning ziconotide, an acid hydrolysis has been observed, reducing the time of use of our admixtures in a significant way, but producing non-toxic degradation products. The degradation was linear in all conditions. Inside the implantable pumps at body temperature turned out to be the best conditions for lower protein breakdown. Finally the degradation process showed a high correlation with the pH and the morphine concentration with a median loss of concentration delay due to degradation of 3.5 days [3; 5] when pH<4.5 and 13 days [13; 24] when pH ≥ 4.5. CONCLUSION: Our admixtures showed different stability depending on the drug concentrations, pH and temperature. The great majority of mixtures in real life in our institution have stability highly compatible with our practice and with the delay between two pump refilling.

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Cav 2.2 Blocker Multitarget Ligands.

N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity.

Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels.

BACKGROUND AND PURPOSE: ω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs). EXPERIMENTAL APPROACH: Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively. KEY RESULTS: R10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ω-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID. CONCLUSIONS AND IMPLICATIONS: R10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding.

In vitro stability of low-concentration ziconotide alone or in admixtures in intrathecal pumps.

OBJECTIVES: Ziconotide is often administered in combination with other analgesics via an intrathecal pump. Studies have established that ziconotide is stable when delivered alone in high concentrations. No stability data are available, however, for ziconotide given in low concentrations and/or with other analgesics as usually occurs in clinical oncology practice. The objective of this study was to assess the in vitro stability of ziconotide alone and combined with other analgesics in intrathecal pumps at 37 °C, as well as in syringes at 5 °C, to evaluate conditions for storing and transporting preparations. MATERIALS AND METHODS: Various ziconotide concentrations (0.1, 0.25, 0.5, and 0.75 µg/mL) were combined with an admixture of ropivacaine (7.5 mg/mL), morphine (7.5 mg/mL), and clonidine (15 µg/mL) in 20-mL intrathecal pumps at 37 °C and in syringes at 5 °C. Solutions of ziconotide alone in concentrations of 0.25, 0.5, 0.75, and 1 µg/mL were introduced into pumps at 37 °C and syringes at 5 °C. Assays were performed using ultra high pressure liquid chromatography. RESULTS: In admixtures, mean ziconotide concentrations decreased linearly to 53.4% (± 3.33%) of baseline after 35 days. When ziconotide was introduced alone in pumps at 37 °C, the residual concentration on day 31 was 35.54% (± 0.04%) with 0.25 µg/mL, 39.37% (± 0.15%) with 0.5 µg/mL, and 44.49% (± 0.18%) with 1 µg/mL. Ziconotide alone or combined with the other analgesics was stable in syringes stored at 5 °C. The preparations complied with the prescriptions, with a mean error of less than 10%, except with the lowest ziconotide concentration (0.1 µg/mL). CONCLUSIONS: At the low ziconotide concentrations studied, the degradation of ziconotide admixed with other drugs was linear and only weakly influenced by the baseline concentration. Linear regression with intrapolation to 30 days showed that the degradation of ziconotide admixed with other drugs was consistent with previously published data.

Mechanism of neurotoxicity of prion and Alzheimer's disease-related proteins: molecular insights from bioinformatically identified ω-conotoxin-like pharmacophores.

Prion diseases are fatal neurodegenerative disorders caused by altered forms of the prion protein (PrPC). It was reported that dysregulation of cellular Ca2+ homeostasis is recurrent in these diseases and that scrapie-infected cells exhibit Ca2+ perturbation via specific impairment of N-type calcium channels. However, it is not known whether such dysfunction is secondary to the broad neuronal damage accompanying prion diseases or whether it underlies pathological interactions of prions with calcium channels. In this research, we examined this latter possibility by searching for channel binding signatures in PrPC through structural comparison with known N-type channel blockers. To this aim, a computational method devised by us to recognize similar distributions of basic residues in protein structures enabled us to find that the bioactive groups representing the pharmacophores of ω-conotoxins GVIA and MVIIA can be overlaid onto similar residues within the PrPC globular domain. This finding, together with the knowledge that Ca2+ homeostasis disruption is common to other neurodegenerative disorders, led us to search for and identify an ω-conotoxin-like pharmacophore also in the Alzheimer's Aß(1-42) peptide. These results point to the potential ability of prions and Aß(1-42) to bind calcium channels as the elusive neurotoxic mechanism common to seemingly unrelated fatal neuropathies.

Effects of Lys2 to Ala2 substitutions on the structure and potency of ω-conotoxins MVIIA and CVID.

Lys2 has previously been implicated as a residue important in binding interactions between omega-conotoxins and the N-type calcium channel. To further assess the importance of this residue, Lys2 to Ala2 derivatives of omega-conotoxins MVIIA and CVID were synthesized and their structures and binding potencies determined. A comparison of the 3D structures of the Ala2 mutants with the parent peptides suggest there are significant structural differences brought about by this substitution. In particular, stabilizing interactions between Lys2 and loop 2 of the omega-conotoxins are removed, leading to greater flexibility in loop 2, which contains Tyr13, a crucial residue for omega-conotoxin binding to the N- and P/Q-type voltage-gated calcium channel (VGCC). The significant drop in binding potencies resulting from replacement of Lys2 thus appears to relate more to entropic factors than to any direct interaction of Lys2 with the VGCC. This has significant implications for the development of a pharmacophore binding model for omega-conotoxins, as removal of Lys2 from consideration suggests that the omega-conotoxins residues that interact with the N-type VGCC reside in loop 2 and 4, and thus cover a significantly smaller and more defined area of the surface of omega-conotoxin than previously thought.

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius.

Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.

Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.