Current Medicinal Chemistry, 2004, 11, 3029-30403029
0929-8673/04 $45.00+.00© 2004 Bentham Science Publishers Ltd.
Ziconotide: Neuronal Calcium Channel Blocker for Treating Severe Chronic
Elan Pharmaceuticals, Inc., 7475 Lusk Boulevard, San Diego, CA, 92121, USA
Abstract: Ziconotide (PRIALT) is a neuroactive peptide in the final stages of clinical development as a novel
non-opioid treatment for severe chronic pain. It is the synthetic equivalent of ω-MVIIA, a component of the
venom of the marine snail, Conus magus. The mechanism of action underlying ziconotide’s therapeutic profile
derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels (N-
VSCCs). Direct blockade of N-VSCCs inhibits the activity of a subset of neurons, including pain-sensing
primary nociceptors. This mechanism of action distinguishes ziconotide from all other analgesics, including
opioid analgesics. In fact, ziconotide is potently anti-nociceptive in animal models of pain in which morphine
exhibits poor anti-nociceptive activity. Moreover, in contrast to opiates, tolerance to ziconotide is not
observed. Clinical studies of ziconotide in more than 2,000 patients reveal important correlations to
ziconotide’s non-clinical pharmacology. For example, ziconotide provides significant pain relief to severe
chronic pain sufferers who have failed to obtain relief from opiate therapy and no evidence of tolerance to
ziconotide is seen in these patients. Contingent on regulatory approval, ziconotide will be the first in a new
class of neurological drugs: the N-type calcium channel blockers, or NCCBs. Its novel mechanism of action as a
non-opioid analgesic suggests ziconotide has the potential to play a valuable role in treatment regimens for
severe chronic pain. If approved for clinical use, ziconotide will further validate the neuroactive venom
peptides as a source of new and useful medicines.
Keywords: ziconotide, PRIALT, SNX-111, N-type calcium channels, pain, analgesia, analgesic, conopeptide, conotoxin.
Ziconotide† is a neuroactive venom peptide currently in
the final stages of clinical development as a non-opioid
treatment for severe chronic pain. It is the synthetic
equivalent of ω-conopeptide MVIIA, a component of the
venom of the piscivorous marine snail, Conus magus.
Omega-MVIIA is one of numerous peptides in C. magus
venom used by this mollusk to subdue its fish prey. The
chemical, biological, pharmacological, and therapeutic
properties of ziconotide have been extensively studied and
reviewed [1, 2, 3, 4]. The mechanism of action underlying
its therapeutic efficacy is the result of a very potent and
highly selective blockade of mammalian neuronal N-type
voltage-sensitive calcium channels (N-VSCCs). Very high
potency and specificity are characteristics common amongst
venom-derived neuroactive peptides and ziconotide’s
development exemplifies the importance of these twin
features in focusing attention on neuroactive peptides as
candidates for human therapeutics.
Ziconotide has now been studied in approximately 2,000
human subjects for its therapeutic potential in two areas of
clinical application [4, 5]: 1) Prevention of ischemic
neurodegeneration in sufferers of stroke and traumatic brain
injury or in patients undergoing coronary artery bypass graft
surgery. 2) Analgesia for patients suffering severe chronic
*Address correspondence to this author at Elan Pharmaceuticals, Inc.,
7475 Lusk Boulevard, San Diego, CA, 92121, USA; Tel: 858.784.6764, E-
†Ziconotide (USAN generic name) is referred to in the literature by
several names, including: CmTx, ω-MVIIA, SNX-111, and PRIALT®
(Elan Pharmaceuticals’ registered brand name for ziconotide).
pain. While the clinical studies of its neuroprotective effects
have been discontinued in phase 2, the development of
ziconotide for its analgesic properties proceeds apace,
including the completion of phase 3 clinical studies.
Calcium-dependent transmitter release at essentially all
chemical synapses in the nervous system is mediated by a
family of VSCCs [see for example 6, 7, 8, 9]. Members of
the family, including the N-VSCC, are expressed on
overlapping subsets of presynaptic terminals [see for
example 7, 10, 11]. A hallmark of the N-VSCC is its
susceptibility to the potent and specific blockade of its
calcium permeability by omega-conopeptides, including
ziconotide [12, 13]. As mentioned above, it is the blockade
of N-VSCCs that underlies ziconotide’s analgesic activity
[14, 15]. The perception of pain usually begins with the
noxious stimulation of primary nociceptive neurons that
innervate peripheral tissues such as skin and internal organs.
These nociceptors convey noxious signals to the spinal cord
through the synapses formed by their central terminals and
postsynaptic terminals of spinal interneurons. Noxious
signals are then transmitted via spinal interneurons to the
brain where they are perceived as pain. Because the central
terminals of primary nociceptors possess N-VSCCs,
blockade of these channels by ziconotide results in the
inhibition of pain . Inhibition of N-VSCCs in synapses
further up the neuraxis and/or inhibition of N-VSCCs on
cell bodies and dendrites of neurons along the nociceptive
pathway may also contribute to the analgesic activity of
ziconotide. However, evidence for these latter sites of action
is less compelling and is a subject for future research.
As a direct blocker of N-VSCCs, ziconotide is not an
opioid analgesic. Studies of ziconotide in animal models of
pain suggest that it may possess valuable therapeutic
3030 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
properties distinct from those of the opiate analgesics. For
example, ziconotide is effective in some animal models in
which the opiate, morphine, exhibits little anti-nociceptive
activity [4, 16] and, in contrast to morphine, tolerance does
not develop with continued administration of ziconotide
. Furthermore, the anti-nociceptive effects of morphine
and ziconotide are additive or even supra-additive [16, 18,
19]. Other sensory modalities such as touch, acute
nociception and proprioception, appear intact in ziconotide-
treated animals . Consistent with these observations in
animals, ziconotide has been shown to provide pain relief to
chronic severe pain patients who have failed to obtain relief
from opiates [5, 20, 21]. In a double-blind, placebo-
controlled phase 3 study, ziconotide’s analgesic efficacy was
significant even though many of the patients continued
treatment with opiates during their treatment with ziconotide
. In addition, no evidence of tolerance to ziconotide was
observed in these patients.
Of the several potential conopeptide-based therapeutics
currently under investigation, ziconotide is the furthest along
in clinical development. If approved, ziconotide will be the
first in a new class of so-called neuronal calcium channel
blockers, or NCCBs, to be marketed for treating
neurological disorders. Its novel mechanism of action
underlies a clinical safety and efficacy profile distinct from
all other existing analgesics, which, in turn, implies a
potentially valuable role in treatment strategies for severe
chronic pain. As such, ziconotide is the harbinger of the
therapeutic use of neuroactive conopeptides as medicines for
a variety of neurological disorders as well as NCCBs as safe
and effective analgesics.
CHEMICAL STRUCTURE AND PROPERTIES
Ziconotide is the synthetic equivalent of ω-conopeptide
MVIIA, first reported as a peptide component of the venom
of Conus magus . Its sequence of 25 amino acids is:
In contrast to many other conopeptides, there are no
unusual post-translationally modified amino acids. The net
positive charge of +6 and the six cysteines are particularly
noteworthy. The pattern of cysteines in the primary sequence
(i.e., C-C-CC-C-C; where the dashes represent intervening
sequences of other amino acids) is characteristic of the “O”
superfamily of conopeptides . Several members of this
superfamily have been isolated and characterized and they all
appear to be ion channel ligands. Within the O superfamily,
ziconotide belongs to the “omega” family comprising
blockers of neuronal voltage-sensitive calcium channels
The carboxyl terminus of ziconotide is amidated and
both the N-terminal and C-terminal amino acids are
cysteines that are covalently linked to two other,
sequentially internal, cysteines via disulfide bridges .
These modifications likely render the peptide less
susceptible to the circulating exopeptidases in the fish that
are the target of C. magus envenomation. Similarly, they
likely lengthen the metabolic lifetime of ziconotide in the
serum and CSF of experimental animals and humans (see
below) compared to linear, unmodified peptides.
The pharmacological activity of the peptide is dependent
on these intact disulfide bonds linked in the disulfide-
bridging pattern of ziconotide as depicted above. These
linkages are the key determinant of the three-dimensional
structure of the molecule. The three-dimensional solution
structure of ziconotide has been solved by magnetic
resonance methods and confirms the C1-C16, C8-C20, and
C15-C25 disulfide linkages that fold the peptide into the
“omega knot” characteristic of the omega family [23, 24]. In
addition to the disulfide bonds, a triple-stranded beta sheet
further stabilizes the structure and may confer additional
resistance to peptidases in addition to possibly enhancing
binding potency and selectivity. The surface of this “mini-
protein” is festooned with the charged and polar side chains
of its amino acids. As a result, ziconotide is highly
The structure of ziconotide has several implications for
its use as a therapeutic. First, the length of the amino acid
sequence and its folding into an active structure present a
challenge for the manufacture of clinical supplies. Thus, the
cost of goods, on a molar basis, is likely significantly
higher for ziconotide than that for small-molecule
therapeutics of average synthetic complexity. Second, the
molecule’s hydrophilicity makes it highly water-soluble and
readily formulatable in aqueous pharmaceutical
presentations. Third, due to the disulfide bonds and the
methionine in position 12, the stability of ziconotide may
be affected by oxidants or reductants, including oxygen .
Fourth, its relatively large size and hydrophilicity limit its
tissue penetration (see below). Thus, ziconotide is most
efficiently used when delivered directly to the compartment
in which its therapeutic molecular target resides. In the case
of treatment for chronic severe pain, this is the spinal cord
and surrounding cerebrospinal fluid (CSF). Once delivered
to a particular compartment, the drug tends not to be cleared
from that compartment by diffusion through the
permeability barriers of that compartment.
In common with ziconotide, most venom peptides under
consideration for therapeutic applications will also very
likely present challenges for manufacturing and possess
relatively low tissue permeability. These factors must be
taken into account when assessing the feasibility of
developing a venom peptide for medical purposes.
IN VITRO AND IN VIVO PHARMACOLOGY
Following its initial isolation from C. magus venom,
ziconotide was first assayed for its biological activity, not in
vitro, but by intracranial injection in mice, where it
produced a characteristic shaking behavior . This result
showed that the peptide was neuroactive in mammals, thus
distinguishing it from many other C. magus venom peptides
that either produced no behavioral response at the dose
administered or produced other behavioral responses,
Ziconotide Current Medicinal Chemistry, 2004, Vol. 11, No. 23 3031
including death. These early investigations suggested
ziconotide possessed a distinct neuropharmacology in
mammals that included lack of potent lethality.
The elicited shaking behavior also allowed ziconotide to
be grouped with other peptides isolated from C. magus and
other Conus species that induced this same behavior .
These shaker peptides were dubbed the “omega” class.
Several of these omega-conopeptides have been used to
elucidate the common pharmacological properties of the
omega family. In particular, ω-GVIA isolated from Conus
geographus was, and continues to be, the archetypical ω-
conopeptide for most non-clinical studies . Virtually all
of the in vitro pharmacological properties first and most
extensively discovered for ω-GVIA are shared by ziconotide.
For example, Kerr and Yoshikami showed that GVIA
inhibited synaptic transmission at the neuromuscular
junction of the frog . The peptide decreased the quantal
content of electrically-stimulated transmitter release, while
quantal amplitude was unaffected, implying a presynaptic
site of action. The effect was reversed by high concentrations
of external Ca++. GVIA also blocked the calcium-dependent
component of the electrically evoked action potential in
isolated embryonic chick sympathetic ganglia. Taken
together, these results strongly suggested that ω-
conopeptides inhibit synaptic transmission by blocking
presynaptic VSCCs and that these ω-conopeptide-sensitive
VSCCs are present on axons and nerve terminals. That
ziconotide in particular blocks presynaptic VSCCs in the
same manner was subsequently confirmed using several
neuronal preparations [27, 9]. The close pharmacological
similarity of ω-GVIA and ziconotide is consistent with the
finding that both peptides compete with each other for a
common high affinity binding site on the N-type of VSCCs
(see below). The microsites of the multi-point binding
pocket for each of these two peptides are overlapping but
probably not identical, however [28, 29].
As the first readily available, highly potent, specific
blockers of a particular subset of neuronal VSCCs, the ω-
conopeptides contributed significantly to the early
appreciation and elucidation of the electrophysiological,
pharmacological, and genetic heterogeneity of the VSCC
family [13, 30]. Several electrophysiologically
distinguishable VSCC phenotypes -- L, N, P, Q, R, and T
-- have been reported . These phenotypes correlate
(though not one-to-one) with the protein products of ten
different genes for the central α1 VSCC subunit. For
example, the Cav2.2 gene gives rise to the α1B subunit that
forms the transmembrane calcium-conducting pore of the N-
VSCC . It was found that N-VSCCs -- originally
defined by their electrophysiological behavior -- are
specifically and potently blocked by ω-conopeptides such as
GVIA and ziconotide . In fact, sympathetic ganglion and
dorsal root ganglion neurons from Cav2.2 knockout mice are
bereft of essentially all GVIA-sensitive calcium currents that,
in contrast, are present in those same kinds of neurons from
wild-type mice . The classical calcium blocking
cardiovascular drugs, such as the dihydropyridines,
benzothiazepines, and phenylalkylamines are potent blockers
of L-type VSCCs, but have essentially no effect on N-
VSCCs at therapeutic doses. Likewise, the ω-conopeptides
do not bind to or block L-VSCCs potently . As
expected, L-type dihydropyridine-sensitive calcium currents
are preserved in the Cav2.2 knock-out mice. These results
essentially prove that the Cav2.2 gene gives rise to the α1B
VSCC protein with its characteristic N-type
electrophysiological phenotype and its selective sensitivity
to N-channel-blocking ω-conopeptides. Thus, sensitivity to
ω-conopeptides such as GVIA and ziconotide is a hallmark
of the N-VSCC phenotype.
As mentioned above, ziconotide possesses very high
(sub-nanomolar) binding affinity for N-VSCCs . On the
other hand, ziconotide has no appreciable affinity for other
ion channels, nor does it bind to µ- or κ-opioid receptors
. Therefore, ziconotide is clearly not an opioid analgesic.
It has also been shown that ziconotide potently inhibits the
calcium current carried by cloned N-type calcium channels
expressed in cultured cells  and blocks transmitter release
in a variety of neuronal preparations . Specific antibodies
raised against peptides of the cloned N-type VSCC
immunoprecipitate the radiolabeled ziconotide binding site
from neuronal tissue homogenates . The staining pattern
of N-channel-specific antibodies is essentially identical to
the distribution of bound radioiodinated ziconotide in
neuronal tissue slices [33, 34, 35, 36]. Taken together, the
above evidence leaves little doubt that ziconotide achieves
its pharmacological effects through the specific and potent
binding to and blocking of N-VSCCs.
All VSCC phenotypic subtypes are represented to some
degree in the nervous system and are expressed in specific
patterns of distribution . These distributions encompass,
on particular subsets of neurons, presynaptic terminals,
axons, cell bodies and dendrites. Ziconotide’s target, the N-
VSCC subtype, contributes, to a greater or lesser degree, to
the complement of VSCCs in specific populations of central
and peripheral neurons. Thus, there are three general levels of
N-VSCC involvement in the function of particular kinds of
neurons and, correspondingly, three general degrees to which
an NCCB can affect the function of those neurons:
1)Neurons that contain predominantly N-type VSCCs
will be effectively inhibited by ziconotide.
2) Neurons that possess a mixed complement of VSCC
subtypes, including N-type, will be partially
inhibited by ziconotide.
3)Neurons that possess few or no N-type VSCCs will
be unaffected by ziconotide.
It is the distribution of N-channels throughout the
nervous system – alone and relative to other VSCC subtypes
-- that determines the clinical safety and efficacy profiles of
ziconotide. Susceptible subsets of neurons that are most
relevant to the pharmacological and therapeutic profile of
ziconotide are discussed briefly below.
Autoradiography of the rat CNS using radioiodinated
ziconotide revealed that N-VSCCs are densely localized in
laminae 1 and 2 of the dorsal horn of the spinal cord, where
primary sensory afferents deliver nociceptive signals from
the periphery to the CNS . This localization contrasts
3032 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
with the broader distribution of P/Q-VSCCs throughout the
spinal cord gray matter, but also including the outer laminae
of the dorsal horn . These distributions were confirmed
by immunohistochemical staining using antibodies
generated against peptides of the α1 subunit proteins of the
N-VSCC (Cav2.2, or α1B) and the P/Q-VSCC (Cav2.1, or
α1A) . One inference from these data is that NCCBs
such as ziconotide would be anti-nociceptive in animal
models of pain and, thus, analgesic in humans. An
additional inference is that if all exposure were limited to the
spinal cord, the therapeutic index of these NCCBs might be
relatively high. Moreover, it might be further inferred that,
while P/Q VSCC blockers might also be analgesic, the
therapeutic index of this class of blockers would be
relatively lower. In fact, some of these predictions appear to
be borne out in studies of the anti-nociceptive effects of
these two kinds of VSCC blockers in animals. That is,
intrathecal ziconotide is potently anti-nociceptive and has a
high safety margin , while intrathecal P- or P/Q-VSCC
blockers are potently lethal and at least some studies show
that they also possess anti-nociceptive activity. Note that i.t.
L-VSCC blockers are reported to have no or minor anti-
nociceptive activity [see for example, 15]. The toxic effects
of P/Q-VSCC blockers may be due to the preponderance of
this VSCC subtype on the cell bodies of skeletal muscle
motor neurons in the cord . Inhibition of the activity of
these neurons would result in motor paralysis and death by
suffocation (see below).
The N subtype is a major VSCC in sympathetic
afferents. These afferents innervate the vasculature and
regulate blood pressure through the release of noradrenaline.
The release of this noradrenaline is mediated by N-VSCCs
. Not surprisingly, several studies show that intravenous
ziconotide elicits a dose-dependent drop in mean arterial
blood pressure (MABP) in conscious rats [see for example,
38]. The dose-response curve is biphasic, however. Only the
first phase, which reduces MABP ~25% with an ED50 of
~9ug/kg, is attributable to inhibition of noradrenaline
release. The second phase reduces MABP at least an
additional ~40% with an ED50 of ~7mg/kg. This phase is
likely due to the non-specific degranulation of mast cells and
consequent release of histamine induced by the polybasic
nature of ziconotide. In this same study, small effects on rat
heart rate due to ziconotide were reported. Ziconotide’s mast
cell degranulating activity serves as an alert for this potential
activity in any polybasic neuroactive venom peptide
therapeutic candidate. Note that intravenous doses of
ziconotide in the mg/kg range give plasma concentrations
that are orders of magnitude above the ED50s determined for
ziconotide in a variety of in vitro functional assays. These
doses are only potentially medically relevant for cases in
which high systemic levels might be desired to drive
ziconotide (or any other polybasic peptidic drug) across the
blood-brain barrier to achieve therapeutic concentrations
The effects of i.v. ziconotide on blood pressure in rats are
consistent with the role of N-VSCCs in sympathetic
regulation of blood pressure as revealed by studies with
Cav2.2 knock-out mice genetically deficient in N-VSCCs
. The NCCB, ω-GVIA, reduces MABP in wild-type
mice by ~30%, but has essentially no effect on MABP in
the N-channel knockout mice. In contrast to its minor effect
in rats, GVIA reduces heart rate in mice by approximately
22%, but, again, has no effect on heart rate in the N-channel
knockouts. A predominant role of N-VSCCs in sympathetic
function is further revealed by assessing the baroreflex in
these knockout mice. This reflex raises blood pressure in
response to lowered blood pressure by stimulating the
release of noradrenaline from sympathetic afferents. Again,
this response is effectively abolished by GVIA in wild-type
mice, while it is unaffected in the knockouts, further
indicating that release of noradrenaline from sympathetic
neurons is N-VSCC-mediated. The role of N-VSCCs and
the effects of omega-conopeptide on cardiac function were
also examined. The positive inotropic effect (increased
strength of heartbeat) of electrical field stimulation of
isolated left atria from wild-type mice was abolished by
GVIA, but GVIA had no effect on atria from knockout mice.
Interestingly, neither the omega-conopeptide nor the
knockout had any appreciable effect on cholinergic,
parasympathetically mediated, negative inotropism. Thus,
the sympathetic and parasympathetic systems represent
subsets of neurons that are, respectively, an example of 1)
neurons that are essentially completely inhibited by
ziconotide and 2) neurons that are essentially unaffected by
In light of the above non-clinical results, it is not
surprising, that in a phase 1 study of the safety and
tolerability of ziconotide in normal volunteers, i.v. drug
produced dose-dependent orthostatic hypotension. Small
decreases in supine blood pressure were also noted. Changes
in diastolic blood pressure and heart rate were small,
variable, and not dose-related. However, sinus bradycardia
was observed in some patients. There were no clinically
significant changes in neurologic or neurocognitive
examinations, routine resting ECGs, EEGs, blood
chemistries, hematologic indices or urinalysis in any
subjects . This phase 1 study constituted the first report
of human exposure to any prospective conopeptide
therapeutic and was the initial confirmation that the
pharmacological effects of ziconotide observed in humans
correlated with those seen in rodents.
The hypotensive effects of i.v. ziconotide has important
consequences for the use of this agent in the treatment of
pain and for the prevention of neurodegeneration following
stroke, traumatic brain injury, or other ischemic insults to
the brain, as discussed briefly later in this review.
The autoradiographic and immunohistochemical studies
cited above also show that both the N- and P/Q-VSCCs are
broadly distributed in the rat brain [36, 33, 40]. Binding
studies show that the density of the P/Q-VSCC in brain
homogenates and synaptosomal preparations is about 1.5 -
10 times greater than that of the N-VSCC across various
brain regions . Thus, P/Q channels appear to be the
predominant mediator of voltage-sensitive, calcium-
dependent transmitter release at brain synapses. In a global
sense, this helps rationalize why NCCBs delivered to the
brain are not lethal, while P/Q blockers similarly delivered
Ziconotide Current Medicinal Chemistry, 2004, Vol. 11, No. 23 3033
are potently lethal. This is consistent with the observation
that the genetic knock-out of the N-VSCC results in a
benign phenotype , while knocking out the P/Q-VSCC
is lethal within 3-4 weeks after birth . In rodents, the
only readily observed behavioral effect of ziconotide and
other NCCB blockers acting centrally is shaking, whether
delivery is i.v., i.c., i.c.v., or i.t. . These non-clinical
results suggested that ziconotide might induce supraspinal
behaviorial effects in humans as well. Supraspinal adverse
effects are indeed observed clinically with intrathecally
administered ziconotide [4, 20] (see below).
Skeletal Neuromuscular Junction
The N-channel-specific ω-conopeptides, such as
ziconotide, block the presynaptic VSCCs at the skeletal
neuromuscular junction (NMJ) in non-mammalian species,
such as those of fish and amphibians . Thus, the
paralytic action of ziconotide is one component of the prey
capture strategy of C. magus. However, ziconotide does not
block the VSCCs at the skeletal NMJ of mammals .
These results highlight the risk in presuming that the
pharmacological effects of neuroactive peptides observed in
non-mammals will translate directly to mammals, including
Staining of isolated mouse NMJ using fluorescently
labeled ω-MVIIC, a P/Q-VSCC-blocking ω-conopeptide,
showed that the predominant VSCC at this synapse is the
P/Q subtype . In the same study, binding of
fluorescently labeled ziconotide could not be detected. These
results are consistent with the potent block of nerve-evoked
muscle contraction by ω-MVIIC and other P/Q-VSCC
blockers [43, 45], while high concentrations of ziconotide
lack any inhibitory effect . Immunohistochemistry using
anti-N-channel and anti-P/Q-channel antibodies echoes this
dearth of N-VSCCs and abundance of P/Q-VSCCs at the rat
NMJ . Thus – critical to its development and use as a
therapeutic -- ziconotide is not paralytic in rats, mice, or
primates [4, 46, 5]. The lack of paralysis in human subjects
following i.v. injection of ziconotide  is consistent with a
relatively ample expression of P/Q-VSCCs but not N-
VSCCs at the human NMJ as shown directly by Protti et al.
The release of hormones from neuroendocrine cells is
mediated by various VSCC subtypes. For example, L-, N-,
P-, Q-, and R-VSCCs mediate catecholamine release from
adrenal chromaffin cells  as well as vasopressin and/or
oxytocin from neurohypophyseal cells . N-VSCCs
contribute fractionally to the calcium currents in these cells
and correspondingly contribute to a portion of the release of
catecholamines and oxytocin, for example. The N-VSCCs in
chromaffin cells have electrophysiological kinetics and ω-
conopeptide pharmacology that differ somewhat from those
of neuronal N-VSCCs . Perhaps due to the relatively
small contribution of N-VSCCs to total hormone release,
there are no reports that systemic administration of
ziconotide or any other NCCB produces detectable
alterations in neuroendocrine function in vivo. It is expected
that anti-nociceptive levels of intrathecally delivered
ziconotide would have little or no direct functional effect on
neuroendocrine glands in the periphery.
A general conclusion may be drawn regarding the
distribution of N-VSCCs across the neuroendocrine system:
The distribution of N-VSCCs is not correlated with the
distribution of any transmitter type or general tissue type.
For example, N-VSCCs mediate some acetylcholine release
in the brain , but not the acetylcholine released at the
skeletal NMJ ; and N-VSCCs mediate the release of
transmitter onto smooth muscle , but not onto skeletal
muscle . Furthermore, N-VSCCs regulate specific pools
of noradrenaline, dopamine, serotonin, acetylcholine,
glutamate and aspartate but P/Q-VSCCs do as well .
The clinical implication of the lack of these correlations is
that the pharmacodynamic profile of ziconotide will differ
substantially from those of drugs based on specific
transmitter types (that is, those that act on particular
receptors or transporters, for example), supporting the notion
that ziconotide and other NCCBs are potentially unique and
useful treatments for neuropathologies.
The peptidic nature of ziconotide has important
implications for its tissue distribution, metabolism, and
clearance, which in turn contribute to the determination of
its potential routes of administration and therapeutic
applications. In rat studies, intravenously injected ziconotide
reached maximal brain concentrations of approximately 0.1
to 0.01% of the total systemic dose [51, 52]. Thus, i.v.
doses in the range of milligrams per kilogram can yield
nanomolar concentrations of ziconotide in the brain. Because
ziconotide exerts its in vitro biological actions in the
nanomolar range , pharmacological effects in the CNS
should be detected with i.v. dosing in this range. In fact, all
of the above is consistent with the ziconotide-induced
shaking behavior observed in rats, which requires milligram
i.v. doses, but only microgram doses administered i.c.v. It
is also consistent with the milligram doses i.v., but
microgram doses i.c.v., necessary to achieve neuroprotection
in rat models of brain ischemia  (see below). Thus,
ziconotide permeates the blood-brain barrier to a similar
degree as other peptides and proteins . Studies of the
distribution of radioiodinated ziconotide following infusion
into the rat brain through a microdialysis probe showed that
ziconotide’s diffusion through the brain parenchyma is slow
and limited – similar to other proteins and peptides that
have been tested [51, 52].
The pharmacokinetic parameters obtained for ziconotide
with i.v. administration in rats and monkeys are also not
unexpected for a peptide of ziconotide’s physicochemical
character . Steady-state volumes of distribution were
approximately 40% of body weight, indicating extravascular
dissemination of ziconotide to both extracellular and
intracellular fluids. Elimination curves contained two
exponential components. The fast component (rat t1/2 = 22
min; monkey t1/2 = 44 min) accounted for approximately
97% of the ziconotide disposition, while the half-life of the
terminal component was 4.6 hours for rat and 6.5 hours for
monkey. The relatively short plasma half-life of the fast
component is likely due to the activity of plasma peptidases.
Pharmacokinetic parameters of intrathecally administered
3034 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
ziconotide have been assessed in chronic pain patients .
Both analgesia and the incidence of adverse neurological
events in the patients in this study were dose-related. In
marked contrast to its short half-life in plasma, the median
half-life in cerebrospinal fluid (CSF) was 4.5 hours and the
clearance was 0.26 ml/min, which is similar to the rate of
turnover of human CSF. The volume of distribution of 99
ml approximates the total volume of human CSF. These
results suggest that ziconotide spreads up and down the
neuraxis from its site of intrathecal infusion and that bulk
CSF flow, rather than metabolism, is the primary
mechanism of clearance of ziconotide from the CSF.
Consistent with low blood-brain barrier permeation, a
pharmacokinetic analysis of the plasma levels of
intrathecally delivered ziconotide could not be performed
because drug plasma levels were below the limit of
Because increases in intracellular free calcium promote
the pathologic processes of ischemia-induced neuronal
injury, agents that suppress the entry of calcium through
either 1) voltage-gated calcium channels or 2) ligand-gated
cation channels are potentially neuroprotective. In fact,
NCCBs could, in principle, achieve neuroprotection by both
mechanisms. That is, NCCBs inhibit calcium influx directly
by blocking N-type VSCCs, as well as indirectly by
blocking the release of excitatory neurotransmitters, such as
glutamate, that activate calcium-conducting post-synaptic
ligand-gated receptor-channels . The neuroprotective
activity of ziconotide has been demonstrated in several
global and focal occlusion models of ischemic brain and
spinal cord damage and in models of traumatic brain injury
in rodents. In the first such study reported, Valentino, et al.
found that ziconotide significantly protected CA1
hippocampal neurons in the rat from degeneration in a dose
dependent manner following transient forebrain ischemia
when administered intravenously
intracerebroventricularly (i.c.v.) . The i.v. doses required
to achieve neuroprotection were more than three orders of
magnitude larger than those required with i.c.v. injection
(mg/kg range vs. ug/kg range). These results are consistent
with the low blood-brain barrier permeability of ziconotide
and support the CNS as the locus of the neuroprotective
effect. Interestingly, the degree of neuroprotection was
significantly greater when administration of ziconotide was
delayed for 6, 12, or 24 hours after the ischemic insult than
when administered only 1 hour after the insult. No
protection was obtained with drug administered 48 hours
after insult. These results are consistent with the additional
observation that ziconotide is approximately 5-fold more
potent when administered 6 hours rather than 1 hour post-
occlusion. It was also shown that maximal neuroprotection
was obtained for at least 12 days following treatment. In
these studies, levels of glutamate in the brain were not
determined before and after ischemia. However, it was noted
that ziconotide did not inhibit potassium depolarization-
induced glutamate release. Buchan et al. demonstrated
similar neuroprotective activity in a rat model of transient
global ischemia . Ziconotide administered 6 or 24 hours
after initiation of reperfusion reduced hippocampal CA1
neuronal injury in rats subjected to forebrain ischemia.
Buchan, et al. also tested the neuroprotective effects of
ziconotide in a transient focal model of ischemic damage
. Significant neuroprotection (reduction of infarct
volume) was observed one day following the ischemic event,
even with a 1 hour delay in administration of drug. Not
surprisingly, the hypotensive effects of i.v. ziconotide and a
concomitant reduction in cerebral blood flow were noted.
Thus, ziconotide was neuroprotective despite the fact that
blood flows were reduced to levels at which brain cells in
the ischemic cortex would not be expected to survive. In a
follow-up study, the permanence of the neuroprotective
effects of ziconotide was examined in the transient forebrain
ischemia model . Ziconotide significantly reduced the
neurodegeneration of hippocampal CA1 neurons at 7 days
post-reperfusion (< 35% cell death), but not at 28 days (>
80% cell death), in contrast to the findings of Valentino, et
al. at 12 days post-reperfusion in a similar model (see
above). In a rat transient middle cerebral artery occlusion
model and using histochemical and magnetic resonance
imaging techniques, Yenari et al. showed significant
reduction in infarct size with ziconotide administered 30
minutes post reperfusion when assessed at 1.5 hours after
reperfusion, and a trend toward neuroprotection when
assessed at 24 hours after reperfusion . Using similar
techniques, but in the rabbit, ziconotide was also shown to
reduce infarct size . Interestingly, no significant
differences in cerebral blood flow or mean arterial blood
pressure was observed between i.v. ziconotide-treated and
saline-treated animals. Similar neuroprotection was obtained
in hypertensive rats subjected to right middle cerebral artery
occlusion . Zhao et al., using a rat focal ischemia
model, showed that ziconotide achieved an 80% reduction of
cortical infarction. In another rat study of the effects of i.v.
ziconotide in a model of focal ischemia, Takizawa, et al.
showed approximately 60% decrease in infarct size in treated
animals . Concomitant with this neuroprotective effect,
extracellular brain glutamate levels were also reduced with
ziconotide treatment. Whether or not the observed
neuroprotective activity resulted from a reduction of
pathological levels of intracellular calcium, and whether this
was due to the direct blockade of N-VSCCs or through a
more general suppression of neuronal activity, including
synaptic transmission, or some combination of these effects,
remains to be determined.
In a rat model of spinal cord ischemia, modest
neuroprotection was achieved with continuous i.t.
administration of ziconotide, as measured by motor
function, spinal neuronal degeneration, and MAP2
immunoreactivity . Based on these findings, the authors
suggested that NCCBs such as ziconotide may provide
spinal neuroprotection for aortic aneurysm surgery patients.
Several studies have demonstrated the neuroprotective
activity of ziconotide in models of traumatic brain injury
(TBI). Because accumulation of calcium in the brain is a
prominent marker associated with cellular damage following
TBI, the effects of i.v. ziconotide on calcium accumulation
were studied in a rat lateral fluid percussion injury model of
TBI . Ziconotide dose-dependently reduced by as much
as 75% injury-induced calcium accumulation in the cerebral
cortex ipsilateral to the injury. Mitochondrial dysfunction is
also correlated with brain damage in rats and humans .
Ziconotide Current Medicinal Chemistry, 2004, Vol. 11, No. 23 3035
Intravenous ziconotide, even when administered 4 hours
post-injury, was effective in significantly improving
mitochondrial function by several biochemical measures. In
a companion study, ziconotide was administered 3, 5, and
24 hours after TBI, and rats were tested for motor and
cognitive performance up to 42 days post-injury. Saline-
treated rats displayed severe motor and cognitive deficits,
while ziconotide-treated animals displayed relatively better
motor and cognitive function .
Consistent with the pharmacokinetic data summarized
above, these studies demonstrated that, while the i.v. route
is relatively inefficient for delivering therapeutic levels of
ziconotide to the brain, the small fraction that does penetrate
the CNS is, nonetheless, efficacious. At the i.v. doses of
ziconotide employed, systemic blood pressure is reduced.
Whether or not this hypotension results in a significant
reduction in cerebral blood flow, ziconotide still confers
significant neuroprotective efficacy.
Beyond outright efficacy, these studies highlight two
additional properties that are likely to be key for predicting
from animal studies the clinical success of ziconotide or
other NCCBs for neuroprotection. First, ziconotide
treatment is neuroprotective up to 24 hours post-injury.
Efficacy with delayed treatment is critical for a
neuroprotective therapeutic because the occurrence of
ischemic insults in humans is usually not predictable and
provision of treatment may be delayed for hours after the
insult. In fact, the neuroprotective efficacy and potency of
ziconotide is increased when treatment is delayed,
suggesting that the as-yet-unknown calcium-dependent step
inhibited by ziconotide occurs many hours after initiation of
the neurodegenerative cascade. Secondly, the neuroprotective
effects of ziconotide may be persistent and appear to last for
at least 12 days following ischemia and at least 42 following
TBI. Thus this neuroprotection may not simply be a delay
in the neurodegenerative process, but actual permanent
The positive neuroprotective activity observed in animal
models prompted the initiation of studies to determine the
neuroprotective efficacy of ziconotide in humans. The i.v.
route was chosen over other more invasive routes. The
disadvantages of the need for considerably larger amounts of
drug, as well as potential issues associated with orthostatic
hypotension, were apparently out-weighed by the
convenience of this route of delivery. In principle,
hypotension induced by a short course of drug treatment
could be managed in a clinical setting populated by
recumbent patients. The results of a phase 1 safety study of
i.v. ziconotide in healthy volunteer subjects to support
subsequent clinical studies of neuroprotective efficacy have
been reported (see above) . Subsequent trials of
ziconotide’s neuroprotective efficacy were suspended in
phase 2 and the results have not, as yet, been published.
The anti-nociceptive activities of ziconotide and its
homologue, GVIA, in rats have been extensively studied and
reported. A few of the key studies are discussed below.
In one of the first studies clearly demonstrating the anti-
nociceptive properties of ziconotide, Malmberg and Yaksh
employed the formalin test to show that intrathecally infused
ziconotide produced a dose-dependent inhibition of both the
phase 1 (acute pain) and phase 2 (persistent pain) of the test
. In contrast, P/Q- and L-VSCC blockers had minimal
effects on either phase of the formalin test at the highest
doses examined. The P-VSCC blocker, omega-agatoxin
IVA, produced a 40% inhibition of phase 1 at the highest
dose tested and phase 2 was substantially suppressed in a
dose-dependent fashion. High doses of ziconotide produced
characteristic shaking behavior, serpentine-like tail
movements and impaired coordination. However, at
antinociceptive doses there was no significant motor effect.
In a similar study, these investigators showed that
ziconotide produced a significant reduction of the response
to formalin at both 2 and 7 days of infusion compared to
saline infusion . In contrast, morphine infusion produced
a significant effect only on day 2, but not on infusion day 7,
indicating that tolerance had developed. The effect of
ziconotide was reversible, as shown by a return to baseline
of nociceptive responses 2 days after termination of the 7-
day infusion. These data indicate that chronic infusion of
ziconotide can produce significant antinociceptive action
without development of tolerance.
Peripheral nerve lesions in rodents can result in
exaggerated pain responses to low intensity mechanical
stimuli (tactile allodynia) and serve as a model for
neuropathic pain in humans. Chaplan, et al. examined the
effects of ziconotide in the spinal nerve ligation model of
neuropathic pain in which tactile allodynia is induced by
tight unilateral ligation of the fifth and sixth lumbar spinal
nerves . Ziconotide was administered via intrathecal,
i.v., or regional nerve block catheters implanted in rats.
Intrathecally delivered ziconotide produced dose-dependent
blockade of tactile allodynia, while intrathecal L-, P/Q-, and
P-VSCC blockers had no effect on pain behavior at the
highest doses examined. No VSCC antagonist suppressed
nociceptive behavior when administered i.v. or when applied
regionally to the injured portion of the nerve. The anti-
nociceptive activities of ziconotide in the formalin test and
the spinal nerve ligation model were confirmed in detail by
Wang, et al. . That is, intrathecal ziconotide is at least
as efficacious and at least 1000 times more potent than
intrathecal morphine in the formalin test and is potently
efficacious in neuropathic pain models where morphine is
much less effective. Ziconotide produces no desensitization
or self-tolerance, in marked contrast to morphine. At doses
above those that are anti-nociceptive, intraspinal ziconotide
elicits a characteristic supra-spinally-mediated shaking, or
tremor. Nakanishi, et al. correlated the anti-nociceptive
activity of N-VSCC blockade with inhibition of glutamate
release . GVIA attenuated phase 1 and 2 flinch responses
evoked by injection of formalin and suppressed the increase
in CSF glutamate after formalin injection into the rat
hindpaw. In this study, morphine had similar effects. Thus,
GVIA appears to reduce formalin-evoked hyperalgesia by
inhibiting N-VSCC-dependent presynaptic glutamate release
from C fibers.
The anti-nociceptive properties of both ziconotide and
GVIA have been tested in rodent models of joint
inflammation. Spinal administration of ziconotide through a
3036 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
microdialysis fiber placed in the dorsal horn, before
induction by kaolin-carrageenan of knee joint inflammation,
prevented nociceptive behavior . In addition, treatment
with ziconotide 4 hours after inflammation reverses
nociceptive behavior. A small reduction in spontaneous
pain-related behaviors (guarding of the limb) also was
observed after pre- or post-treatment with ziconotide. In
contrast, a P/Q-VSCC blocker was anti-nociceptive with pre-
treatment, but had no effect when administered after
induction of inflammation. L-VSCC blockers had no anti-
nociceptive effects. None of the VSCC blockers reduced
joint swelling under any conditions. Sluka concluded that
N-VSCCs contribute to both the development and
maintenance of hyperalgesia associated with joint
inflammation, while P-VSCCs are only involved during
development of hyperalgesia. Nebe, et al. showed that i.t.
administration of GVIA reduced spinal neuronal responses to
innocuous and noxious pressure applied to the knee. Effects
were observed both in rats with normal knees and in rats in
which knee inflammation by intra-articular injection of
mustard oil had induced a state of hyperexcitability in spinal
neurons . When GVIA was applied onto the spinal cord
above the recording site, the responses to articular
stimulation were reduced. Thus, the spinal application of
GVIA reduced, but did not completely prevent, the fast and
slow development of neuronal hyperexcitability of spinal
cord neurons. In a similar study in which knee joint
inflammation was induced by injection of kaolin-
carrageenan, a P-VSCC blocker applied to the spinal cord
surface above the recorded neuron prevented the increase of
the neuronal responses to innocuous pressure onto the knee
and to innocuous and noxious pressure onto the ankle .
This suggested that P-VSCCs, as well as N-VSCCs, in the
spinal cord are involved in the generation and maintenance
of inflammation-evoked hyperexcitability of spinal cord
Horvath, et al. examined the ability of ziconotide to
inhibit the transmission of noxious stimuli from the
mesentery in a rat model of visceral pain . In this model,
transient changes in mean arterial blood pressure in response
to mechanical pressure or to the application of acetic acid
were taken as an indication of nociception. Hypertensive
reflexes were dose-dependently reduced by intrathecal
morphine or clonidine, but were left unaltered by intrathecal
administration of ziconotide or the L-VSCC blocker,
verapamil. However, ziconotide markedly enhanced the
effectiveness of all doses of clonidine against both types of
mesenteric stimuli. Interestingly, ziconotide reduced the
ability of morphine to inhibit mechanically evoked reflexes,
while there was no statistically significant effect in
chemonociception. The authors suggest combined
administration of clonidine with ziconotide might lead to
effective control of visceral pain and with reduced side
Taken together, the above results clearly demonstrate
that, with intrathecal delivery, ziconotide is potently
effective in several rodent models of persistent pain,
inflammatory pain and neuropathic pain.
In three different strains of Cav2.2 genetic knockout mice
deficient in N-type VSCCs, the transgenic animals display a
hypoalgesic phenotype in several models of pain [70, 71,
72, 73]. Omega-conopeptide-sensitive N-type calcium
currents in neurons from these mice are completely
eliminated. These results firmly establish that blockade of
N-VSCC is the molecular locus of the anti-nociceptive
activity of ziconotide.
The positive results in the animal studies described
above provided the foundation for the study of the analgesic
efficacy of ziconotide in the clinic. Just as with the pre-
clinical work, because of its low tissue penetrability and its
potent sympathetically mediated hypotensive effects when
administered systemically, ziconotide is administered to
human subjects by continuous infusion by an external or an
implanted pump via an intrathecal catheter. An initial open-
label phase1/phase2 study preliminarily assessed the safety,
tolerability, and analgesic potential of ziconotide . The
study included patients with severe, chronic pain as a
consequence of cancer, AIDS, spinal cord injury, thalamic
pain and brachial plexus avulsion. All had previously failed
to obtain relief from opiate therapy, including in some cases,
intrathecal opiates. Of the 24 patients who completed the
study, visual analog scale of pain intensity (VASPI) scores
of 19 were reduced by an average of 43%. In 15 patients,
concomitant opioid use was reduced by at least 50%. In
contrast to morphine, ziconotide did not produce respiratory
depression and no evidence of tolerance to ziconotide was
detected. The most common adverse effects were nystagmus,
confusion, nausea, dizziness, headache and gait disturbance.
These effects are presumed to be due to the diffusion of the
drug to the brain from its site of infusion in the spinal cord,
and were reversed upon discontinuation or reduction of
dosing. Importantly, no generalized insensitivities to non-
painful stimuli were reported by the study subjects.
Encouraged by these results, randomized, double-blind
and placebo-controlled phase 3 studies of ziconotide were
initiated [5, 74]. The results from one of these studies
treating severe, chronic pain in 111 patients with cancer or
AIDS have been reported . All patients entered in the
study had failed to obtain adequate pain relief from analgesic
therapies including oral and/or intrathecal opiates. Despite
the apparently inadequate analgesic efficacy, at the start of
the study, 92% of the patients were taking opioids.
Intrathecal ziconotide was titrated upwards in dose over 5-6
days to the point of analgesic effect, or to a maximum
allowed dose if no analgesia was obtained. Similar to the
open-label study, the main measure of analgesic efficacy was
the mean percentage change in VASPI from baseline prior to
dosing, to that at the end of the titration period. Mean scores
improved by 53% in the ziconotide group compared with
18% in the placebo group. The VASPI is a scale commonly
used in assessments of pain therapy, in which patients rate
their pain on a scale of 0 mm to 100 mm corresponding to a
range of no pain to worst imaginable pain, respectively. The
53% improvement in mean VASPI score translates into a
reduction of mean score from 74mm to 35mm for the
treatment group. Pain relief was reported to be modest or
complete in 53% of ziconotide-treated patients, while only
17% of the placebo-treated patients reported modest pain
relief and none reported complete relief. The level of
concomitant opiate use was decreased by 10% in the
Ziconotide Current Medicinal Chemistry, 2004, Vol. 11, No. 23 3037
ziconotide group, but increased by 5% in the placebo group.
Also similar to the results from the open-label study,
ziconotide treatment was associated with a number of
adverse events: abnormal gait, dizziness, nystagmus,
confusion, somnolence, fever, postural hypotension, urinary
retention, nausea and vomiting. Again, most of these are
thought to be a consequence of the movement of ziconotide
rostrally up the neuraxis leading to block of N-VSCCs in
the brain. According to the authors, further clinical studies –
both completed and on-going -- using lower dosages of
ziconotide and longer durations of treatment will better
define the long-term risk-benefit profile of ziconotide therapy
for severe chronic pain.
The safety and analgesic efficacy of ziconotide when
administered intrathecally to patients with acute
postoperative pain were assessed in a randomized, double-
blind, pilot study including patients undergoing elective
total abdominal hysterectomy, radical prostatectomy or total
hip replacement . After intrathecal injection of local
anesthetic and before surgical incision, a continuous
intrathecal infusion of either placebo or 1 of 2 doses of
ziconotide was started and continued for 48 to 72 hours
postoperatively. Primary and secondary efficacy was
measured by mean daily patient controlled analgesia (PCA)
opiate consumption and VASPI scores. In the 26 patients
evaluated for efficacy, mean daily PCA opiate consumption
was less in patients receiving ziconotide than in placebo-
treated patients. VASPI scores during the first 8 hours
postoperatively were markedly lower in ziconotide-treated
than in placebo-treated patients. In 4 of 6 patients receiving
the high-dose of ziconotide, adverse events, such as
dizziness, blurred vision, nystagmus and sedation were
observed. After ziconotide discontinuation, these symptoms
resolved. Thus, ziconotide showed analgesic activity, as
reflected in decreased PCA opiate consumption and lower
VASPI scores with an acceptable side-effect profile in the
low-dose ziconotide group.
MECHANISM OF ACTION – IMPLICATIONS FOR
As discussed above, ziconotide very likely achieves its
analgesic efficacy through selective blockade of the N-
VSCCs on the spinal presynaptic terminals of nociceptive
neurons [2, 4, 17, 73]. Similarly, opiates achieve a
substantial portion of their analgesic efficacy through the
inhibition of N-VSCCs on nociceptor terminals as well [see
for example, 75]. How, then, is it possible that the highly
specific NCCB, ziconotide, is effective in pain for patients
who fail therapy with opiates and/or continue on oral opiate
therapy concomitant with ziconotide treatment? The answer
lies in the molecular and cellular mechanisms of action that
confer differential pharmacologies to these two agents and,
in principle, allow the additivity of their analgesic activities.
That is, opiates indirectly modulate N-VSCCs through a G-
protein-coupled mechanism inititated by binding to µ opiate
receptors , while ziconotide directly inhibits N-VSCCs
by binding to and blocking the Ca++ permeability of the
channel itself . Opiate modulation results in only a
reduction of the probability of opening of N-VSSCs, but not
in the essentially complete block achieved with a pore
blocker such as ziconotide. Moreover, not all presynaptic N-
VSCCs are productively linked to G-protein-coupled opiate
receptor modulation. For example, in rats, N-VSCCs
mediate excitatory neurotransmitter release from both C-fiber
and A-fiber nociceptors, but opiates modulate this release
predominantly from C-fiber terminals only . As a result,
morphine and the ziconotide analogue, omega-conopeptide
GVIA, have differential effects on A-fiber and C-fiber-
mediated pain behavior, which leads to the additive or super-
additive anti-nociceptive activities of these two agents [16,
18, 77]. In further support of the notion that only a subset of
N channels are modulated by opiates, morphine inhibits N-
channel calcium current in only about two-thirds of
dissociated rat DRG neurons, while (by definition) omega-
conopeptide GVIA blocks all such current .
Perhaps the best-known example of a lack of coupling
between opiate receptors and some N-VSCCs is the
phenomenon of opiate tolerance or desensitization, in which
repeated application of opiates results in decreased analgesic
efficacy. While the complete set of molecular mechanisms
underlying this tolerance remains to be elucidated, it is
correlated with the loss of opiate receptor-mediated G-
protein-coupled inhibition of N-VSCC activity . What
fraction of additional ziconotide efficacy in the ziconotide-
responsive, opiate-resistant patients is due to inhibition of
N-VSCCs that are never coupled to opiate receptors and
what fraction is due to the inhibition of N-VSCCs
uncoupled from opiate receptors following the development
opiate tolerance in these patients remains a matter of
conjecture. The efficacy of ziconotide in opiate-resistant
patients was anticipated by the results of several rodent
studies that found that the analgesic activities of morphine
and ziconotide (or GVIA) are additive and that these NCCBs
retain analgesic activity even in morphine-tolerant animals
The direct antagonism of N-VSCCs by ziconotide also
explains the lack of tolerance to ziconotide in experimental
animals and pain patients, and is consistent with the general
observation that tolerance is more likely to develop to an
agonist than to an antagonist. In order to blunt the effects of
an opiate such as morphine, nociceptors simply uncouple N-
VSCCs from modulation by opiate receptors. However, no
such simple molecular maneuver is available to nociceptors
to reverse the action of a direct calcium channel antagonist,
such as ziconotide.
A further factor underlying the differential
pharmacologies of ziconotide and opiates stems from the
multiple modes of action of opiates compared to the highly
selective action of ziconotide. These additional modes of
action include modulation of potassium channels, other
voltage-sensitive calcium channel subtypes and glutamate
Thus, ziconotide’s novel and distinct pharmacology
potentially gives rise to three properties of ziconotide that
are consistent with its observed effects on patients: 1)
efficacy in patients who have failed opiate therapy, 2)
additive efficacy in patients being treated concomitantly with
opiates, even those who may be experiencing opiate
tolerance, and 3) lack of tolerance to ziconotide. As a result,
ziconotide may have a unique and useful place in our
pharmacopeia of pain therapies -- with the two
mechanistically distinct classes of N-channel inhibitors,
3038 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
ziconotide and the opiates, being used effectively in
combination in some chronic pain patients to enhance
analgesic activity and minimize adverse effects.
As the first NCCB to be tested in humans, ziconotide
has been shown both non-clinically and clinically to be an
effective analgesic. The intrathecal route of administration
and dose-limiting adverse effects will, however, limit its use
to a subpopulation of pain sufferers. The molecular diversity
of N-VSCCs offers the possibility that a second-generation
NCCB might overcome one or both of these limitations. A
major source of this molecular diversity arises from
alternatively spliced N-VSCC mRNAs, which yield N-
VSCCs with distinct neuronal localizations and, perhaps,
functional properties [80, 81]. The functional properties of
N-VSCCs can also be differentially affected by selective
association with auxiliary subunits for which there are
multiple genes and multiple splice variants, as well . All
these structurally variant multimolecular N-VSCC
complexes have the potential for possessing differential
pharmacologies, including differential sensitivities to the
numerous N-VSCC-blocking omega-conopeptides that exist
and/or those yet to be discovered or engineered, as well. For
example, one ω-conopeptide, CVID or AM-336, which is
very similar in amino acid sequence to ziconotide, is anti-
nociceptive in rats but is reported to have a smaller effect on
blood pressure and produce less shaking activity than
ziconotide at anti-nociceptive doses . It is speculated that
this is due to a higher potency against an N-VSCC splice
variant that populates primary nociceptive neurons relative to
its potency against N-VSCC splice variant(s) predominating
on sympathetic afferents or neurons in the brain. Thus, while
recognizing that less than 0.1% of the total dose is likely to
be centrally available, systemically administered AM-336
might be a feasible analgesic. Alternatively, if administered
intrathecally for pain, AM-336 might have a higher
therapeutic index than ziconotide. Furthermore, a relative
lack of sympathetic effects, if true, may allow the systemic
use of an N-VSCC-blocking ω-conopeptide such as AM-336
for neuroprotective therapy. AM-336 is currently in phase 2
clinical trials in Australia for chronic pain. Another newly-
discovered VSCC-blocking ω-conopeptide with an emerging
pharmacological profile of interest is CNVIIA . In
contrast to GVIA and ziconotide, this conopeptide does not
block the VSCCs at the frog neuromuscular junction. It will
be interesting to watch the N-VSCC variant blocking
selectivity, if any, of CNVIIA, as it unfolds.
The key molecular features that endow ziconotide and
other omega-conopeptides with high affinity and high
selectivity for N-VSCCs have been determined [85, 86]. In
addition, the three-dimensional structures of several omega-
conopeptides have been solved [23, 24, 87]. As the
physiological roles of the various N-VSCC splice variants
become better understood, the possibility of rational design
of safer, more effective splice variant-selective conopeptide-
based NCCBs becomes more likely.
This is also true for the design and development of small
molecule blockers of N-VSCCs. Several have been reported
and appear to have pharmacologies distinct from ziconotide
[88, 89, 90, 91]. Some of the observed differential
pharmacology could be due to N-VSCC splice variant
selectivity that might herald the development of orally-
available, safer, and more effective small molecule NCCBs.
Ziconotide is the furthest along in development of any of
the many neuroactive conopeptides and points the way for
the development of other neuroactive peptides as
therapeutics. Ziconotide also represents the first generation
of the NCCB therapeutic class. The generations of NCCBs
that follow will almost certainly include analgesics with
improved efficacy, safety and/or pharmacokinetic properties
but may also include neuroprotectants and neuroleptics with
novel therapeutic profiles. The development of ziconotide
clearly reveals the challenges inherent in developing peptide
therapeutics, but also highlights the unique mechanisms of
action and exquisite target specificity commonly found in
neuroactive peptides that may be exploited to bring relief to
sufferers of a variety of neuropathologies.
Terlau, H.; Olivera, B.M., Physiol. Rev., 2004, 84(1), 41-68.
Miljanich, G.P.; Ramachandran, J., Annu. Rev. Pharmacol.
Toxicol. 1995, 35, 707-734.
Newcomb, R.N.; Miljanich, G.P., In The Handbook of
Neurotoxicology; Massaro, Ed.; Humana Press Inc.: Totowa, NJ,
2002; Vol. 1, pp. 617-651.
Bowersox, S.S.; Luther, R., Toxicon. 1998, 36(11), 1651-1658.
Staats, P.S.; Yearwood, T.; Charapata, S.G.; Presley, R.W.;
Wallace, M.S.; Byas-Smith, M.; Fisher, R.; Bryce, D.A.; Mangieri,
E.A.; Luther, R.R.; Mayo, M.; McGuire, D.; Ellis, D., JAMA 2004,
Wu, L.G.; Borst, J.G.; Sakmann, B., Proc. Natl. Acad. Sci. U. S. A.
1998, 95(8), 4720-4725.
Murakami, N.; Ishibashi, H.; Katsurabayashi, S,; Akaike, N.,
Brain Res. 2002, 951(1),121-129.
Martinez-Pinna, J.; Lamas, J.A.; Gallego, R., Brain Res. 2002,
Doroshenko, P.A.; Woppmann, A.; Miljanich, G.; Augustine, G.J.,
Neuropharmacology 1997, 36(6), 865-872.
Timmermann, D.B.; Westenbroek, R.E.; Schousboe, A.; Catterall,
W.A., J. Neurosci. Res. 2002, 67(1), 48-61.
Turner, T.J.; Dunlap, K., Neuropharmacology. 1995, 34(11),
McCleskey, E.W.; Fox, A.P.; Feldman, D.H.; Cruz, L.J.; Olivera,
B.M.; Tsien, R.W.; Yoshikami, D., Proc. Natl. Acad. Sci. U. S. A.
1987, 84(12), 4327-4331.
Olivera, B.M.; Miljanich, G.P.; Ramachandran, J.; Adams, M.E.,
Annu. Rev. Biochem. 1994, 63, 823-867.
Malmberg, A.B.; Yaksh, T.L., J. Neurosci. 1994, 14(8), 4882-
Chaplan, S.R.; Pogrel, J.W.; Yaksh, T.L., J. Pharmacol. Exp. Ther.
1994, 269(3), 1117-1123.
Pirec, V.; Laurito, C.E.; Lu, Y.; Yeomans, D.C., Anesth. Analg.
2001, 92(1), 239-243.
Malmberg, A.B.; Yaksh, T.L., Pain 1995, 60(1), 83-90.
Wang, Y.X.; Gao, D.; Pettus, M.; Phillips, C.; Bowersox, S.S., Pain
2000, 84(2-3), 271-281.
Omote, K.; Kawamata, M.; Satoh, O.; Iwasaki, H.; Namiki, A.,
Anesthesiology 1996, 84(3), 636-643.
Atanassoff, P.G.; Hartmannsgruber, M.W.; Thrasher, J.;
Wermeling, D.; Longton, W.; Gaeta, R.; Singh, T.; Mayo, M.;
McGuire, D.; Luther, R.R., Reg. Anesth. Pain Med. 2000, 25(3),
Wermeling, D.; Drass, M.; Ellis, D.; Mayo, M.; McGuire, D.;
O'Connell, D.; Hale, V.; Chao, S., J. Clin. Pharmacol. 2003, 43(6),
ZiconotideCurrent Medicinal Chemistry, 2004, Vol. 11, No. 23 3039
 Olivera, B.M.; Gray, W.R.; Zeikus, R.; McIntosh, J.M.; Varga, J.;
Rivier, J.; de Santos, V.; Cruz, L.J., Science 1985, 230(4732),
Kohno, T.; Kim, J.I.; Kobayashi, K.; Kodera, Y.; Maeda, T.; Sato,
K., Biochemistry 1995, 34(32), 10256-10265.
Basus, V.J,; Nadasdi, L.; Ramachandran, J.; Miljanich, G.P., FEBS
Lett. 1995, 370(3), 163-169.
Li, S.; Schoneich, C.; Borchardt, R.T., Pharm. Res. 1995, 12(3),
Kerr, L.M.; Yoshikami, D., Nature 1984, 308(5956), 282-284.
Yeager, R.E,; Yoshikami, D.; Rivier, J.; Cruz, L.J.; Miljanich,
G.P., J. Neurosci. 1987, 7(8), 2390-2396.
Feng, Z.P.; Doering, C.J.; Winkfein, R.J.; Beedle, A.M.; Spafford,
J.D.; Zamponi, G.W., J. Biol. Chem. 2003, 278(22), 20171-20178.
Kristipati, R.; Nadasdi, L.; Tarczy-Hornoch, K.; Lau, K.;
Miljanich, G.P.; Ramachandran, J.; Bell, J.R., Mol. Cell Neurosci.
1994, 5(3), 219-228.
Kurihara, T.; Tanabe, T., Nippon Yakurigaku Zasshi. 2003, 121(4),
Miller, R.J., Trends Neurosci. 2001, 24(8), 445-449.
Ino, M.; Yoshinaga, T.; Wakamori, M.; Miyamoto, N.; Takahashi,
E.; Sonoda, J.; Kagaya, T.; Oki, T.; Nagasu, T.; Nishizawa, Y.;
Tanaka, I.; Imoto, K.; Aizawa, S.; Koch, S.; Schwartz, A.;
Niidome, T.; Sawada, K.; Mori, Y., Proc. Natl. Acad. Sci. U. S. A.
2001, 98(9), 5323-5328.
Westenbroek, R.E.; Hell, J.W.; Warner, C.; Dubel, S.J.; Snutch,
T.P.; Catterall, W.A., Neuron 1992, 9(6), 1099-1115.
Takemura, M.; Kiyama, H.; Fukui, H.; Tohyama, M.; Wada, H.,
Brain Res. 1988, 451(1-2), 386-389.
Kerr, L.M.; Filloux, F.; Olivera, B.M.; Jackson, H.; Wamsley, J.K.,
Eur. J. Pharmacol. 1988, 146(1), 181-183.
Gohil, K.; Bell, J.R.; Ramachandran, J.; Miljanich, G.P., Brain Res.
1994, 653(1-2), 258-266.
Westenbroek, R.E.; Hoskins, L.; Catterall, W.A., J. Neurosci.
1998, 18(16), 6319-6330.
Bowersox, S.S.; Singh, T.; Nadasdi, L.; Zukowska-Grojec, Z.;
Valentino, K.; Hoffman, B.B., J. Cardiovasc. Pharmacol. 1992,
McGuire, D.; Bowersox, S.; Fellmann, J.D.; Luther, R.R., J.
Cardiovasc. Pharmacol. 1997, 30(3), 400-403.
Westenbroek, R.E.; Sakurai, T.; Elliott, E.M.; Hell, J.W.; Starr,
T.V.; Snutch, T.P.; Catterall, W.A., J. Neurosci. 1995, 15(10),
Jun, K.; Piedras-Renteria, E.S.; Smith, S.M.; Wheeler, D.B.; Lee,
S.B.; Lee, T.G.; Chin, H.; Adams, M.E.; Scheller, R.H.; Tsien,
R.W.; Shin, H.S., Proc. Natl. Acad. Sci. U. S. A. 1999, 96(26),
Olivera, B.M.; Cruz, L.J.; Yoshikami, D., Curr. Opin. Neurobiol.
1999, 9(6), 772-777.
Bowersox, S.S.; Miljanich, G.P.; Sugiura, Y.; Li, C.; Nadasdi, L.;
Hoffman, B.B.; Ramachandran, J.; Ko, C.P., J. Pharmacol. Exp.
Ther. 1995, 273(1), 248-256.
Sugiura, Y.; Woppmann, A.; Miljanich, G.P.; Ko, C.P., J.
Neurocytol. 1995, 24(1), 15-27.
Protti, D.A.; Uchitel, O.D., Neuroreport 1993, 5(3), 333-336.
Hillyard, D.R.; Monje, V.D.; Mintz, I.M.; Bean, B.P.; Nadasdi, L.;
Ramachandran, J.; Miljanich, G.; Azimi-Zoonooz, A.; McIntosh,
J.M.; Cruz, L.J.; Imperial, J.S.; Olivera, B.M., Neuron 1992, 9(1),
Protti, D.A.; Reisin, R.; Mackinley, T.A.; Uchitel, O.D., Neurology
1996, 46(5), 1391-1396.
Albillos, A.; Neher, E.; Moser, T., J. Neurosci. 2000, 20(22),
Wang, G.; Dayanithi, G.; Newcomb, R.; Lemos, J.R., J. Neurosci.
1999, 19(21), 9235-9241.
Artalejo, C.R.; Perlman, R.L.; Fox, A.P., Neuron 1992, 8(1), 85-95.
Valentino, K.; Newcomb, R.; Gadbois, T.; Singh, T.; Bowersox, S.;
Bitner, S.; Justice, A.; Yamashiro, D.; Hoffman, B.B.; Ciaranello,
R.; Miljanich. G.; Ramachandran, J., Proc. Natl. Acad. Sci. U. S.
A. 1993, 90(16), 7894-7897.
Newcomb, R.; Abbruscato, T.J.; Singh, T.; Nadasdi, L.; Davis,
T.P.; Miljanich, G., Peptides 2000, 21(4), 491-501.
Gaur, S.; Newcomb, R.; Rivnay, B.; Bell, J.R.; Yamashiro, D.;
Ramachandran, J.; Miljanich, G.P., Neuropharmacology 1994,
Bowersox, S.; Mandema, J.; Tarczy-Hornoch, K.; Miljanich, G.;
Luther, R.R., Drug Metab. Dispos. 1997, 25(3), 379-383.
 Takizawa, S.; Matsushima, K.; Fujita, H.; Nanri, K.; Ogawa, S.;
Shinohara, Y., J. Cereb. Blood Flow Metab. 1995, 15(4), 611-618.
Buchan, A.M.; Gertler, S.Z.; Li, H.; Xue, D.; Huang, Z.G.;
Chaundy, K.E.; Barnes, K.; Lesiuk, H.J., J. Cereb. Blood Flow
Metab. 1994, 14(6), 903-910.
Colbourne, F.; Li, H.; Buchan, A.M.; Clemens, J.A., Stroke 1999,
Yenari, M.A.; Palmer, J.T.; Sun, G.H.; de Crespigny, A.; Mosely,
M.E.; Steinberg, G.K., Brain Res. 1996, 739(1-2), 36-45.
Perez-Pinzon, M.A.; Yenari, M.A.; Sun, G.H.; Kunis, D.M.;
Steinberg, G.K., J. Neurol. Sci. 1997, 153(1), 25-31.
Zhao, Q.; Smith, M.L.; Siesjo, B.K., Acta Physiol. Scand. 1994,
Burns, L.H.; Jin, Z.; Bowersox, S.S., J. Vasc. Surg. 1999, 30(2),
Samii, A.; Badie, H.; Fu. K.; Luther, R.R.; Hovda, D.A., J.
Neurotrauma 1999, 16(10), 879-892.
Verweij, B.H.; Muizelaar, J.P.; Vinas, F.C.; Peterson, P.L.; Xiong,
Y.; Lee, C.P., J. Neurosurg. 2000, 93(5), 829-834.
Berman, R.F.; Verweij, B.H.; Muizelaar, J.P., J. Neurosurg. 2000,
Nakanishi, O.; Ishikawa, T.; Imamura, Y., Cell Mol. Neurobiol.
1999, 19(2), 191-197.
Sluka, K.A., J. Pharmacol. Exp. Ther. 1998, 287(1), 232-237.
Nebe, J.; Vanegas, H.; Schaible, H.G., Exp. Brain Res. 1998,
Nebe, J.; Vanegas, H.; Neugebauer, V.; Schaible, H.G., Eur. J.
Neurosci. 1997, 9(10), 2193-2201.
Horvath, G.; Brodacz, B.; Holzer-Petsche, U., Pain 2001, 93(1),
Saegusa, H.; Kurihara, T.; Zong, S.; Kazuno, A.; Matsuda, Y.;
Nonaka, T.; Han, W.; Toriyama, H.; Tanabe, T., EMBO J. 2001,
Hatakeyama, S.; Wakamori, M.; Ino, M.; Miyamoto, N.;
Takahashi, E.; Yoshinaga, T.; Sawada, K.; Imoto, K.; Tanaka, I.;
Yoshizawa, T.; Nishizawa, Y.; Mori, Y.; Niidome, T.; Shoji, S.,
Neuroreport 2001, 12(11), 2423-2427.
Kim, C.; Jun, K.; Lee, T.; Kim, S.S.; McEnery, M.W.; Chin, H.;
Kim, H.L.; Park, J.M.; Kim, D.K.; Jung, S.J.; Kim, J.; Shin, H.S.,
Mol. Cell. Neurosci. 2001, 18(2), 235-245.
Saegusa, H.; Matsuda, Y.; Tanabe, T., Neurosci. Res. 2002, 43(1),
Mathur, V.S., Seminars in Anesthesia, Perioperative Medicine and
Pain 2000, 19(2), 67-75.
Nestler, E.J.; Alreja, M.; Aghajanian, G.K., Brain Res. Bull. 1994,
Attali, B.; Saya, D.; Nah, S.Y.; Vogel, Z., J. Biol. Chem. 1989,
Omote, K.; Kawamata, M.; Satoh, O.; Iwasaki, H.; Namiki, A.,
Anesthesiology 1996, 84(3), 636-643.
Bohn, L.M.; Gainetdinov, R.R.; Lin, F.T.; Lefkowitz, R.J.; Caron,
M.G., Nature 2000, 408(6813), 720-723.
Law, P.Y.; Wong, Y.H.; Loh, H.H., Annu. Rev. Pharmacol.
Toxicol. 2000, 40, 389-430.
Bell, T.J.; Thaler, C.; Castiglioni, A.J.; Helton, T.D.; Lipscombe,
D., Neuron 2004, 41(1), 127-138.
Zamponi, G.W.; McCleskey, E.W., Neuron 2004, 41(1), 3-4.
Augustine, G.J., Curr. Opin. Neurobiol. 2001, 11(3), 320-326.
Scott, D.A.; Wright, C.E.; Angus, J.A., Eur. J. Pharmacol. 2002,
Favreau, P.; Gilles, N.; Lamthanh, H.; Bournaud, R.; Shimahara,
T.; Bouet, F.; Laboute, P.; Letourneux, Y.; Menez, A.; Molgo, J.;
Le Gall, F., Biochemistry 2001, 40(48), 14567-14575.
Nadasdi, L.; Yamashiro, D.; Chung, D.; Tarczy-Hornoch, K.;
Adriaenssens, P.; Ramachandran, J., Biochemistry 1995, 34(25),
Nielsen, K.J. Adams, D.; Thomas, L.; Bond, T.; Alewood, P.F.;
Craik, D.J.; Lewis, R.J., J. Mol. Biol. 1999, 289(5), 1405-1421.
MacLachlan, L.K.; Middleton, D.A.; Edwards, A.J.; Reid, D.G.,
Methods Mol. Biol. 1997, 60, 337-362.
Song, Y.; Bowersox, S.S.; Connor, D.T.; Dooley, D.J.; Lotarski,
S.M.; Malone, T.; Miljanich, G.; Millerman, E.; Rafferty, M.F.;
Rock, D.; Roth, B.D.; Schmidt, J.; Stoehr, S.; Szoke, B.G.; Taylor,
C.; Vartanian, M.; Wang, Y.X., J. Med. Chem. 2000, 43(19),
3040 Current Medicinal Chemistry, 2004, Vol. 11, No. 23G.P. Miljanich
Seko, T.; Kato, M.; Kohno, H.; Ono, S.; Hashimura, K.; Takenobu,
Y.; Takimizu, H.; Nakai, K.; Maegawa, H.; Katsube, N.; Toda,
M., Bioorg. Med. Chem. Lett. 2002, 12(17), 2267-2269.
Hu, L.Y.; Ryder, T.R.; Rafferty, M.F.; Taylor, C.P.; Feng, M.R.;
Kuo, B.S.; Lotarski, S.M.; Miljanich, G.P.; Millerman, E.; Siebers,
K.M.; Szoke, B.G., Bioorg. Med. Chem. 2000, 8(6), 1203-1212.
Menzler, S.; Bikker, J.A.; Suman-Chauhan, N.; Horwell, D.C.,
Bioorg. Med. Chem. Lett. 2000, 10(4), 345-347.