Further insights into the antinociceptive potential of a peptide disrupting the n-type calcium channel-crmp-2 signaling complex hypoxia anoxia

The N-type voltage-gated calcium channel (ca v2.2) has gained immense prominence in the treatment of chronic pain. While decreased channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse side effects. Targeting modulators of channel activity may facilitate improved analgesic properties associated with channel block and a broader therapeutic window. A novel interaction between ca v2.2 and collapsin response mediator protein 2 (CRMP-2) positively regulates channel function by increasing surface trafficking. We recently identified a CRMP-2 peptide (TAT-CBD3), which effectively blocks this interaction, reduces or completely reverses pain behavior in a number of inflammatory and neuropathic models.Hypoxia anoxia


importantly, TAT-CBD3 did not produce many of the typical side effects often observed with ca v2.2 inhibitors. Notably chronic pain mechanisms offer unique challenges as they often encompass a mix of both neuropathic and inflammatory elements, whereby inflammation likely causes damage to the neuron leading to neuropathic pain, and neuronal injury may produce inflammatory reactions. To this end, we sought to further disseminate the ability of TAT-CBD3 to alter behavioral outcomes in two additional rodent pain models. While we observed that TAT-CBD3 reversed mechanical hypersensitivity associated with a model of chronic inflammatory pain due to lysophosphatidylcholine-induced sciatic nerve focal demyelination (LPC), injury to the tibial nerve (TNI) failed to respond to drug treatment.Hypoxia anoxia moreover, a single amino acid mutation within the CBD3 sequence demonstrated amplified ca v2.2 binding and dramatically increased efficacy in an animal model of migraine. Taken together, TAT-CBD3 potentially represents a novel class of therapeutics targeting channel regulation as opposed to the channel itself.

CRMP-2, a novel target in pain research.

CRMP-2 is an axonal growth/guidance protein recently identified to interact with ca v2.2. 3 this interaction leads to increased calcium current density through upregulation of channel surface expression. Ultimately, the interaction between CRMP-2 and ca v2.2 promotes increased neurotransmitter release from both central and peripheral synapses. 3 – 5 therefore, we hypothesized that blocking this interaction could serve as a novel therapeutic target in the treatment of chronic pain conditions ( fig. 1A).Hypoxia anoxia in order to target this protein/protein interaction, we developed a 15 amino acid peptide (CBD3) containing the region of CRMP-2 responsible for binding ca v2.2. Consistent with the role of CRMP-2 in ca v2.2 trafficking, co-expression of CBD3 reduced ca v2.2 surface expression as well as subsequent calcium current density. Due to the dependence of synaptic transmission between nociceptors and dorsal horn neurons on ca v2.2, we hypothesized that modulation by CBD3 may have analgesic properties in vivo. Fusion of the HIV-1 trans-activator of transcription (TAT) domain to CBD3 resulted in a cell-permeant version (TAT-CBD3) allowing for systemic administration. TAT-CBD3 proved effective in ameliorating the hypersensitivity associated with various rodent models of chronic inflammatory and neuropathic pain states 6 – 8 ( fig. 1B).Hypoxia anoxia

Clinical efficacy is only achieved through compliance.

While pharmacologic inhibition of ca v2.2 is analgesic in nature, it is riddled with adverse side effects associated with the channel’s additional physiologic functions. The N-type specific synthetic blocker ω-conotoxin, prialt (intrathecal ziconotide), is FDA-approved for the treatment of severe intractable pain; however, its efficacy is confounded by a narrow therapeutic window and limited method of delivery. 9 , 10 side effects associated with prialt treatment range from mild to severe including, but not limited to, memory dysfunction, ataxia and decreased sympathetic tone. 9 , 11 despite profound effects on pain behavior, TAT-CBD3 did not affect memory retrieval, motor coordination or depression-associated behaviors ( table 1).Hypoxia anoxia interestingly, TAT-CBD3 was also slightly anxiolytic, which is consistent with observations from ca v2.2 knock-out mice. 12 an area of potential concern with TAT-CBD3 treatment may be the effect on the sympathetic nervous system. Unlike release from other synapses, which can involve multiple calcium channel subtypes, norephinephrine release from sympathetic neurons is mostly dependent upon the N-type channel. 13 , 14 indeed, ziconotide inhibits sympathetic norephinephrine release, 15 which is likely the mechanism underlying decreases in sympathetic tone observed during treatment. 10 , 16 in contrast, i.P. Injection of TAT-CBD3 (10 mg/kg) did not alter mean arterial pressure, heart rate, or core body temperature ( fig. 2).Hypoxia anoxia despite a relative lack of adverse side effects, animals receiving doses of TAT-CBD3 higher than that required to achieve analgesic effects (10 mg/kg) demonstrated a transient tail kinking and whole body contortion immediately after injection. The underlying cause and physiologic consequences of these episodes are unknown.

Speed of action.

One of the more striking properties of TAT-CBD3 is the speed with which the analgesic effects are observed in vivo. 17 A fluorescently-tagged TAT-CBD3 was readily observed in both sensory and motor neurons within 15 min of systemic administration. Additionally, decreases in calcium current density were observed in isolated drgs following a 15-min application of TAT-CBD3.Hypoxia anoxia taken together, these findings suggest TAT-CBD3 is capable of transduction into target tissues and is able to exert effects within a short time-frame. Given the proposed mechanism of TAT-CBD3, the decrease in calcium current density seen after a 15-min application suggests that ca v2.2 trafficking is a highly dynamic process. While it is not impossible, such a high turnover rate of surface-expressed ca v2.2 is surprising. It is possible that CRMP-2 not only aids in the membrane trafficking of ca v2.2 but also stabilizes the channel within the membrane. If the latter were the case, blocking the interaction between these two proteins may effectively lead to loss of channels from the membrane as well as decreased forward trafficking, potentially accounting for the fast onset of the analgesic effect.Hypoxia anoxia

Alternatively, TAT-CBD3 could also be preventing an unknown effect of CRMP-2 on ca v2.2 function. During our initial studies of this interaction, CRMP-2 did not appear to alter biophysical properties of the channel such as voltage dependence of activation/inactivation. 3 however, further analysis has revealed that CRMP-2 may affect inactivation in a subtler manner. We have recently examined the effect of CRMP-2 on calcium channel preferential closed-state inactivation. Use-dependent accumulation of non L-type channels in an inactivated state was determined by subjecting hippocampal neurons to brief depolarizing pulses to +20 mv, delivered at either 10 hz or 20 hz from a holding potential of −80 mv.Hypoxia anoxia the extent of inactivation can be determined by comparing the peak amplitude of the first pulse to the last pulse in the series. Overexpression of CRMP-2-EGFP prevented activity dependent accumulation in an inactivated state in the 10 hz and 20 hz stimulation paradigms ( fig. 3), suggesting that CRMP-2 may also increase calcium influx during periods of high activity by preventing accumulation of channels in an inactivated state. As certain pain conditions have been associated with hyperexcitability of sensory neurons, 18 it is possible that TAT-CBD3 could allow for increased accumulation of inactivated ca v2.2 in these neurons.

Pain models.

There is promise in the efficacy demonstrated by TAT-CBD3 in a variety of pain models.Hypoxia anoxia while TAT-CBD3 attenuated flinching responses in both phases of the formalin test, the effect was much larger in the first phase. This is somewhat surprising as the majority of studies reported a preferential effect of prialt on the second phase treatment 10 ( table 1). As both agents share a common target, ca v2.2, the incongruity seen within the formalin test may be attributed to differences in route of administration. It is not surprising that the effect of prialt is mostly seen within the second phase as it was delivered centrally, via intrathecal injection. TAT-CBD3, on the other hand, was delivered locally via intraplantar injection into the dorsal hind paw. Subsequently, the differences in the effects seen in phases 1 and 2 could be due to distribution of the peptide.Hypoxia anoxia while we are currently investigating the biodistribution of TAT-CBD3 following intraplantar injection, it is likely that the compound is concentrated within peripheral nociceptors. We expect that systemic administration of TAT-CBD3 would lead to a larger attenuation during the second phase.

The pronounced ability of intraplantar TAT-CBD3 to reduce phase 1 of the formalin test is intriguing as the exact role of ca v2.2 in this paradigm is unknown. 19 while we are confident that sensory nerves exhibit uptake of TAT-CBD3, it is unknown whether the peptide is able to reach the soma or central terminals of the DRG following distal target administration. The existence of peripheral voltage-gated calcium channels and their role on peripheral release has not been demonstrated.Hypoxia anoxia injection of the N-type specific blocker SNX-111 into the hind paw reduced hyperalgesia associated with sciatic nerve ligation, suggesting an involvement of peripheral ca v2.2 in some chronic pain states. 20 we cannot rule out the possibility that TAT-CBD3 disrupts interaction with other CRMP-2 binding partners. CRMP-2 has also been shown to interact with the NR2B subunit of the N-methyl- D-aspartate (NMDA) receptor; 21 yet, the domain on CRMP-2 responsible for mediating this interaction is unknown. Recent work in our laboratory suggests that TAT-CBD3 also disrupts this interaction (brittain and khanna, unpublished results). While the functional effect of the CRMP-2/NR2B interaction has yet to be determined, it is possible that TAT-CBD3 given via intraplantar injection may affect peripheral NMDA receptors.Hypoxia anoxia interestingly, subcutaneous formalin injection induces the release of glutamate into the hind paw 22 and subsequent activation of NMDA receptors contributes to the pain behaviors associated with this model. 23 despite a potential interaction with NR2B-containing NMDA receptors, intraperitoneal injection of TAT-CBD3 did not alter spatial memory retrieval, even at higher doses. 6

Route of administration is an important aspect of therapeutic treatment. The ability to administer an agent in various ways can be clinically advantageous. Systemic administration of TAT-CBD3 reversed tactile hypersensitivity in a model of aids therapy-induced peripheral neuropathy using zalcitibine (ddc). 24 , 25 we have since tested systemic TAT-CBD3 treatment in two additional neuropathic pain models.Hypoxia anoxia one model involves the non-traumatic topical application of lysophosphatidylcholine (LPC), which leads to focal demyelination of afferent nerve fibers without axonal loss. 26 this injury is associated with increased behavioral reflex responsiveness to mechanical stimuli in mice and rats. 26 , 27 LPC-induced sciatic nerve demyelination resulted in persistent mechanical hypersensitivity, which was effectively reversed following TAT-CBD3 treatment (10 mg/kg, i.P.) ( fig. 4). Up until this point we had determined the ability of TAT-CBD3 to attenuate non-traumatic neuropathic pain behaviors; however, its efficacy in traumatic neuropathic pain had not been evaluated. Ligation of the tibial nerve, a branch of the sciatic nerve, induces sustained mechanical allodynia and spontaneous pain. 28 while robust pain behaviors were observed two weeks following the injury, TAT-CBD3 treatment (10 mg/kg, i.P.) had no effect on mechanical hypersensitivity in these animals ( fig. 4).Hypoxia anoxia mechanistic differences between injury- and chemical-induced neuropathy may account for the lack of effect following tibial nerve injury. While the ddc and LPC models are commonly regarded as models of neuropathic pain, it is possible that the inflammatory system contributes differently in these models compared to direct nerve injury. Indeed, there is abundant variation even within direct injury models (reviewed in ref. 29). Ongoing research is focusing on potential differences in tibial nerve injury-induced pain states, which may account for the resistance to TAT-CBD3 treatment.

Optimizing the peptide: initial studies.

Single amino-acid mutation scans of the CBD3 sequence revealed potential opportunities for optimization of ca v2.2 binding.Hypoxia anoxia mutation of an alanine at the sixth position to a lysine resulted in the highest level of binding ( fig. 5A), ~3-fold higher than the native CBD3 sequence (not shown). A TAT-conjugated peptide harboring this mutation (TAT-CBD3 A6K) was tested for efficacy in an animal model of migraine. Changes in meningeal blood flow in response to 100 nm capsaicin were assayed using laser doppler flowmetry as previously described in references 6 and 30. The peptide was administered directly to the dura 15 min prior to nasal administration of capsaicin ( fig. 5B). TAT-CBD3 A6K dramatically reduced capsaicin-evoked changes in blood flow [cap, 35 ± 5% (n = 9) vs. TAT-CBD3 A6K, 7 ± 5% (n = 5)]. Basal blood flow was not altered by the mutant CBD3 peptide compared to saline [saline, −1 ± 2% (n = 9) vs.Hypoxia anoxia TAT-CBD3 A6K, 0 ± 3% (n = 5)] ( fig. 5C and D). This single amino acid mutation resulted in ~80% reduction in capsaicin-evoked blood flow, compared to the native CBD3 peptide which previously decreased the response to capsaicin by ~64% at similar concentrations. 6 the higher extent of ca v2.2 binding exhibited by TAT-CBD3 A6K may allow for a lower concentration threshold for analgesic efficacy, thereby further reducing the potential for off-target effects.

Sympathetic activity.

Sprague dawley rats (250–275 g) were were placed under general inhaled isoflurane anesthesia for assessment of sympathetic activity. The femoral artery was cannulated with a 5-cm segment of polytetrafluoroethylene tubing (AWG28, TW; zeus inc; orangeburg, SC).Hypoxia anoxia the arterial line was attached by a connector to PE-90 tubing that ran directly to a honeywell high sensitivity pressure transducer for continuous recording of pulsatile arterial blood pressure using a powerlab 8/30 data acquisition system and labchart software (AD instruments, mountain view, CA, USA). Heart rate and mean arterial pressure (defined as 1/3 systolic pressure + 2/3 diastolic pressure) were derived from the pulsatile arterial pressure waveform using labchart 7 (version 7.2.2) software. Core body temperature was recorded using a rectal temperature probe connected to the powerlab system. Throughout the experiments, body temperature was maintained at 36–38°C with a heating blanket connected to a homeothermic temperature control unit (harvard apparatus; holliston, MA).Hypoxia anoxia