In vivo silencing of the CaV3.2 T-type calcium channels in sensory neurons alleviates hyperalgesia in rats with streptozocin-induced diabetic neuropathy

Earlier, we showed that streptozocin (STZ)-induced type 1 diabetes in rats leads to the development of painful peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia and mechanical allodynia accompanied by significant enhancement of T-type calcium currents (T-currents) and cellular excitability in medium-sized dorsal root ganglion (DRG) neurons. Here, we studied the in vivo and in vitro effects of gene-silencing therapy specific for the Ca_V3.2 isoform of T-channels, on thermal and mechanical hypersensitivities, and T-current expression in small- and medium-sized DRG neurons of STZ-treated rats. We found that silencing of the T-channel Ca_V3.2 isoform using antisense oligonucleotides, had a profound and selective anti-hyperalgesic effect in diabetic rats and is accompanied by significant down-regulation of T-currents in DRG neurons. Anti-hyperalgesic effects of Ca_V3.2 antisense oligonucleotides in diabetic rats were similar in models of rapid and slow onset of hyperglycemia following intravenous and intraperitoneal injections of STZ, respectively. Furthermore, treatments of diabetic rats with daily insulin injections reversed T-current alterations in DRG neurons in parallel with reversal of thermal and mechanical hypersensitivities in vivo. This confirms that Ca_V3.2 T-channels, important signal amplifiers in peripheral sensory neurons, may contribute to the cellular hyperexcitability that ultimately leads to the development of painful PDN.     

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (Na_V1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased Na_V1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting Na_V1.7 alpha subunit mRNA attenuated the expression of Na_V1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective Na_V1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the Na_V1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances Na_V1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of Na_V1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that Na_V1.7 is a target for the treatment of chronic muscle pain.PerspectiveWe demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of Na_V1.7 in nociceptors.     

Changes in the expression of NaV1.7, NaV1.8 and NaV1.9 in a distinct population of dorsal root ganglia innervating the rat knee joint in a model of chronic inflammatory joint pain

Voltage‐gated sodium channels play an essential role in regulating the excitability of nociceptive primary afferent neurones. In particular the tetrodotoxin‐sensitive (TTX‐S) Na_V1.7 and the tetrodotoxin‐resistant (TTX‐R) Na_V1.8 and Na_V1.9 channels have been suggested to play a role in inflammatory pain. Previous work has revealed acute administration of inflammatory mediators, such as Freund's complete adjuvant (FCA) or carrageenan caused an upregulation in the levels of Na_V1.7 and Na_V1.8 protein in DRG (dorsal root ganglia) tissue up to 4 days post‐insult. In the present study, the expression of Na_V1.7, Na_V1.8 and Na_V1.9 was examined over a 28 day timecourse during a rat model of FCA‐induced chronic inflammatory joint pain. Using the retrograde tracer Fast Blue (FB) and specific Na_V1.7, Na_V1.8 and Na_V1.9 sodium channel antibodies, immunohistochemical staining techniques were used to study sodium channel expression in a distinct population of L3–L5 knee joint afferent DRGs. In the ganglia, counts were made of positively labelled cells in the FB population. The results demonstrate that, following FCA injection, Na_V1.9 expression is upregulated at days 14, 21 and 28 post‐FCA, with Na_V1.7 and Na_V1.8 showing increased channel expression at days 14 and 28. These observations are accompanied by a unilateral joint hypersensitivity in the FCA‐injected knee indicated by a behavioural shift in weight distribution measured using an incapacitance tester. The increased presence of these channels suggests that Na_V1.7, Na_V1.8 and Na_V1.9 play a role, at least in part, in the maintenance of chronic inflammatory pain several weeks after the initial insult.     

Full-length membrane-bound tumor necrosis factor-α acts through tumor necrosis factor receptor 2 to modify phenotype of sensory neurons

Neuropathic pain resulting from spinal hemisection or selective spinal nerve ligation is characterized by an increase in membrane-bound tumor necrosis factor-alpha (mTNFα) in spinal microglia without detectable release of soluble TNFα (sTNFα). In tissue culture, we showed that a full-length transmembrane cleavage-resistant TNFα (CRTNFα) construct can act through cell–cell contact to activate neighboring microglia. We undertook the current study to test the hypothesis that mTNFα expressed in microglia might also affect the phenotype of primary sensory afferents, by determining the effect of CRTNFα expressed from COS-7 cells on gene expression in primary dorsal root ganglia (DRG) neurons. Co-culture of DRG neurons with CRTNFα-expressing COS-7 cells resulted in a significant increase in the expression of voltage-gated sodium channel isoforms Na_V1.7 and Na_V1.8, and voltage-gated calcium channel subunit Ca_V3.2 at both mRNA and protein levels, and enhanced CCL2 expression and release from the DRG neurons. Exposure to sTNFα produced an increase only in CCL2 expression and release. Treatment of the cells with an siRNA against tumor necrosis factor receptor 2 (TNFR2) significantly reduced CRTNFα-induced gene expression changes in DRG neurons, whereas administration of CCR2 inhibitor had no significant effect on CRTNFα-induced increase in gene expression and CCL2 release in DRG neurons. Taken together, the results of this study suggest that mTNFα expressed in spinal microglia can facilitate pain signaling by up-regulating the expression of cation channels and CCL2 in DRG neurons in a TNFR2-dependent manner.     

Characterization of recombinant human bone morphogenetic protein‐2 delivery from injectable hyaluronan‐based hydrogels by means of 125I‐radiolabelling

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP‐2) from a hyaluronan‐based hydrogel system. The in vitro release of BMP‐2 from similar hydrogels has previously been studied by enzyme‐linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, ¹²⁵I radiolabelling was used instead to monitor BMP‐2 in vitro and in vivo. To minimize protein loss during handling, ¹²⁵I‐BMP‐2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP‐2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast‐release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP‐2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP‐2 delivery system. Copyright © 2012 John Wiley & Sons, Ltd.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

A novel Tc‐99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model

The serine–aspartic acid–valine (SDV) peptide binds specifically to integrin α_Vβ₃. In the present study, we successfully developed a TAMRA–GHEG–ECG–SDV peptide labeled with both Tc‐99 m and TAMRA to target the integrin α_Vβ₃ of tumor cells; furthermore, we evaluated the diagnostic performance of Tc‐99 m TAMRA–GHEG–ECG–SDV as a dual‐modality imaging agent for tumor of the murine model. TAMRA–GHEG–ECG–SDV was synthesized using Fmoc solid‐phase peptide synthesis. Radiolabeling of TAMRA–GHEG–ECG–SDV with Tc‐99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT‐1080 and HT‐29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc‐99 m, the Tc‐99 m TAMRA–GHEG–ECG–SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐1080 tumor (integrin α_Vβ₃ positive) and low uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐29 tumor (integrin α_Vβ₃ negative) were demonstrated. A competition study revealed that HT‐1080 tumor uptake was effectively blocked by the co‐injection of an excess concentration of SDV. Specific uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV in the integrin α_Vβ₃‐positive tumor. Tc‐99 m TAMRA–GHEG–ECG–SDV could be a good candidate for a dual‐modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd.     

A dual CT‐MR dendrimer contrast agent as a surrogate marker for convection‐enhanced delivery of intracerebral macromolecular therapeutic agents

The feasibility of using Gd dendrimer‐based macromolecules (Gd‐G8 dendrimer) as a dual CT and MR contrast agent for monitoring convection‐enhanced delivery of therapy in the brain is evaluated both in vitro and in vivo with optimal dosing established. In vitro CT attenuation values of the Gd‐based agents (～6.0 HU mM ^?1) were ～1.6 times greater than iodine‐based agents and the attenuation of the Gd‐DTPA was comparable to Gd‐G8 dendrimer. Visible enhancement was observed on both CT and MR using Gd‐G8 dendrimer over a range of 23–78 mM ; however, a concentration of at least 47 mM in Gd was required for adequate delineation of the injection site on both CT and MR. MR offers greater sensitivity than CT in estimating the volume of distribution (V_d) and effectively quantified the agent's concentration and diffusion using T₁ mapping at much lower concentrations of Gd (<10 mM in [Gd]). Copyright © 2008 John Wiley & Sons, Ltd.     

Treatment of growth plate injury using IGF‐I‐loaded PLGA scaffolds

Growth plate fracture can lead to retarded growth and unequal limb length, which may have a lifelong effect on a person's physical stature. The goal of this research was to develop an in vivo tissue‐engineering approach for the treatment of growth plate injury via localized delivery of insulin‐like growth factor I (IGF‐I) from cell‐free poly(lactic‐co‐glycolic acid) (PLGA) scaffolds. Mass loss and drug release studies were conducted to study the scaffold degradation and IGF‐I release patterns. In vitro cell studies showed that rat bone marrow stromal cells seeded on the porous scaffolds colonized the pores and deposited matrix within the scaffolds. These in vitro evaluations were followed by a proof‐of‐concept animal study involving implantation of scaffolds in proximal tibial growth plate defects in New Zealand white rabbits. Histological analysis of tissue sections from the in vivo studies showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF‐I‐releasing scaffolds; in contrast, only bone was formed in empty defects and those treated with IGF‐free scaffolds. The present findings show the potential for treating growth plate injury using in vivo tissue engineering techniques. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Visualization of delayed release of compounds from pH‐sensitive capsules in vitro and in vivo in a hamster model

Delayed controlled release is an innovative strategy to locally administer therapeutic compounds (e.g. chemotherapeutics, antibodies etc.). This would improve efficiency and reduce side effects compared with systemic administration. To enable the evaluation of the efficacy of controlled release strategies both in vitro and in vivo, we investigated the release of contrast agents (¹⁹ F‐FDG and BaSO₄) to the intestinal tract from capsules coated with pH‐sensitive polymers (EUDRAGIT L‐100) by using two complementary techniques, i.e. ¹⁹ F magnetic resonance imaging (MRI) and computed tomography (CT). Using in vitro ¹⁹ F‐MRI, we were able to non‐destructively and dynamically establish a time window of 2 h during which the capsules are resistant to low pH. With ¹⁹ F‐MRI, we could establish the exact time point when the capsules became water permeable, before physical degradation of the capsule. This was complemented by CT imaging, which provided longitudinal information on physical degradation of the capsule at low pH that was only seen after 230 min. After oral administration to hamsters, ¹⁹ F‐MRI visualized the early event whereby the capsule becomes water permeable after 2 h. Additionally, using CT, the integrity and location (stomach and small intestines) of the capsule after administration could be monitored. In conclusion, we propose combined ¹⁹ F‐MRI and CT to non‐invasively visualize the different temporal and spatial events regarding the release of compounds, both in an in vitro setting and in the gastrointestinal tract of small animal models. This multimodal imaging approach will enable the in vitro and in vivo evaluation of further technical improvements to controlled release strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

A‐Fibers Mediate Cold Hyperalgesia in Patients with Oxaliplatin‐Induced Neuropathy

Cold hyperalgesia is a common side effect of oxaliplatin treatment; still, the pathophysiological and molecular mechanisms as well as the contribution of different primary afferent fiber systems are unclear. Therefore, patients with oxaliplatin‐induced acute neuropathy with (n = 6) and without (n = 7) cold hyperalgesia were tested by applying a preferential blockade of peripheral myelinated A‐fiber afferents in combination with quantitative sensory testing. Additionally, an interview‐based questionnaire assessed the severity of symptoms and the impact on daily activities. Results indicate a deficit of cold perception in patients without cold hyperalgesia compared to patients with cold hyperalgesia prior to A‐fiber blockade. In patients with cold hyperalgesia, a preferential blockade of A‐fibers abolished cold hyperalgesia. This suggests that oxaliplatin‐induced cold hyperalgesia is mediated by A‐fibers and that a deficit in A‐fiber function might prevent the development of cold hyperalgesia. The work supports findings in rodents and in human sural nerve biopsies indicating that oxaliplatin interferes with axonal ion conductance in intact A‐fibers by sensitizing potassium and/or sodium channels. Drugs that act on these molecular targets might be of potential value to treat oxaliplatin‐induced cold hyperalgesia.     

Knockdown of sodium channel NaV1.6 blocks mechanical pain and abnormal bursting activity of afferent neurons in inflamed sensory ganglia

Inflammatory processes in the sensory ganglia contribute to many forms of chronic pain. We previously showed that local inflammation of the lumbar sensory ganglia rapidly leads to prolonged mechanical pain behaviors and high levels of spontaneous bursting activity in myelinated cells. Abnormal spontaneous activity of sensory neurons occurs early in many preclinical pain models and initiates many other pathological changes, but its molecular basis is not well understood. The sodium channel isoform Na_V1.6 can underlie repetitive firing and excitatory persistent and resurgent currents. We used in vivo knockdown of this channel via local injection of siRNA to examine its role in chronic pain after local inflammation of the rat lumbar sensory ganglia. In normal dorsal root ganglion (DRG), quantitative polymerase chain reaction showed that cells capable of firing repetitively had significantly higher relative expression of Na_V1.6. In inflamed DRG, spontaneously active bursting cells expressed high levels of Na_V1.6 immunoreactivity. In vivo knockdown of Na_V1.6 locally in the lumbar DRG at the time of DRG inflammation completely blocked development of pain behaviors and abnormal spontaneous activity, while having only minor effects on unmyelinated C cells. Current research on isoform-specific sodium channel blockers for chronic pain is largely focused on Na_V1.8 because it is present primarily in unmyelinated C fiber nociceptors, or on Na_V1.7 because lack of this channel causes congenital indifference to pain. However, the results suggest that Na_V1.6 may be a useful therapeutic target for chronic pain and that some pain conditions may be mediated primarily by myelinated A fiber sensory neurons.     

Prevention of oxaliplatin‐induced mechanical allodynia and neurodegeneration by neurotropin in the rat model

Oxaliplatin is a key drug for colorectal cancer, but it causes acute peripheral neuropathy (triggered by cold) and chronic neuropathy (sensory and motor neuropathy) in patients. Neurotropin, a non‐protein extract from the inflamed rabbit skin inoculated with vaccinia virus, has been used to treat various chronic pains. In the present study, we investigated the effect of neurotropin on the oxaliplatin‐induced neuropathy in rats. Repeated administration of oxaliplatin caused cold hyperalgesia from Day 5 to Day 29 and mechanical allodynia from Day 15 to Day 47. Repeated administration of neurotropin relieved the oxaliplatin‐induced mechanical allodynia but not cold hyperalgesia, and inhibited the oxaliplatin‐induced axonal degeneration in rat sciatic nerve. Neurotropin also inhibited the oxaliplatin‐induced neurite degeneration in cultured pheochromocytoma 12 (PC12) and rat dorsal root ganglion (DRG) cells. On the other hand, neurotropin did not affect the oxaliplatin‐induced cell injury in rat DRG cells. These results suggest that repeated administration of neurotropin relieves the oxaliplatin‐induced mechanical allodynia by inhibiting the axonal degeneration and it is useful for the treatment of oxaliplatin‐induced neuropathy clinically.     

Abdominal hyperalgesia in secretory phospholipase A2‐induced rat pancreatitis: Distinct roles of NK1 receptors

We investigated the potential of secretory phospholipase A₂ (sPLA₂)‐induced pancreatitis to promote abdominal hyperalgesia, as well as to depolarize sensory fibres in vitro using a grease‐gap technique. Pancreatitis was induced by the injection of sPLA₂ from Crotalus durissus terrificus (sPLA₂ Cdt, 300μgkg^?1) venom into the common bile duct of rats. Pancreatic inflammatory signs, serum amylase levels and abdominal hyperalgesia were evaluated in rats treated or not with SR140333, a tachykinin NK₁ receptor antagonist. Injection of sPLA₂ Cdt caused pancreatic oedema formation and increased pancreatic neutrophil infiltration and serum amylase at 4 h, which returned to normality by 24 h, except for the neutrophil infiltration, which was still increased at this time point. Animals injected with sPLA₂ exhibited a lower withdrawal threshold to electronic von Frey stimulation in the upper abdominal region at 4 h, but not 24 h, post‐injection when compared with saline‐injected rats. Pre‐treatment of animals with SR140333 significantly reduced the sPLA₂ Cdt‐induced abdominal hyperalgesia, without affecting the other parameters. Neither sPLA₂ Cdt nor sPLA₂ from Naja mocambique mocambique venom depolarized capsaicin‐sensitive sensory fibres from rat vagus nerve, but they decreased the propagated compound action potentials in both A and C fibres. These data show for the first time that NK₁ receptors play an important role in the early abdominal hyperalgesia in a rat model of sPLA₂‐induced pancreatitis, suggesting that these receptors are of importance in the development of pain in the pancreatitis condition. We also provide evidence that sPLA₂s do not directly depolarize sensory fibres in vitro.     

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na_vs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na_vs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na_vs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury–induced neuropathic pain was used, and an Na_v1.7-specific inhibitor, ProTxII, allowed the isolation of Na_v1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na_v1.7 and Na_v1.8 currents. The redistribution of Na_v1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L^–/–). SNS-Nedd4L^–/– mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na_v1.7 and Na_v1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na_vs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.     

Specific E‐selectin targeting with a superparamagnetic MRI contrast agent

Targeting of the endothelial inflammatory adhesion molecule E‐selectin by magnetic resonance imaging (MRI) was performed with a superparamagnetic contrast agent in the context of in vitro and in vivo models of inflammation. The specific contrast agent was obtained by grafting a synthetic mimetic of sialyl Lewis^x (sLe^x), a natural ligand of E‐selectin expressed on leukocytes, on the dextran coating of ultrasmall particles of iron oxide (USPIO). This new contrast agent, called USPIO‐g‐sLe^x, was tested, in vitro, on cultured human umbilical vein endothelial cells (HUVECs) stimulated to express inflammatory adhesion molecules, and in vivo, on a mouse model of hepatitis. In vitro, HUVECs were stimulated with the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) and were then incubated with USPIO‐g‐sLe^x or ungrafted USPIO. In vivo, hepatitis was induced on NMRI mice by injection of concanavalin A (Con A). USPIO‐g‐sLe^x and ungrafted USPIO were injected intravenously. In vitro results showed an extensive retention of USPIO‐g‐sLe^x on TNF‐α stimulated HUVECs. Image intensity and R₂ measurements performed on T₂‐weighted MR images demonstrated a significantly higher binding of USPIO‐g‐sLe^x on stimulated HUVECs. In vivo, USPIO are known to pass through the fenestrae of the liver and to be captured by Kupffer cells, inducing a loss of signal intensity on T₂‐weighted MR images. Unexpectedly, when injected to Con A‐treated mice, USPIO‐g‐sLe^x induced a significantly lower attenuation of liver signal intensity than USPIO or USPIO‐g‐sLe^x injected to healthy mice, or USPIO injected to Con A‐treated mice, suggesting that the specific contrast media is retained extracellularly by an interaction with E‐selectin overexpressed on the vascular endothelium. Both in vitro and in vivo results therefore indicate that USPIO‐g‐sLe^x is recognizing endothelial E‐selectin. USPIO‐g‐sLe^x is thus well suited for the MRI diagnosis of inflammation and for the in vitro evaluation of endothelial cells activation. Copyright © 2006 John Wiley & Sons, Ltd.     

Herbal medicine Shakuyaku‐kanzo‐to reduces paclitaxel‐induced painful peripheral neuropathy in mice

Objectives: Paclitaxel is widely used in cancer chemotherapy for the treatment of solid tumors such as breast, ovarian and lung cancer. However, it sometimes induces moderate to severe muscle pain, and impairs the patients’ quality of life. An appropriate method for relieving this pain is not well established. Shakuyaku‐kanzo‐to, a herbal medicine, is known to relieve menstrual pain, muscle spasm, and muscle pain, and its effectiveness is expected. To ascertain the effectiveness of Shakuyaku‐kanzo‐to on paclitaxel‐induced pain, we investigated the effects of Shakuyaku‐kanzo‐to and its constituent herbal medicines in a mouse model. Methods: Seven‐week‐old male ddY mice were used. To make a mouse model of paclitaxel‐induced pain, different single, intraperitoneally injected doses of this drug were tested in various groups of mice, and the optimal dose was determined. To estimate the effects of Shakuyaku‐kanzo‐to, the constituent herbal medicines Shakuyaku and Kanzo, and loxoprofen sodium as a non‐steroidal anti‐inflammatory drug on paclitaxel‐induced pain, mechanical allodynia and hyperalgesia of the hind paw were assessed. Results: Paclitaxel administered at a dose of 10mg/kg or more produced allodynia and hyperalgesia; the time courses were similar to those of pain after paclitaxel administration in cancer patients. Shakuyaku‐kanzo‐to significantly relieved the allodynia and hyperalgesia induced by paclitaxel (10mg/kg). Shakuyaku and Kanzo inhibited the allodynia and hyperalgesia to some extent, but not significantly, while loxoprofen sodium was without effects. Conclusions: A single administration of paclitaxel (10mg/kg) produced allodynia and hyperalgesia in mice, suggesting that it could be used as an animal model resembling the painful conditions observed in humans medicated with this drug. Using this model, Shakuyaku‐kanzo‐to was shown to relieve paclitaxel‐induced painful peripheral neuropathy.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.