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The burst release of small molecular water-soluble drugs is a major problem when pursuing their long-acting formulations. Although various types of carrier materials have been developed for tackling this problem, it is still a big challenge to prevent water-soluble small molecules from fast release and diffusion. In this study, a biomineralization strategy based upon a self-assembling peptide is proposed for the slow release of lidocaine, a classic anesthetic with high solubility and a very small molecular weight. A bolaamphiphilic peptide was designed to self-assemble and produce negatively charged nanofibers, which were used as the template to absorb positively charged lidocaine molecules through an electrostatic interaction. The biomineralization of lidocaine was then induced by adjusting the pH, which lead to the formation of lidocaine microcrystals with a homogenous size. The microcrystals were incorporated into a hyaluronic acid hydrogel to form an injectable formulation. This formulation slowly released lidocaine and generate a prolonged anesthetic and analgesic effect in rodent models. Due to the constrained local and plasma lidocaine concentration, as well as the biocompatibility and biodegradability of the peptide materials, this formulation also showed considerable safety. These results suggest that nanofiber assisted biomineralization can provide a potential strategy for the fabrication of long-acting formulations for small molecular water-soluble drugs. STATEMENT OF SIGNIFICANCE: Long-acting formulations are highly pursued to achieve stronger therapeutic effect, or to avoid repeated administration of drugs, especially through painful injection. Using carrier materials to slow down the release of bioactive molecules is a common strategy to reach this goal. However, for many water-soluble small molecular drugs currently used in clinic, it is notoriously difficult to slow down their release and diffusion. This study proposes a novel strategy based on a controllable mineralization process using self-assembling peptide nanofibers as the template. Taking lidocaine as an example, we showed how peptide-drug microcrystals with well-controlled size and shape could be obtained, which exhibit significantly prolonged anesthetic and analgesic effect. As a proof-of-concept study, this work proposes a promising strategy to control the release of water-soluble small molecular drugs.
Learn More >Policies to address opioid-related harms include strategies to reduce opioid prescribing for new and ongoing pain management. Concerns have been raised that people with chronic non-cancer pain (CNCP) may be adversely affected by prescribing restrictions, and by involuntary tapering and cessation of opioids. We describe self-reported challenges obtaining prescription opioids among people prescribed opioids long-term for CNCP and explore associations with participant and treatment characteristics.
Learn More >Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin-stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G-protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down-regulation increases BK receptor desensitization in real-time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2-sensitive G-protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.
Learn More >Measurement of macrophage activation and its modulation for immune regulation is of great interest to arrest inflammatory responses associated with degeneration of intervertebral discs that cause chronic back pain, and with transplants that face immune rejection. Due to the phenotypic plasticity of macrophages that serve multiple immune functions, the net disease outcome is determined by a balance of subpopulations with competing functions, highlighting the need for single-cell methods to quantify heterogeneity in their activation phenotypes. However, since macrophage activation can follow several signaling pathways, cytometry after fluorescent staining of markers with antibodies does not often provide dose-dependent information on activation dynamics. We present high throughput single-cell impedance cytometry for multiparametric measurement of biophysical changes to individual macrophages for quantifying activation in a dose and duration dependent manner, without relying on a particular signaling pathway. Impedance phase metrics measured at two frequencies and the electrical diameter from impedance magnitude at lower frequencies are used in tandem to benchmark macrophage activation by degenerated discs against that from lipopolysaccharide stimulation at varying dose and duration levels, so that reversal of the activation state by curcumin can be ascertained. This label-free single-cell measurement method can form the basis for platforms to screen therapies for inflammation, thereby addressing the chronic problem of back pain.
Learn More >P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.
Learn More >Neuropathic and inflammatory pain are major clinical challenges due to their ambiguous mechanisms and limited treatment approaches. N-methyl-D-aspartate receptor (NMDAR) and calcium-calmodulin-dependent protein kinase II (CaMKII) are responsible for nerve system sensation and are required for the induction and maintenance of pain. However, the roles of NMDAR and CaMKII in regulating orofacial pain are still less well known. Here, we established a neuropathic pain model by transecting a mouse inferior alveolar nerve (IAN) and an inflammatory pain model by injecting complete Freunds adjuvant (CFA) into its whisker pad. The Cre/loxp site-specific recombination system was used to conditionally knock out (KO) NR2B in the trigeminal ganglion (TG). Von Frey filament behavioral tests showed that IANX and CFA-induced mechanical allodynia were altered in NR2B-deficient mice. CFA upregulated CaMKIIα and CaMKIIβ in the mouse TG and spinal trigeminal caudate nucleus (SpVc). CaMKIIα first decreased and then increased in the TG after IANX, and CaMKIIβ decreased in the TG and SpVc. CFA and IANX both greatly enhanced the expression of phospho (p)-NR2B, p-CaMKII, cyclic adenosine monophosphate (cAMP), p-ERK, and p-cAMP response element binding protein (CREB) in the TG and SpVc. These neurochemical signal pathway alterations were reversed by the conditional KO of NR2B and inhibition of CaMKII. Similarly, IANX- and CFA-related behavioral alterations were reversed by intra-ganglionic (i.g.) -application of inhibitors of CaMKII, cAMP, and ERK. These findings revealed novel molecular signaling pathways (NR2B-CaMKII-cAMP-ERK-CREB) in TG- and SpVc-derived latent subsequent peripheral and spinal central sensitization under nerve injury and inflammation, which might be beneficial for the treatment of orofacial allodynia.
Learn More >Monoamine oxidase (MAO) inhibitors have been investigated for the treatment of neuropathic pain. Here, we assessed the antiallodynic effects of a novel MAO-B inhibitor, KDS2010, on paclitaxel (PTX)-induced mechanical hypersensitivity. Oral administration of KDS2010 effectively relieved PTX-induced mechanical hypersensitivity in a dose-dependent manner. KDS2010 (25 mg/Kg) significantly prevented and suppressed PTX-induced pain responses with minimal effects on the body weight, motor activity, and working memory. KDS2010 significantly reduced reactive astrocytosis and reactive oxygen species (ROS) level in the L4-L6 spinal cord of PTX-treated mice. Furthermore, KDS2010 reversed the attenuation of GABAergic spontaneous inhibitory postsynaptic current (sIPSC) frequency in spinal dorsal horn neurons, although it failed to restore the reduced tonic GABA inhibition nor the increased GABA transporter 1 (GAT1) expression in PTX-treated mice. In addition, bath application of a reactive oxygen species (ROS) scavenger (PBN) restored the sIPSC frequency in PTX-treated mice but not in control and PTX + KDS2010-treated mice. These results indicated that the antiallodynic effect of KDS2010 is not due to a MAO-B-dependent GABA production. Finally, PBN alone also exerted a similar analgesic effect as KDS2010, but a co-treatment of PBN with KDS2010 showed no additive effect, suggesting that inhibition of MAO-B-dependent ROS production is responsible for the analgesic effect by KDS2010 on PTX-induced allodynia. Overall, KDS2010 attenuated PTX-induced pain behaviors by restoring the altered ROS level and GABAergic inhibitory signaling in the spinal cord, suggesting that KDS2010 is a promising therapeutic strategy for chemotherapy-induced peripheral neuropathy.
Learn More >Migraine headaches in children may cause attacks that require abortive treatment. This study evaluated the incidence and efficacy of medications used for relieving migraine headache attacks in the pediatric population in Israel. Children 6-18 years of age who were diagnosed in our pediatric neurology clinic as having migraine headaches were enrolled into the study. Children and their parents recorded the children response to abortive treatment during consecutive migraine attacks. Fifty children, with 116 migraine attacks, were included in the study (30 females; mean age 12; range 6-18). Forty-seven (94%) reported on abortive treatment on the first migraine attack, 43 (86%) on a second migraine attack and 26 (52%) on a third migraine attack. During the first recorded migraine attack, 41 children (87.5%) reported taking only one type of medication for each headache episode, mainly ibuprofen or acetaminophen; less than a quarter used dipyrone (metamizol). Overall the improvement rate after two hours was 65.4% ± 27 for ibuprofen, 59.8 ± 35.3 for acetaminophen and 50.9 ± 27.4 for dipyrone without statistical difference. However, in the first recorded headache episode, males had a significantly better response to acetaminophen, compared to ibuprofen (95% ± 28 vs 75 ± 20). In conclusion, Children with migraine in Israel mainly use a single medication for each headache episode. Ibuprofen is the most commonly used abortive treatment; however, acetaminophen was associated with a better response among some of our patients.
Learn More >Prediction errors (PEs) are generated when there are differences between an expected and an actual event or sensory input. The insula is a key brain region involved in pain processing, and studies have shown that the insula encodes the magnitude of an unexpected outcome (unsigned PEs). In addition to signaling this general magnitude information, PEs can give specific information on the direction of this deviation-i.e., whether an event is better or worse than expected. It is unclear whether the unsigned PE responses in the insula are selective for pain or reflective of a more general processing of aversive events irrespective of modality. It is also unknown whether the insula can process signed PEs at all. Understanding these specific mechanisms has implications for understanding how pain is processed in the brain in both health and in chronic pain conditions. In this study, 47 participants learned associations between 2 conditioned stimuli (CS) with 4 unconditioned stimuli (US; painful heat or loud sound, of one low and one high intensity each) while undergoing functional magnetic resonance imaging (fMRI) and skin conductance response (SCR) measurements. We demonstrate that activation in the anterior insula correlated with unsigned intensity PEs, irrespective of modality, indicating an unspecific aversive surprise signal. Conversely, signed intensity PE signals were modality specific, with signed PEs following pain but not sound located in the dorsal posterior insula, an area implicated in pain intensity processing. Previous studies have identified abnormal insula function and abnormal learning as potential causes of pain chronification. Our findings link these results and suggest that a misrepresentation of learning relevant PEs in the insular cortex may serve as an underlying factor in chronic pain.
Learn More >Chronic pain, cognitive deficits, and pain-related disability are inter-related. The prevalence of chronic pain and undiagnosed cognitive difficulties in middle age and older adults is increasing. Of the cognitive systems, executive function and episodic memory are most relevant to chronic pain. We examined the hypothesis that cognitive screening composite scores for executive function and memory would negatively associate with pain intensity and pain disability in a group of middle aged and older adults with knee pain, with or at risk for osteoarthritis.
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