Cutaneous targets for topical pain medications in patients with neuropathic pain: individual differential expression of biomarkers supports the need for personalized medicine.

Introduction: Numerous potential cutaneous targets exist for treating chronic pain with topically applied active pharmaceutical ingredients. This preliminary human skin tissue investigation was undertaken to characterize several key biomarkers in keratinocytes and provide proof-of-principle data to support clinical development of topical compounded formulations for peripheral neuropathic pain syndromes, such as postherpetic neuralgia (PHN). Objectives: The study intended to identify objective biomarkers in PHN skin on a patient-by-patient personalized medicine platform. The totality of biopsy biomarker data can provide a tissue basis for directing individualized compounded topical preparations to optimize treatment efficacy. Methods: Referencing 5 of the most common actives used in topical pain relief formulations (ketamine, gabapentin, clonidine, baclofen, and lidocaine), and 3 well-established cutaneous mediators (ie, neuropeptides, cannabinoids, and vanilloids), comprehensive immunolabeling was used to quantify receptor biomarkers in skin biopsy samples taken from ipsilateral (pain) and contralateral (nonpain) dermatomes of patients with PHN. Results: Epidermal keratinocyte labeling patterns were significantly different among the cohort for each biomarker, consistent with potential mechanisms of action among keratinocytes. Importantly, the total biomarker panel indicates that the enriched PHN cohort contains distinct subgroups. Conclusion: The heterogeneity of the cohort differences may explain studies that have not shown statistical group benefit from topically administered compounded therapies. Rather, the essential need for individual tissue biomarker evaluations is evident, particularly as a means to direct a more accurately targeted topical personalized medicine approach and generate positive clinical results.

Cutaneous nerve fiber and peripheral Nav1.7 assessment in a large cohort of patients with postherpetic neuralgia.

ABSTRACT: The mechanisms of pain in postherpetic neuralgia (PHN) are still unclear, with some studies showing loss of cutaneous sensory nerve fibers that seemed to correlate with pain level. We report results of skin biopsies and correlations with baseline pain scores, mechanical hyperalgesia, and the Neuropathic Pain Symptom Inventory (NPSI) in 294 patients who participated in a clinical trial of TV-45070, a topical semiselective sodium 1.7 channel (Nav1.7) blocker. Intraepidermal nerve fibers and subepidermal Nav1.7 immunolabeled fibers were quantified in skin punch biopsies from the area of maximal PHN pain, as well as from the contralateral, homologous (mirror image) region. Across the entire study population, a 20% reduction in nerve fibers on the PHN-affected side compared with that in the contralateral side was noted; however, the reduction was much higher in older individuals, approaching 40% in those aged 70 years or older. There was a decrease in contralateral fiber counts as well, also noted in prior biopsy studies, the mechanism of which is not fully clear. Nav1.7-positive immunolabeling was present in approximately one-third of subepidermal nerve fibers and did not differ on the PHN-affected vs contralateral sides. Using cluster analysis, 2 groups could be identified, with the first cluster showing higher baseline pain, higher NPSI scores for squeezing and cold-induced pain, higher nerve fiber density, and higher Nav1.7 expression. While Nav1.7 varies from patient to patient, it does not seem to be a key pathophysiological driver of PHN pain. Individual differences in Nav1.7 expression, however, may determine the intensity and sensory aspects of pain.

A peripheral CB2 cannabinoid receptor mechanism suppresses chemotherapy-induced peripheral neuropathy: evidence from a CB2 reporter mouse.

ABSTRACT: CB2 cannabinoid receptors (CB2) are a promising therapeutic target that lacks unwanted side effects of CB1 activation. However, the cell types expressing CB2 that mediate these effects remain poorly understood. We used transgenic mice with CB2 promoter-driven expression of enhanced green fluorescent protein (EGFP) to study cell types that express CB2 and suppress neuropathic nociception in a mouse model of chemotherapy-induced peripheral neuropathy. Structurally distinct CB2 agonists (AM1710 and LY2828360) suppressed paclitaxel-induced mechanical and cold allodynia in CB2EGFP reporter mice with established neuropathy. Antiallodynic effects of AM1710 were blocked by SR144528, a CB2 antagonist with limited CNS penetration. Intraplantar AM1710 administration suppressed paclitaxel-induced neuropathic nociception in CB2EGFP but not CB2 knockout mice, consistent with a local site of antiallodynic action. mRNA expression levels of the anti-inflammatory cytokine interleukin-10 were elevated in the lumbar spinal cord after intraplantar AM1710 injection along with the proinflammatory cytokine tumor necrosis factor alpha and chemokine monocyte chemoattractant protein-1. CB2EGFP, but not wildtype mice, exhibited anti-GFP immunoreactivity in the spleen. However, the anti-GFP signal was below the threshold for detection in the spinal cord and brain of either vehicle-treated or paclitaxel-treated CB2EGFP mice. EGFP fluorescence was coexpressed with CB2 immunolabeling in stratified patterns among epidermal keratinocytes. EGFP fluorescence was also expressed in dendritic cells in the dermis, Langerhans cells in the epidermis, and Merkel cells. Quantification of the EGFP signal revealed that Langerhans cells were dynamically increased in the epidermis after paclitaxel treatment. Our studies implicate CB2 expressed in previously unrecognized populations of skin cells as a potential target for suppressing chemotherapy-induced neuropathic nociception.

Keratinocyte Biomarkers Distinguish Painful Diabetic Peripheral Neuropathy Patients and Correlate With Topical Lidocaine Responsiveness.

This study investigated quantifiable measures of cutaneous innervation and algesic keratinocyte biomarkers to determine correlations with clinical measures of patient pain perception, with the intent to better discriminate between diabetic patients with painful diabetic peripheral neuropathy (PDPN) compared to patients with low-pain diabetic peripheral neuropathy (lpDPN) or healthy control subjects. A secondary objective was to determine if topical treatment with a 5% lidocaine patch resulted in correlative changes among the quantifiable biomarkers and clinical measures of pain perception, indicative of potential PDPN pain relief. This open-label proof-of-principle clinical research study consisted of a pre-treatment skin biopsy, a 4-week topical 5% lidocaine patch treatment regimen for all patients and controls, and a post-treatment skin biopsy. Clinical measures of pain and functional interference were used to monitor patient symptoms and response for correlation with quantitative skin biopsy biomarkers of innervation (PGP9.5 and CGRP), and epidermal keratinocyte biomarkers (Nav1.6, Nav1.7, CGRP). Importantly, comparable significant losses of epidermal neural innervation (intraepidermal nerve fibers; IENF) and dermal innervation were observed among PDPN and lpDPN patients compared with control subjects, indicating that innervation loss alone may not be the driver of pain in diabetic neuropathy. In pre-treatment biopsies, keratinocyte Nav1.6, Nav1.7, and CGRP immunolabeling were all significantly increased among PDPN patients compared with control subjects. Importantly, no keratinocyte biomarkers were significantly increased among the lpDPN group compared with control. In post-treatment biopsies, the keratinocyte Nav1.6, Nav1.7, and CGRP immunolabeling intensities were no longer different between control, lpDPN, or PDPN cohorts, indicating that lidocaine treatment modified the PDPN-related keratinocyte increases. Analysis of the PDPN responder population demonstrated that increased pretreatment keratinocyte biomarker immunolabeling for Nav1.6, Nav1.7, and CGRP correlated with positive outcomes to topical lidocaine treatment. Epidermal keratinocytes modulate the signaling of IENF, and several analgesic and algesic signaling systems have been identified. These results further implicate epidermal signaling mechanisms as modulators of neuropathic pain conditions, highlight a novel potential mode of action for topical treatments, and demonstrate the utility of comprehensive skin biopsy evaluation to identify novel biomarkers in clinical pain studies.

Human-like cutaneous neuropathologies associated with a porcine model of peripheral neuritis: A translational platform for neuropathic pain.

Despite enormous investment in research and development of novel treatments, there remains a lack of predictable, effective, and safe therapeutics for human chronic neuropathic pain (NP) afflictions. NP continues to increase among the population and treatments remain a major unmet public health care need. In recent years, numerous costly (time and money) failures have occurred attempting to translate successful animal pain model results, typically using rodents, to human clinical trials. These continued failures point to the essential need for better animal models of human pain conditions. To address this challenge, we have previously developed a peripheral neuritis trauma (PNT) model of chronic pain induced by a proximal sciatic nerve irritation in pigs, which have a body size, metabolism, skin structure, and cutaneous innervation more similar to humans. Here, we set out to determine the extent that the PNT model presents with cutaneous neuropathologies consistent with those associated with human chronic NP afflictions. Exactly as is performed in human skin biopsies, extensive quantitative multi-molecular immunofluorescence analyses of porcine skin biopsies were performed to assess cutaneous innervation and skin structure. ChemoMorphometric Analysis (CMA) results demonstrated a significant reduction in small caliber intraepidermal nerve fiber (IENF) innervation, altered dermal vascular innervation, and aberrant analgesic/algesic neurochemical properties among epidermal keratinocytes, which are implicated in modulating sensory innervation. These comprehensive pathologic changes very closely resemble those observed from CMA of human skin biopsies collected from NP afflictions. The results indicate that the porcine PNT model is more appropriate for translational NP research compared with commonly utilized rodent models. Because the PNT model creates cutaneous innervation and keratinocyte immunolabeling alterations consistent with human NP conditions, use of this animal model for NP testing and treatment response characteristics will likely provide more realistic results to direct successful translation to humans.

The evolution and multi-molecular properties of NF1 cutaneous neurofibromas originating from C-fiber sensory endings and terminal Schwann cells at normal sites of sensory terminations in the skin.

In addition to large plexiform neurofibromas (pNF), NF1 patients are frequently disfigured by cutaneous neurofibromas (cNF) and are often afflicted with chronic pain and itch even from seemingly normal skin areas. Both pNFs and cNF consist primarily of benign hyperproliferating nonmyelinating Schwann cells (nSC). While pNF clearly arise within deep nerves and plexuses, the role of cutaneous innervation in the origin of cNF and in chronic itch and pain is unknown. First, we conducted a comprehensive, multi-molecular, immunofluorescence (IF) analyses on 3mm punch biopsies from three separate locations in normal appearing, cNF-free skin in 19 NF1 patients and skin of 16 normal subjects. At least one biopsy in 17 NF1 patients had previously undescribed micro-lesions consisting of a small, dense cluster of nonpeptidergic C-fiber endings and the affiliated nSC consistently adjoining adnexal structures-dermal papillae, hair follicles, sweat glands, sweat ducts, and arterioles-where C-fiber endings normally terminate. Similar micro-lesions were detected in hind paw skin of mice with conditionally-induced SC Nf1-/- mutations. Hypothesizing that these microlesions were pre-cNF origins of cNF, we subsequently analyzed numerous overt, small cNF (s-cNF, 3-6 mm) and discovered that each had an adnexal structure at the epicenter of vastly increased nonpeptidergic C-fiber terminals, accompanied by excessive nSC. The IF and functional genomics assays indicated that neurturin (NTRN) and artemin (ARTN) signaling through cRET kinase and GFRα2 and GFRα3 co-receptors on the aberrant C-fiber endings and nSC may mutually promote the onset of pre-cNF and their evolution to s-cNF. Moreover, TrpA1 and TrpV1 receptors may, respectively, mediate symptoms of chronic itch and pain. These newly discovered molecular characteristics might be targeted to suppress the development of cNF and to treat chronic itch and pain symptoms in NF1 patients.