NRD.E1, an innovative non-opioid therapy for painful diabetic peripheral neuropathy-A randomized proof of concept study.

BACKGROUND: Painful diabetic peripheral neuropathy (PDPN) affects up to 26% of patients with diabetes mellitus, with major impacts on their general health and well-being. Most available drugs fail to deliver acceptable pain reduction in the majority of patients and are often poorly tolerated. NRD.E1 is a novel product that has shown anti-nociceptive preclinical effects and good tolerability in healthy volunteer studies. METHODS: This phase 2a, randomized, dose-finding, Proof of Concept study enrolled patients with PDPN of ≥3 months duration. After at least one treatment-free week (WO week), 88 patients entered a 1-week single-blind (SB)-placebo run-in period, followed by 3 weeks' double-blind (DB) treatment, during which they received NRD.E1 at 10, 40 or 150 mg/day or placebo. RESULTS: The primary endpoint (change from SB-placebo run-in week to week 3 in weekly mean of daily average numerical rating scale [NRS] pain intensity) showed clinically relevant placebo-corrected treatment effect pain reductions at 40 mg and 150 mg/day of 0.82 (95% CI: 0.07, 1.58, p = 0.034) and 0.66 (95% CI: -0.03, 1.35; p = 0.061) NRS points, respectively, though did not meet the pre-specified value of p = 0.016 required due to multiplicity. An additional post hoc endpoint looking at the change from WO baseline to week 3 in weekly mean of daily average NRS showed the placebo-corrected treatment effect was 1.46 (95% CI: 0.26, 2.66), and 1.20 (95% CI: 0.10, 2.29) NRS points, respectively. Secondary and post hoc analyses of NRS pain data (including 30 & 50% responder rate and NNT), sleep interference, Short-form McGill pain questionnaire (especially pain intensity assessed on Visual Analogue Scale), Patient's and Clinician's Global Impression of Change showed effects consistent with the primary findings. NRD.E1 was well tolerated, with only headache reported in more than two patients and more frequently on NRD.E1 than placebo. CONCLUSIONS: The data suggest that NRD.E1 potentially represents a novel non-opioid therapeutic option for patients with PDPN, with at least similar efficacy and better tolerability than available therapies, justifying its further evaluation in larger-scale confirmatory studies. SIGNIFICANCE: NRD.E1 is a novel non-opioid therapeutic which is being developed for the treatment of PDPN. In this randomized, controlled, dose-finding, Proof of Concept study, NRD.E1 induced a clinically relevant pain reduction and it was well tolerated. Available data suggest that NRD.E1 has at least similar efficacy and better tolerability than the currently available therapies, potentially offering a promising new therapeutic option to patients with PDPN and possibly other neuropathic pain indications.

First-in-Human Single-Ascending-Dose, Multiple-Dose, and Food Interaction Studies of NRD.E1, an Innovative Nonopioid Therapy for Painful Diabetic Peripheral Neuropathy.

Painful diabetic peripheral neuropathy is characterized by burning, stabbing, or electric shock-type pain, which severely impacts day-to-day functioning and quality of life. Here, we report the results of 3 phase I studies with NRD135S.E1 (referred to as NRD.E1), a new, orally available chemical entity, presently developed for the treatment of painful diabetic peripheral neuropathy. The first study was a first-in-human, randomized, placebo-controlled, single-ascending-dose study, where NRD.E1 was administered to healthy male subjects in single dosages ranging from 300 to 1200 mg. The second study was a randomized, placebo-controlled multiple-dose study, where healthy male subjects received 300 mg of NRD.E1 once daily for 5 consecutive days. The third study was an open-label food interaction study in healthy men and women following a crossover design, where NRD.E1 was administered under fed and fasted conditions at 40 mg. The studies revealed dose-dependent absorption, increased exposure to NRD.E1 when administered with food, and no relevant accumulation after once-daily administration. All 3 phase I studies consistently showed rapid absorption of orally administered NRD.E1 followed by fast elimination, mainly via metabolization (glucuronidation), and small secondary increases in plasma concentrations. NRD.E1 was well tolerated, with no subject discontinuation due to treatment-emergent adverse events in any study.

Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum.

Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum. We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium (LjTbx20, LjTbx4/5, and LjIsl1/2A), establishment of left-right heart asymmetry (LjPitxA), and partitioning of the heart tube (LjTbx2/3A), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A-positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.

A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning.

Embryogenesis depends on self-regulatory interactions between spatially separated signaling centers, but few of these are well understood. Limb development is regulated by epithelial-mesenchymal (e-m) feedback loops between sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist Gremlin1 (GREM1). By combining mouse molecular genetics with mathematical modeling, we showed that BMP4 first initiates and SHH then propagates e-m feedback signaling through differential transcriptional regulation of Grem1 to control digit specification. This switch occurs by linking a fast BMP4/GREM1 module to the slower SHH/GREM1/FGF e-m feedback loop. This self-regulatory signaling network results in robust regulation of distal limb development that is able to compensate for variations by interconnectivity among the three signaling pathways.

Application of sonic hedgehog to the developing chick limb.

Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polarizing region signaling. The polarizing region (or zone of polarizing activity) is involved in one of the best known cell-cell interactions in vertebrate embryos and is pivotal in controlling digit number and pattern. At later stages of limb development, the application of Shh protein to the regions between digit primordia can induce changes in digit morphogenesis.

Identification and developmental expression of two Tbx1/10-related genes in the agnathan Lethenteron japonicum.

We have identified two Tbx1/10-related genes, LjTbx1/10A and LjTbx1/10B, from the Japanese river lamprey Lethenteron japonicum. We used in situ hybridization to characterize their expression pattern during embryonic development. LjTbx1/10A and LjTbx1/10B shared common expression in the pharyngeal arches and otic vesicle, although their levels and timing of expression differed markedly. LjTbx1/10A was highly expressed in the mesodermal core of pharyngeal arches and the adjacent endoderm throughout pharyngeal arch development, whereas LjTbx1/10B expression was only transiently upregulated in forming pharyngeal pouches. LjTbx1/10A transcripts first appeared at stage 25 in otic vesicles, whereas LjTbx1/10B transcripts could already be detected in the developing otic placode at stage 20. These results suggest that lamprey LjTbx1/10A and LjTbx1/10B may play distinct roles in the patterning and development of the pharyngeal apparatus. It appears that lamprey Tbx1/10 genes have undergone subfunctionalization independent from gnathostomes, with regard to both regulation and function.

Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis.

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.

Manipulations of PKA in chick limb development reveal roles in digit patterning including a positive role in Sonic Hedgehog signaling.

Sonic Hedgehog (Shh) signaling by the polarizing region, at the posterior of the vertebrate limb bud, is pivotal in determining digit number and identity. Shh establishes a gradient of the bifunctional transcriptional effector, Gli3, with high levels of full-length activator (Gli3A) in the posterior bud, where digits form, and high levels of shorter repressor (Gli3R) in the anterior. Repressor formation depends on protein kinase A (PKA), but in Drosophila, PKA also plays a role in activator function. Increasing PKA levels in chick limb development using Forskolin had no effect on posterior polarizing activity but weak polarizing activity, based on ligand-independent Shh signaling, was induced in anterior limb bud cells resulting in extra digits. Manipulating PKA activity levels directly with a retrovirus expressing activated PKA induced extra digits similar to those induced by Forskolin treatment suggesting that PKA may have a previously unrecognized positive role in Shh signaling in vertebrate limbs. Expressing dominant negative PKA also induced extra, sometimes multiple digits, from anterior limb bud demonstrating the negative role in Shh signaling. PKA levels in the limb bud are high posteriorly and low anteriorly, suggesting that PKA activity may influence the outcome of Shh signaling in normal development.

Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients.

Defects in long-range regulatory elements have recently emerged as previously underestimated factors in the genesis of human congenital disorders. Léri-Weill dyschondrosteosis is a dominant skeletal malformation syndrome caused by mutations in the short stature homeobox gene SHOX. We have analysed four families with Léri-Weill dyschondrosteosis with deletions in the pseudoautosomal region but still with an intact SHOX coding region. Using fluorescence in situ hybridization and single nucleotide polymorphism studies, we identified an interval of approximately 200 kb that was deleted in all tested affected family members but retained in the unaffected members and in 100 control individuals. Comparative genomic analysis of this interval revealed eight highly conserved non-genic elements between 48 and 215 kb downstream of the SHOX gene. As mice do not have a Shox gene, we analysed the enhancer potential in chicken embryos using a green fluorescent protein reporter construct driven by the beta-globin promoter, by in ovo electroporation of the limb bud. We observed cis-regulatory activity in three of the eight non-genic elements in the developing limbs arguing for an extensive control region of this gene. These findings are consistent with the idea that the deleted region in the affected families contains several distinct elements that regulate Shox expression in the developing limb. Furthermore, the deletion of these elements in humans generates a phenotype apparently undistinguishable to those patients identified with mutations in the SHOX coding region and, for the first time, demonstrates the potential of an in vivo assay in chicken to monitor putative enhancer activity in relation to human disease.

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements.

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri-Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2. Shox is expressed in central regions of early chick limb buds and proximal two thirds of later limbs, whereas Shox2 is expressed more posteriorly in the proximal third of the limb bud. Shox expression is inhibited distally by signals from the apical ectodermal ridge, both Fgfs and Bmps, and proximally by retinoic acid signaling. We tested Shox functions by overexpression in embryos and micromass cultures. Shox-infected chick limbs had normal proximo-distal patterning but the length of skeletal elements was consistently increased. Primary chick limb bud cell cultures infected with Shox showed an initial increase in cartilage nodules but these did not enlarge. These results fit well with the proposed role of Shox in cartilage and bone differentiation and suggest chick embryos as a useful model to study further the role of Shox in limb development.