DF2726A, a New Il-8 Signaling Inhibitor, Is Able to Counteract Chemotherapy-induced Neuropathic Pain

DF2726A, a new IL-8 signaling inhibitor, is able to counteract chemotherapy-induced neuropathic pain.

Introduction

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Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side effect of several antineoplastics and a main cause of sensory disturbances in cancer survivors, negatively impacting patients’ quality of life [19,31]. Chemotherapeutic agents associated with neurotoxicity include taxanes, platinum-based drugs, vinca alkaloids, thalidomide and bortezomib [20]. To date, there are no available drugs for the prevention and/or treatment of CIPN [19,32]. Therefore, a deeper understanding of the pathophysiology of CIPN to better identify new pharmacological targets and more specific treatments is mandatory.

CIPN affects approximately 70% of patients receiving chemotherapeutic treatments, and its development is directly related to the specific drug or combination of anticancer agents used, dosing regimen, and clinical conditions [31].

Clinically, CIPN is characterized by a series of sensory symptoms including paraesthesia and dysesthesia manifested as numbness, tingling and altered touch perception as well as mechanical or thermal allodynia and hyperalgesia [36]. Although the pathogenesis of CIPN has not been fully elucidated, it is noteworthy that the overall neuropathy symptom profile appears to be substantially shared across different classes of chemotherapeutic agents including taxanes, platinum, proteasome inhibitors, and vinca alkaloids. Peripheral nerve degeneration or small fiber neuropathy is generally accepted as the underlying mechanism in the development of CIPN [5,7], but several studies report  that neuropathic pain caused by anticancer agents may occur early after the first infusion in the absence of damage to intra-epidermal nerve fibers (IENFs) or axonal degeneration in peripheral nerves [27,33].

Recent evidence has contributed to clarify the determinant role of cytokines and chemokines in the process leading to neuronal hyperexcitability. Exposure to chemotherapeutics such as paclitaxel [35] and oxaliplatin [7] consistently increased secretion of pro-inflammatory cytokines (TNFα, IL-1β and IL-6) and downregulated anti-inflammatory cytokines (IL-10 and IL-4) in spinal astrocytes and dorsal root ganglia (DRGs), triggering alterations in peripheral neuropathic pathways.

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Paclitaxel was also associated with upregulation of Toll-like receptor 4 (TLR4) signaling, inducing higher expression of CCL2 [18] as well as recruitment of macrophages and pro-inflammatory T cells in DRGs [38]. In addition, preclinical in vivo results showed CCL2 overexpression in paclitaxel-induced spinal astrocytes and DRGs [13]. Similarly, a very recent report described the induction of CCL2/CCR2 pathway in oxaliplatin-induced DRGs, increasing mechanical and cold allodynia, and hypersensitivity of spinal astrocytes [15]. Several other studies found that both paclitaxel and oxaliplatin induced analogous mechanisms in the pathogenesis of neuropathic pain including spinal astrocyte activation, loss of IENFs, and mechanical and cold hypersensitivity [2,4,14].

We recently reported the efficacy of reparixin, an inhibitor of CXCR1/2, in paclitaxel-induced CIPN in rats, identifying the activation of the IL-8/CXCR1/2 axis as a mechanism strictly implicated in the induction and maintenance of paclitaxel induced-neuropathy. In the present study, we investigated a novel selective inhibitor of IL-8 receptors, DF2726A, suitable for chronic oral administration, extending the relevance of the activation of IL-8 pathway to the class of platinum chemotherapeutics. Based on our results, we suggest that DF2726A might be a promising candidate for clinical treatment of CIPN conditions due to its efficacy and optimized pharmacokinetic/pharmacodynamic profile.

Methods

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See Supplementary methods for the in vitro pharmacological and physicochemical characterization and for pharmacokinetic studies.

Animals

Behavioural experiments were performed on male Wistar rats (200-250 g, Harlan, Italy) housed in the animal care facility at the Department of Pharmacy of the University of Naples Federico II, Italy. Animals were housed, in a group of five, in a room with controlled temperature (22±1°C), humidity (60 ± 10%) and light (12 h per day); food and water were available ad libitum. All animals were weighted on the day of each treatment. All behavioural tests were performed between 09:00 and 17:00 h, and the animals were used only once. Animal care and manipulations were conducted in conformity with International and National law and policies (EU Directive 2010/63/EU for animal experiments, ARRIVE guidelines, and the Basel declaration including the 3R concept). The procedures reported here were approved by the Institutional Committee on the Ethics of Animal Experiments (CVS) of the University of Naples Federico II and by Ministero della Salute under protocol no. 2014-00884607. Rats were randomized and divided into equal-size groups (n=10 group) not predetermined by a statistical method. After completion of experiments, animals were sacrificed by cervical dislocation.

Drugadministration

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DF2726A was dissolved in saline and administered at the dose of 30 mg/2 ml/kg/os for 24 consecutive days starting 3 days before oxaliplatin administration and continuing for a further 21 days after the first administration of oxaliplatin. During this period, the compound was given 2 h after oxaliplatin treatment.

Induction of neuropathy by oxaliplatin

Rats received i.p. injections of oxaliplatin (Tocris) (2.4 mg/kg/day) or vehicle (5% glucose, 0.5 mL/rat), administered for 5 consecutive days every week for 3 weeks as described [6].

Behavioural testing was performed prior to oxaliplatin/vehicle administration (day -1) in order to determine the basal values of  mechanical and cold nociceptive thresholds, and again on 3, 5, 7, 10, 14 and 21 days following oxaliplatin/vehicle injection as shown in the schematic below.

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Mechanical allodynia

To assess for changes in sensation or in the development of mechanical allodynia, sensitivity to tactile stimulation was measured using the Dynamic Plantar Aesthesiometer (DPA, Ugo Basile, Italy), which is an automated version of the von Frey hair assessment [17]. Animals were individually placed in Plexiglas boxes (30 × 30 × 25 cm) with a mesh metal floor covered by a plastic dome that enabled the animal to walk freely, but not to jump. When a trial is initiated, the device raises the filament to touch the foot and progressively increases force until the animal withdraws its foot, or until it reaches a maximum of 50 g of force (cut-off). The DPA automatically records the force at which the foot is withdrawn. This test does not require any special pre-training, but just an acclimation period to the environment and testing procedure. Each paw was tested twice per session and the test was performed on both paws on the day before (day -1) and then  3, 5, 7, 10, 14and 21days after first administration of oxaliplatin or vehicle. No consistent left and right differences were observed. The means of paw withdrawal (expressed in grams) were calculated from an average of four separate measurements.

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Cold allodynia

Cold sensitivity was measured as the number of foot withdrawal responses after application of acetone to the dorsal surface of the paw [9,27]. Animals were individually placed in Plexiglas boxes (30 × 30 × 25 cm). A drop of acetone (25 μL) was applied to the dorsal surface of paws with a syringe connected to a thin polyethylene tube while the rats were standing on a metal mesh. A brisk foot withdrawal response, after the spread of acetone over the dorsal surface of the paw, was considered as a sign of cold allodynia. The procedure was repeated three times at 5 min intervals on both paws. The mean of paw withdrawal (expressed in numbers) was determined from an average of six separate measurements. Cold responses were measured before (day -1) and then 3, 5, 7, 10, 14and 21days after first administration of oxaliplatin or vehicle. No consistent left and right differences were observed.

Immunohistochemistry

Animals were sacrificed and a biopsy (3 mm) was taken from pad of the right hind paw. Biopsies were immediately placed in Zamboni’s fixative, where they were left at 4°C. Tissues were then transferred to 20% sucrose for at least 24 h and used for immunohistochemistry analysis. Specimens were frozen in Optimal Cutting Temperature compound (OCT, Sakura Finetek Inc, CA, USA) and sliced into 20 μm thick serial coronal sections by cryostat AMES LAB-TEK (Westmont, Illinois, USA).

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In brief, sections were blocked with phosphate buffer saline (PBS) containing 10% BSA and 0.3% Tween-20, for 1 h at RT and incubated, overnight at 4°C, with the primary antibodies: IL-8 (mouse, 1:25, R&D Systems Inc. Minneapolis, MN, USA), PGP9.5 (rabbit, 1:1000, AbD Serotec, NC, USA), acetylated alpha tubulin (mouse, 1:250, Abcam Cambridge, MA, USA) and collagen IV (goat, 1:25, Southern Biotech, Birmingham, AL, USA). Sections were then rinsed in PBS several times before incubation for 2 h at RT with secondary antibodies, donkey AlexaFluor 488 anti-rabbit or donkey AlexaFluor 633 (1:2000), goat anti-mouse conjugated with Alexafluor 488 (1:2000; Life Technologies, Camarillo, CA, USA). After extensive washing, coverslips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories Burlingame, CA, USA) and then observed at a Leica TCS SP5 confocal microscope (Leica, Mannheim, Germany).

Quantification

Five slices were examined for each animal. For each slide, 4 fields were counted. For quantitative evaluation of IL-8/CINC-1 (the homolog of IL-8 in rat) and PDG95, photomicrographs for each condition were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA) as previously described [1]. Briefly, the pictures were converted into gray scale, and arbitrary units were assigned: 0=black (i.e. absence of signal) and 255=white. The signal was first measured on the tissue in an area devoid of signal at visual inspection and assumed as background; the threshold was then set at 1.5 times the background and the surface area and mean gray intensity were measured for all areas above the threshold. To obtain the signal intensity (in arbitrary units), the background was subtracted from the mean gray intensity and the result was multiplied by the surface area above threshold. This value was divided by the surface area of tissue section to calculate the signal intensity per unit surface area.

Cell culture and drug treatments

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F11 hybridoma cells (ECACC 08062601), chosen as a model of DRG neurons [3,26] were cultivated in DMEM (Euroclone, Milan, Italy) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin (Euroclone, MI, Italy) and 1% glutamine (Euroclone) at 37°C, in humidified 95% air with 5% CO2 atmosphere. For all the experiments, cells were used at 18th passage. For immunofluorescence analysis, cells were seeded on coverslips at 1 × 104 cells/cm2 for 24 h. Cells were then differentiated with rat NGF (rNGF; Sigma-Aldrich). rNGF was dissolved in DMEM with 1% penicillin/streptomycin and 1% glutamine (FBS free) at a final concentration of 50 ng/ml. Medium was replaced every 3 days until complete differentiation, which occurred after 7 days.

Following neuronal differentiation, neurons were treated for 24 h with DF2726A (1 μM final concentration), oxaliplatin (Sigma-Aldrich; 20 µM final concentration), or the combination of the two molecules.

DF2726A stock solution (4.3 mM) was prepared freshly by dissolving 1.5 mg of DF2726A in 1 ml PBS, 20 µl NaOH 1N and 30 µl HEPES 1M.

Oxaliplatin stock solution (40 mM) was prepared by dissolving the powder in DMSO, and aliquots were stored at -20°C.

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MTS assay

Cell viability was determined at 24 h using Cell Titer One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA), a colorimetric method based on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenil)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). The quantity of formazan formed, as a function of viability, was measured at 492 nm using an Infinite F200 ELISA plate reader (Tecan, Männedorf, Switzerland). The assay was performed in quintuplicate. The results were expressed as absorbance at 492 nm.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT and permeabilized in methanol for 5 min at -20°C. Cells were then blocked with PBS containing 4% BSA for 30 min and incubated with the following primary antibodies diluted in the blocking solution overnight at 4°C: rabbit acetylated α-tubulin (1:4000; Cell Signaling Technology, Inc., Danvers, MA, USA). Cells were then rinsed in PBS several times before incubation with secondary antibodies: goat anti-rabbit conjugated with Alexafluor 633 (1:2000; Life Technologies, CA, USA) for 30 min at RT. After extensive washing, coverslips were mounted with Vectashield mounting medium (Vector Laboratories Burlingame, CA, USA) with DAPI and then observed at a Leica TCS SP5 confocal microscope (Leica).

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Western blotting

Control and treated cells were collected and lysed in ice-cold RIPA buffer (PBS pH 7.4 containing 0.5% sodium deoxycolate, 1% Igepal, 0.1% SDS, 5 mM EDTA, 1% protease and phosphatase inhibitor cocktails; Sigma-Aldrich). Protein lysates (30 μg) were separated on 8–12% SDS-polyacrilamide gel and electroblotted onto polyvinyldifluoride membrane (PVDF; Sigma-Aldrich). Nonspecific binding sites were blocked by 5% non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) in Tris buffered saline (TBS: 20 mM Tris- HCl, pH 7.4, containing 150 mM NaCl) for 30 min at RT. Membranes were then incubated overnight at 4°C with the following primary antibodies, diluted with TBS containing 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk: rabbit acetylated α-tubulin (1:3000; Cell Signaling Technology), rabbit p-FAK (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA ), rabbit p-JAK2 (1:500; Santa Cruz Biotechnology), rabbit PI3K (1:500; Abcam, Cambridge, UK), rabbit p-cortactin (1:1000; Abcam), rabbit GAPDH (1:5000; Santa Cruz Biotechnology), goat p-STAT3 (1:200; Santa Cruz Biotechnology), rabbit p-Akt (1:1000; Immunological Sciences, Rome, Italy), goat COX2 (1:500; Santa Cruz Biotechnology), p-ERK1/2 (1:500 Santa Cruz Biotechnology). As secondary antibodies, peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:10000; Vector Laboratories) and anti-goat (1:1000; Santa Cruz Biotechnology) were used. Immunoreactive bands were visualized by Pierce ECL Substrate (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The relative densities of immunoreactive bands were determined and normalized with GAPDH, using ImageJ software. Values were given as relative units (RU).

RNA isolation and quantitative real-time PCR

Total RNA was extracted by Trizol reagent (Life Technologies, Lofer, Austria), according to the manufacturer’s instructions. Total RNA concentration was determined spectrophotometrically in RNAase-free water, and 1 µg aliquots of total RNA were reverse transcribed into cDNA using ProtoScript First Strand cDNA Syntesis Kit (New England BioLabs, Ipswich, MA, USA). RT-PCR was carried out using Thermo System (ThermoFisher Scientific), in a total volume of 20 μl containing EagleTaq Universal Master Mix (ROX; Roche, Penzberg, Germany), DEPC water and 5 μl cDNA. Prime Time qPCR Assay kits TRPV1 qRnoCIP0024978 and TRPV4 qRnoCIP0027857 were purchased from Biorad (Bio-rad Laboratories). Triplicate samples were run for each gene. The reference gene GAPDH qRnoCIP0050838 was used as an internal control to normalize the expression of target genes. Relative expression levels were calculated for each sample after normalization against the reference gene, using the ΔΔCt method for comparing relative fold expression differences.

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Statistics

For the in vivo results, all data were presented as mean ± SEM. Data analysis was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). The significance of differences between groups was determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests for multiple comparisons. The level of significance was set at P < 0.05.

For the in vitro and ex vivo results (rat paw), data were expressed as mean ± SEM.  Statistical analysis was performed by Student’s unpaired t test. The level of significance was set at P < 0.05.

Results

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DF2726A in vitro characterization

The main in vitro pharmacological and physicochemical results are summarized in Table 1.

In vitro pharmacological characterization showed that DF2726A did not inhibit spontaneous human polimorphonuclear neutrophil (hPMN) migration per se, but was equally efficacious in inhibiting IL-8- and CXCL1-induced hPMN chemotaxis with IC50 values calculated in 10 ± 5 nM and 8 ± 3 nM for IL-8 and CXCL1, respectively.

Counterscreens demonstrated > 100-fold selectivity for 55 targets among GCPRs, ion channels, enzymes and transporters (see Supplementary Information).

DF2726A displayed excellent physicochemical and in vitro ADME properties, with good aqueous solubility and low lipophilicity.

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High plasma protein binding in three species (human, dog and rat) was observed with values higher than 94%. Furthermore, DF2726A showed excellent stability toward liver microsomes in human, dog and rat, no inhibition of selected CYP-enzymes involved in drug-drug interactions and no CYP-induction. The permeability in the Caco-2 cell assay was good with no P-glycoprotein (P-gp) inhibition. DF2726A exhibited no hERG inhibition in a patch-clamp assay (IC50 > 1 mM) and was devoid of potential cardiovascular liability. The compound did not show any in vitro genotoxiticy as demonstrated in the AMES Test. Finally, DF2726A did not inhibit COX-1 and COX-2 when tested at 50 µM.

Pharmacokinetic studies

Pharmacokinetic studies of DF2726A in Sprague Dawley rats showed a slow oral absorption (Cmax was reached at 6 h post dosing), high plasma exposure, a long half-life and low clearance (Table 2). The oral elimination t_1/2 of DF2726A was similar to intravenous elimination t_1/2 (16.2 h vs 15.2 h). The relatively low volume of distribution for DF2726A can be explained by the high plasma protein binding observed in the rat (97.5%).

Interestingly, DF2726A showed a very high bioavailability (96.8%) and a suitable pharmacokinetic profile for oral treatment.

DF2726A prevented oxaliplatin-induced peripheral neuropathy

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In control animals administered with oxaliplatin vehicle i.p, the paw withdrawal threshold remained unchanged during the whole experimental period, with a trend corresponding to that observed in naïve animals receiving no treatment (data not shown). In contrast, oxaliplatin-saline treated animals showed marked changes in paw withdrawal responses due to neurological toxicity.

In cold allodynia experiments, no paw withdrawal response was induced by acetone in control animals, indicating that acetone-evoked cold stimulation is not noxious in non-neuropathic rats. Conversely, the paw withdrawal threshold was significantly increased at all experimental time-points in oxaliplatin-saline-treated animals (Fig. 1A). In particular, animals treated with oxaliplatin-saline showed a significant, persistent cold allodynia from day 3 to day 21 post-initial oxaliplatin dosing.  DF2726A treatment produced a marked attenuation of cold allodynia starting from day 5 and persisting until the final test day (Fig. 1A).

In DPA test, the paw withdrawal threshold of oxaliplatin-saline-treated animals resulted significantly reduced only at 14 and 21 days after the first oxaliplatin treatment. DF2726A showed a significant anti-allodynic effect at 14 and 21 days (Fig. 1B).

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In control animals (i.e., no oxaliplatin administration), DF2726A treatment induced no changes in cold and mechanical sensitivity (data not shown).

The beneficial effect of DF2726A was not limited to the oxaliplatin-induced CIPN model; paclitaxel-evoked cold and mechanical allodynia in rats was also prevented by administration of DF2726A (Supplementary Fig. 1).

DF2726A prevented oxaliplatin-induced loss of IENFs

Loss of IENFs was reported to play a critical role in the development of various neuropathies occurring in response to chemotherapeutic agents, such as oxaliplatin [4]. The immunofluorescence assay of PGP9.5 (a marker of IENFs) and collagen IV showed damage in IENFs extending from derma into epidermis in oxaliplatin-saline-treated compared to control rats (Fig. 2A). Interestingly, DF2726A treatment strongly counteracted this effect, preventing the loss of epidermal innervation (Fig. 2A). In addition, immunofluorescence assay showed that oxaliplatin treatment increased IL-8/CINC1 signaling in the dermis. DF2726A treatment significantly attenuated this phenomenon, inducing a protective effect on nerve fibers (Fig. 2B). Lastly, anti-acetylated α-tubulin was evaluated in untreated, oxaliplatin- and DF2726A-treated nerve fibers. Immunofluorescence images showed that oxaliplatin treatment increased acetylated α-tubulin levels, while DF2726A completely restored control conditions (Fig. 2C). Taken together, these data show that oxaliplatin causes a pronounced decrease in IENFs, which was prevented by DF2726A administration.

In vitro models

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The viability assay in neuronal cells after different treatments showed that oxaliplatin and DF2726A were ineffective in modulating neuronal cell viability at a concentration range of 5-60 µM and 5-20 µM, respectively (Supplementary Fig. 2). For all the other experiments, DF2726A was used at a final concentration of 1 µM, and oxaliplatin at a final concentration of 20 µM. For comparison, paclitaxel alone or in combination with DF2726A was also used. Paclitaxel was ineffective in modulating cell viability in the concentration range 5-20 nM (Supplementary Fig. 2).

Acetylated α-tubulin (a marker of stable microtubules) was analyzed by immunofluorescence in neurons after the different treatments. In untreated cells and in cells treated with DF2726A, acetylated α-tubulin was moderately present. In contrast, both paclitaxel and oxaliplatin-treated neurons showed an increase in acetylated α-tubulin levels (Supplementary Fig. 3 and Fig. 3, respectively). Specifically, fluorescence intensity in neurites was stronger, neurite diameter was greater, and cytoskeleton organization was more evident. Interestingly, acetylated α-tubulin in oxaliplatin+DF2726A-treated neurons was decreased compared to that in control conditions, indicating that the presence of DF2726A counteracts chemotherapy-mediated effects (Fig. 3). The same effect was observed for paclitaxel+DF2726A treatment (Supplementary Fig. 3). In addition, we observed a greater thickness in paclitaxel-treated neurites compared to control, while under combined treatment with DF2726A cells appeared  as control cells (Supplementary Fig. 3).

Acetylated α-tubulin was also assayed by western blotting. In agreement with morphological data, both paclitaxel and oxaliplatin determined a significant increase in acetylated α-tubulin, while in combination with DF2726A the protein expression level was comparable to that of control cells (Supplementary Fig. 3 and Fig. 3, respectively).

DF2726A counteracted oxaliplatin-induced neurotoxic pathways

We investigated the effect of DF2726A on expression of the active form of the protein of focal adhesion (p-FAK), which is involved in microtubule stabilization. p-FAK levels were significantly increased by both paclitaxel and oxaliplatin treatment, but decreased in the presence of DF2726A (Supplementary Fig. 4 and Fig. 4, respectively). In addition, we analyzed the active form of JAK2 (p-JAK2), involved in p-STAT3 signaling, which in turn has a function in neuropathic pain and synaptic plasticity [23,37]. Both paclitaxel and oxaliplatin treatment increased p-JAK2, while combination with DF2726A restored control conditions (Supplementary Fig. 4 and Fig. 4, respectively). p-STAT3 protein levels under the different conditions were also investigated (Supplementary Fig. 4 and Fig. 4, respectively). p-STAT3 levels increased after treatment with both chemotherapeutic agents compared to control, while DF2726A counteracted this effect (Supplementary Fig. 4 and Fig. 4, respectively). We also investigated PI3K/p-cortactin pathway, which is involved in axonal arborization and synaptic plasticity. Both paclitaxel and oxaliplatin strongly upregulated PI3K/p-cortactin expression, while combination treatment with DF2726A attenuated this effect (Supplementary Fig. 4 and Fig. 4, respectively). All together, these data suggest a common pathway activated by both taxane and platinum agents, and that DF2726A is able to counteract the effects of both drugs.

Activation of MAPK pathways is known to contribute to cellular damage. Oxaliplatin activates p38 and ERK1/2and promotes apoptosis in DRG neurons [28,30]. In our experimental conditions, oxaliplatin strongly upregulated the active form of ERK1/2 (p-ERK1/2) and increased PI3K and p-Akt, while in combination with DF2726A protein levels were comparable to those of control cells (Fig. 5). Lastly, oxaliplatin robustly upregulated COX2 protein levels, while in combination with DF2726A COX2 expression levels were comparable to the control (Fig. 5). Oxaliplatin also affects TRVP receptors, which are involved in hypernociception. Specifically, TRVP1 and TRPV4 play a role in mechanical and thermal allodynia, respectively [10,22]. We therefore assayed TRVP1 and TRVP4 receptor gene expression by real-time PCR. Oxaliplatin significantly increased the expression of both receptors, while DF2726A restored control values (Fig. 6).

Discussion

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CIPN is a common adverse effect and the main dose-limiting toxicity associated with selected chemotherapeutic agents, including paclitaxel and oxaliplatin [19]. Although major efforts are now focusing on preventing the development of taxane- and platinum-induced acute and delayed CIPN without impacting clinical outcomes, no drugs have yet been approved for prophylaxis and/or treatment of CIPN. Current therapeutic strategies are based on the continuous modification of chemotherapy regimens to manage the symptoms of devastating side effects and to improve patients’ quality of life.

Platinum compounds bind to nuclear DNA of cancer cells, leading to aberrant re-entry into cell cycle, apoptosis and tumor degradation. Paclitaxel and oxaliplatin are the two chemotherapeutics most frequently associated with the development of severe untreatable CIPN. Oxaliplatin, a third-generation platinum chemotherapeutic agent, causes severe acute and chronic peripheral neuropathies, including cold allodynia, loss of sensitivity and motor disturbances [7,15,19]. The pathophysiological mechanisms of paclitaxel- and oxaliplatin-induced neuropathy are not completely understood. However, a large body of evidence describes an associative and causal relationship between activation of the immune system following taxane and platinum therapy and development of CIPN symptoms [35,38]. Several preclinical studies demonstrated that DRGs and spinal cord astrocytes treated with paclitaxel and oxaliplatin display higher levels of pro-inflammatory chemokines and cytokines (CCL2, CCL3, TNF-α, IL-6, IL1β and IL-8), as well as decreased anti-inflammatory cytokine expression (IL-10 and IL-4), causing sensitization of nociceptors, mechanical hyperalgesia and loss of IENFs [2,4]. A recent controlled clinical trial found an association between higher serum levels of pro-inflammatory cytokines and severity of neuropathy induced by oxaliplatin in patients with colon cancer [12]. However, the exact mechanisms linking systemic cytokine and chemokine levels following chemotherapy with the development of CIPN symptoms remain to be elucidated.

Consistent with the above studies, our previous findings showed the significant effect of reparixin in attenuating the development of CIPN in sensitive neurons, suggesting a potential therapeutic role for chemokine inhibitors in prophylaxis and treatment of paclitaxel-induced CIPN [6]. We contributed to clarify the role of paclitaxel-induced IL-8 expression as a trigger of the progressive neural toxicity mediated by activation of the pathways responsible for microtubule stabilization, axonal arborization and synaptic plasticity.

In the present study, we evaluated the efficacy of DF2726A, a novel optimized selective dual inhibitor of CXCR1 and CXCR2, in an oxaliplatin-induced neuropathic pain model, confirming the key role of these pharmacological targets and the additional importance of pharmacokinetic/pharmacodynamic optimization. Importantly, this work also extended our mechanistic and pharmacological studies of the paclitaxel-induced model, showing that the mechanisms underlying the neurotoxicity of the two drugs, unrelated to their chemotherapeutic action, are largely conserved and imply a key involvement of IL-8 receptors.

As previously reported for reparixin [6], we found that DF2726A acts as a non-competitive allosteric modulator of CXCR1/2 (data not shown). DF2726A is a potent dual inhibitor of CXCR1/2 (IC50), with a good cross-reactivity versus rat and mouse, and an optimal selectivity profile versus a large panel of unrelated receptors. In line with the objectives of the lead optimization program, DF2726A exhibited improved features compared to reparixin, such as a longer half-life (16.2 h vs 1.78 h p.o. administration), a reduced plasma protein binding and better oral bioavailability. The optimization of the pharmacokinetic/pharmacodynamic profile was found to be associated with greater in vivo efficacy of DF2726A compared to that previously reported for reparixin in counteracting the increased sensitivity to tactile and cold stimuli induced by oxaliplatin and paclitaxel. The results reported here describe for the first time the efficacy of CXCR1/2 inhibition in in vivo models of oxaliplatin-induced peripheral neuropathy. DF2726A administration in rats afforded an attenuated level of behavioral hypersensitivity, suggesting that IL-8 signaling contributes to both taxane- and platinum-induced CIPN. In line with the in vivo effects, we observed that oxaliplatin led to a strong decrease in the number of IENFs, which was significantly counteracted by DF2726A. Although the exact mechanism of epidermal denervation is poorly understood, some studies suggested that it is linked to both paclitaxel- and oxaliplatin-induced neurological toxicity [2,4], and is associated with increased levels of multiple pro-inflammatory cytokines [21,35]. Interestingly, we showed that IENF loss corresponds to a local increase in IL-8 induced by oxaliplatin, and that DF2726A treatment strongly attenuated this phenomenon, suggesting its important protective role in nerve fibers.

In order to dissect the signal transduction pathways underlying the function of DF2726A in alleviating both paclitaxel- and oxaliplatin-induced neuropathic pain, we assessed the effects of IL-8-induced signaling in DRG-derived neuron cultures. In cancer, alterations in microtubule stability, including acetylation of tubulin, are reported to influence cellular responses to chemotherapeutics, tumor development, cellular trafficking and survival [6,25]. Although not classified as antimicrotubule agents, platinum compound agents are also associated with the alteration of assembly processes and signaling functions of tubulin, as well as changes in cytoskeletal and axonal functions, all of which lead to neurotoxicity [11,20,24,34]. It was previously reported that DRG neurons express IL-8 receptor [6]. We investigated the effect of DF2726A inhibition on both taxane- and platinum-induced tubulin acetylation in DRG-derived neurons. In our experimental conditions, both paclitaxel and oxaliplatin induced an increase in α-acetylated tubulin in DRGs, an effect efficiently counteracted by exposure to DF2726A. Under both paclitaxel and oxaliplatin treatment, DRG-derived neurons expressed higher levels of p-FAK involved in microtubule stabilization, and activated the PI3K/p-cortactin pathway, leading to terminal axonal arborization and synaptic plasticity. Additionally, paclitaxel and oxaliplatin exposure increased expression of p-JAK2, thereby triggering p-STAT3, known to have a function in neuropathic pain and synaptic plasticity. In line with our previous findings [6], we demonstrated that CXCR1/2 inhibition by DF2726A is effective in reducing p-STAT3, p-FAK and PI3K/p-cortactin activation induced by both taxane and platinum agents, by modulating key features of chemotherapy-associated neurotoxicity. The activation of COX2 and ERK1/2 pathways can further contribute to cellular damage and neuropathic pain [28,29]. Oxaliplatin treatment was reported to induce peripheral neuropathy by triggering the COX2 and p-ERK1/2 cascade pathways [8,16,30]. Notably, our study corroborated these findings and showed that DF2726A significantly reduces COX2 and p-ERK1/2 protein expression by counteracting their oxaliplatin-induced activation, thus suggesting a pivotal role for CXCR1/2 in activating the COX2/ERK cascade.

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We also investigated the effect of CXCR1/2 inhibition on gene expression of TRP receptors. Recent studies showed that expression of TRP receptors, including TRVP1 and TRVP4, was strongly increased in DRG and spinal cord neurons after oxaliplatin treatment, sensitizing to thermal hyperalgesia [10,22]. Interestingly, we demonstrated that DF2726A is able to counteract upregulation of TRP receptor gene expression involved in oxaliplatin-induced neuropathic pain, supporting its potential use in combination with chemotherapeutics in future clinical trials.

Our findings show that IL-8 and its receptors CXCR1/2 are critically involved in the development of neurotoxic activity associated with both paclitaxel and oxaliplatin treatment in DRGs, and CXCL8 blocking by DF2726A significantly counteracts their effects in vitro and in vivo.

Taken together, the results of our mechanistic studies suggest that IL-8 expressed in peripheral neurons may trigger a neuro-inflammatory reaction, resulting in a progressive neural sensitization via activation of key pathways involved in  microtubule stabilization, terminal axonal arborization and synaptic plasticity, and that the progressive accumulation of IL-8 may be partially responsible for the progressive epidermal denervation associated with chemotherapeutic treatments.

In conclusion, we show for the first time in a model of both paclitaxel- and oxaliplatin-related CIPN that activation of IL-8 signaling is involved in the development of peripheral neuropathies, and that interfering with this signal using the novel compound DF2726A may be a useful strategy to alleviate chemotherapy-induced neurotoxicity.

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