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

Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side effect of several anti-neoplastics and a main cause of sensory disturbances in cancer survivors, negatively impacting patients’ quality of life. Peripheral nerve degeneration or small fibre neuropathy is generally accepted as the underlying mechanism in the development of CIPN. Recent evidence has contributed to clarify the determinant role of cytokines and chemokines in the process leading to neuronal hyperexcitability. Exposure to oxaliplatin triggers alterations in peripheral neuropathic pathways previously linked to IL-8 pathway. We investigated a novel selective inhibitor of IL-8 receptors, DF2726A, and showed its effects in counteracting CINP pathways, 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.

incubated at 37°C for 15 min in the presence or absence of different concentrations of DF2726A or vehicle were seeded in the upper compartment. The two compartments were separated by 5 µm presize polycarbonate filter (polyvinylpyrrolidone-free for PMN chemotaxis). The chamber was incubated at 37°C in air with 5% CO2 for 45 min (PMNs), or 2 h (monocytes). At the end of incubation, filters containing migrated cells were removed, fixed, stained with Diff-Quik and 5 oil immersion fields at high magnification (100X; Zeiss microscope) were counted after sample coding.

Physicochemical characterization
The main physicochemical properties of the compound (pKa, logD7.4, logP and solubility) were determined using the SiriusT3 apparatus (Sirius Analytical Instruments Ltd., East Sussex, UK) equipped with an Ag/AgCl double junction reference pH electrode, a Sirius D-PAS spectrometer and a turbidity-sensing device. The titration experiments were conducted in 0.15 M KCl solution under argon atmosphere at a temperature of 25 ± 1°C.

Selectivity
DF2726A was tested at Eurofins Cerep SA (France) by radioligand binding assays to assess the offtarget activities towards a panel of GPCRs, enzymes, ion channels, transporters and nuclear receptors. All the selected targets are recommended by four major pharmaceutical companies 3 .
DF2726A was dissolved in DMSO to achieve 10 mM stock solution, which was diluted with water/HBSS to a final concentration of 10 μM. Cell membrane homogenates (48 μg protein) were incubated for 60 min at 22°C with the respective reference compound in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2 and 1 mM EDTA. After incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard Instruments, Meriden, CT, USA) presoaked with 0.3% polyethylenimine (PEI) and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard Instruments). The filters were dried, then counted for radioactivity in a scintillation counter (Topcount, Packard Instruments) using a scintillation cocktail (Microscint-O, Packard Instruments).
The results were expressed as the percentage inhibition of the control radioligand-specific binding.
The compounds were tested at a single concentration of 10 µM in triplicate.
TRPM8-, TRPA1-, TRPV1-, TRPV4-and Nav1.7-expressing HEK-293 cells were analyzed in order to study the response to the compounds using a Ca2+ mobilization-dependent fluorescence signal in 384 MTP format. Cells were seeded at 10,000 cells per well in 384 MTP in complete medium (25 µl well-1). Twenty-four hours after seeding, the medium was removed and cells were loaded with 20 μL/well of the Fluo-8 NW dye solution. The dye-loaded cell plates were incubated for 1 h at RT. Test compounds at 3X-concentration in 1.5% DMSO Tyrode's buffer were added to the wells of an assay plate, in 10 μL volume (for a final DMSO concentration of 0.5%) and read by the FLIPRTETRA plate. The kinetic response was monitored by the instrument over a period of 3 mi (180 seconds). A second injection of 10 μL well-1 of reference agonists (Capsaicin, GSK1016790A, Isothiocyanate and Veratridine for TRPA1, TRPV1, TRPV4 and Nav1.7, respectevely) at 4X-concentration in assay buffer (EC80) was added by the FLIPRTETRA. The signal of the emitted fluorescence was recorded for an additional 3 min.
DF2726A was tested at 8 concentrations in quadruplicate (30 μM was the highest tested concentration) to determinate the IC50 towards the panel of ion channels. The compound curve fitting profile on each dose-response was performed with the Condoseo module of Genedata Screener 13.0.5.
DF2726A was tested on TRPM8, TRPV1, TRPV4, TRPA1 and Nav1.7 ion channels in agonist and antagonist mode. Similarly, 50 µL of incubated PBS were added to 50 µL of blank plasma and 300 µL of acetonitrile with Verapamil. Samples were centrifuged at 12000 x g for 5 min and surnatants were transferred to vials for LC-MS/MS analysis.

COX1-2 assay
DF2726A was tested to evaluate the activity towards human COX1 and COX2 by measuring the formation of PGE2 from arachidonic acid using a recombinant enzyme isolated from transfected Sf-9 cells.
The test compound, reference compound or water (control) were pre-incubated for 20 min at RT with the enzyme (≈ 5 µg for COX1 assay and ≈ 0.2 µg for COX2 assay) in a buffer containing 90 mM Tris-HCl (pH 8.0), 1.98 mM phenol and 1.02 µM hematine. Thereafter, the reaction was initiated by adding 4 µM (COX1) or 2 µM (COX2) of arachidonic acid and the mixture was incubated for 5 min at RT. For basal control measurements, arachidonic acid was omitted from the reaction mixture. Following incubation, the reaction was stopped by the addition of 1 M HCl then 1 M Tris/HCl (pH 8.0) followed by cooling to 4°C.
The fluorescence acceptor (d2 labeled PGE2) and the fluorescence donor (anti-PGE2 antibody labeled with europium Cryptate) were then added. After 120 min, the fluorescence transfer corresponding to the amount of residual PGE2 was measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as a percentage inhibition of the control enzyme activity. The standard inhibitory reference compound was Diclofenac, which was tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value was calculated. DF2726A was tested at a single concentration of 50 µM in triplicate.

hERG inhibition
The human ether-a-go-go related gene (hERG) inhibition assay was performed by Eurofins Panlabs Inc, USA. DF2726A was tested to assess the potential interaction on hERG channel stably transfected in Chinese hamster ovary (CHO-K1) cell line. Single cell ionic currents were measured in the automated patch clamp configuration at room temperature by using QPatch 16 instrument.
The compound was tested at 5 increasing concentrations (0.01, 0.05, 0.1, 0.3 and 1 mM) in triplicate to obtain the IC50 values.

AMES assay
DF2726A was tested to investigate the potential gene mutation induction by means of the Salmonella Typhimurium reverse mutation assay in TA98, TA100, TA1535 and TA1537 strains.
The assay was performed with and without liver microsomal activation by using rat liver S9 fraction. The compound was tested at the following concentrations: 100 µM, 50 µM, 10 µM, 5 µM (n = 12). The bacterial plates were incubated with the test compound for 96 hours, after which bacterial growth was measured spectrophotometrically using a pH indicator that changes color in response to the acidification of the media due to bacterial growth. To prevent false negatives due to bactericidal or bacteriostatic effects, a bacterial cytotoxicity assay was conducted in parallel with the Ames fluctuation assay (8 concentrations with 100 μM as the highest concentration and n = 3).
Four reference compounds (quercetin, streptozotocin, aminoanthracene and aminoacridine) were included in all assays.
On day of treatment, an exact amount of DF2726A was dissolved in the appropriate volume of vehicle to obtain a final concentration of 2.0 mg/mL (IV) and 1 mg/mL (OS). Both of the formulations were filtered at 0.45 μm filter before use in animals.

Drug Administration
DF2726A was administered at a dose of 30 mg/2ml/kg/os for 14 consecutive days starting 3 days before paclitaxel administration and continuing for 11 days after the first administration of paclitaxel.

Induction of neuropathy by paclitaxel
Rats received 4 once daily intraperitoneal (i.p.) injections of paclitaxel (Tocris, Italy) (2 mg/kg/day i.p.; cumulative dose of 8 mg/kg i.p.) or vehicle (saline, 1ml/kg/day i.p.), administered on alternate days (days 0, 2, 4, and 6) as described 4 . Behavioral testing was performed prior to paclitaxel/vehicle administration (day-1) in order to determine the basal values of the mechanical and cold nociceptive thresholds, and again on 5, 7, 10 and 14 days following paclitaxel/vehicle injection as described 5 .

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). Animals were placed in a chamber with a mesh metal floor covered by a plastic dome that enabled the animal to walk freely, but not to jump. The mechanical stimulus was then delivered in the mid-plantar skin of the hind paw. The cut-off was fixed at 50 g. Testing was performed on both paws before (day -1) and then on 5, 7, 10 and 14 days after paclitaxel administration.

Cold allodynia
Cold sensitivity was measured as the number of foot withdrawal responses after application of acetone to the dorsal surface of the paw 6 . A drop of acetone 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. Data represent mean of 3 measurements performed at an interval of approximately 5 min.
Cold responses were measured on both paws before (day -1) and then on 5, 7, 10 and 14 days after Paclitaxel stock solution (10 mM) was prepared by dissolving the powder in DMSO, and aliquots were stored at -20°C.