Andrea Aramini

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL‐8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential.

Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

Significance Persistent pain in inflammatory and neuropathic conditions is often refractory to conventional analgesic therapy, with most patients suffering with unrelieved pain and serious treatment-related side effects. There is still a tremendous need to identify novel therapeutics for pain control with innovative biological mechanisms and minimal side effects. In this paper we challenge the hypothesis that a conserved structural motif across the G protein-coupled receptor family plays a regulatory role in the negative modulation of receptor activation and use a multidisciplinary approach to the rational drug design and characterization of a novel potent allosteric inhibitor of the C5a anaphylatoxin receptor (C5aR), thus providing a new promising avenue for the improvement of pharmacotherapy of chronic pain. Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR−/− mice compared with WT mice. Furthermore, treatment of C5aR−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.

DF2755A, a novel non-competitive allosteric inhibitor of CXCR1/2, reduces inflammatory and post-operative pain

The activation of CXCR1/2 has been implicated in the genesis of inflammatory and postoperative pain. Here, we investigated a novel orally acting allosteric inhibitor of CXCR1/2 (DF2755A) and evaluated its antinociceptive effect in several models of inflammatory and post-operatory pain. DF2755A was tested in vitro for efficacy in the chemotaxis assay, selectivity and toxicity. In vivo, C57Bl/6 mice were treated orally with DF2755A and the following experiments were performed: pharmacokinetic profile; inflammatory hyperalgesia models using electronic pressure meter test; neutrophil migration assay assessed by myeloperoxidase assay. DF2755A selectively inhibited neutrophil chemotaxis induced by CXCR1/2 ligands without effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF2755A on CXCL8-induced chemotaxis. DF2755A given orally was well absorbed (88.2%), and it was able to reduce, in a dose (3-30mg/kg)-dependent manner, inflammatory hyperalgesia induced by carrageenan, LPS and CXCL1/KC as well as neutrophil recruitment and IL-1β production. In addition, DF2755A was able to reduce post-incisional nociception. Therapeutic treatment with DF2755A reduced CFA-induced inflammatory hyperalgesia even when injected intrathecally. The present results indicate that DF2755A is a novel selective allosteric inhibitor of CXCR1/2 with a favorable oral pharmacokinetic profile. Furthermore, the results might suggest that DF2755A might be a candidate of a novel therapeutic option to control inflammatory and post-operative pain.

Structure-Activity Relationship of novel phenylacetic CXCR1 inhibitors.

We reported recently the Structure-Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.

DFL23448, A Novel Transient Receptor Potential Melastin 8–Selective Ion Channel Antagonist, Modifies Bladder Function and Reduces Bladder Overactivity in Awake Rats

The transient receptor potential melastin 8 ion channel (TRPM8) is implicated in bladder sensing but limited information on TRPM8 antagonists in bladder overactivity is available. This study characterizes a new TRPM8-selective antagonist (DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol]) and evaluates it in cold-induced behavioral tests and tests on bladder function and experimental bladder overactivity in vivo in rats. DFL23448 displayed IC50 values of 10 and 21 nM in hTRPM8 human embryonic kidney 293 cells activated by Cooling Agent 10 or cold, but it had limited activity (IC50 > 10 μM) at transient receptor potential vanilloids TRPV1, TRPA1, or TRPV4 or at various G protein–coupled receptors. In rats, DFL23448 administered intravenously or orally had a half-life of 37 minutes or 4.9 hours, respectively. DLF23448 (10 mg/kg i.v.) reduced icilin-induced “wet dog–like” shakes in rats. Intravesical DFL23448 (10 mg/l), but not vehicle, increased micturition intervals, micturition volume, and bladder capacity. During bladder overactivity by intravesical prostaglandin E2 (PGE2), vehicle controls exhibited reductions in micturition intervals, micturition volumes, and bladder capacity by 37%–39%, whereas the same parameters only decreased by 12%–15% (P < 0.05–0.01 versus vehicle) in DFL23448-treated rats. In vehicle-treated rats, but not in DFL23448-treated rats, intravesical PGE2 increased bladder pressures. Intravenous DFL23448 at 10 mg/kg, but not 1 mg/kg DFL23448 or vehicle, increased micturition intervals, micturition volumes, and bladder capacity. During bladder overactivity by intravesical PGE2, micturition intervals, micturition volumes, and bladder capacity decreased in vehicle– and 1 mg/kg DFL23448–treated rats, but not in 10 mg/kg DFL23448–treated rats. Bladder pressures increased less in rats treated with DFL23448 10 mg/kg than in vehicle– or 1 mg/kg DFL23448–treated rats. DFL23448 (10 mg/kg i.v.), but not vehicle, prevented cold stress–induced bladder overactivity. Our results support a role for bladder TRPM8-mediated signals in experimental bladder overactivity.

Development and validation of an LC-MS/MS method for determination of methanesulfonamide in human urine.

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantification of methanesulfonamide (MSA) in human urine. MSA is a potential in vivo metabolite of reparixin, a specific inhibitor of the CXCL8 biological activity. In this study, a simple derivatization procedure with a new reagent, N-(4-methanesulfonyl-benzoyl)-imidazole, was set up to enable MSA and the internal standard (I.S.), ethanesulfonamide (ESA), to be analysed by LC-MS/MS. After derivatization, samples were evaporated and reconstituted in 30% acetonitrile, aq. MSA and I.S. derivatives were separated by reversed phased HPLC (high performance liquid chromatography) on a Luna 5micro C18 column and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MR M) in the negative ion mode. The most intense [M-H](-) MRM transition of derivatized MSA at m/z 276.2-->197.2 was used for quantitation and the transition at m/z 290.2-->211.2 was used to monitor derivatized ESA. The method was linear over the concentration range from 1 to 100 microg/ml, with a lower limit of quantitation of 1 microg/ml. The intra- and inter-day precisions were less than 5.5% and 10.1%, respectively, and the accuracies were between -4.0% and +11.3%. The method was successfully applied to quantify levels of MSA in human urine after intravenous administration of reparixin to healthy volunteers.

Antinociceptive effect of two novel transient receptor potential melastatin 8 antagonists in acute and chronic pain models in rat

Transient receptor potential (TRP) channels are a superfamily of non‐selective cation permeable channels involved in peripheral sensory signalling. Animal studies have shown that several TRPs are important players in pain modulation. Among them, the TRP melastatin 8 (TRPM8) has elicited more interest for its controversial role in nociception. This channel, expressed by a subpopulation of sensory neurons in dorsal root ganglia (DRG) and trigeminal ganglia (TG), is activated by cold temperatures and cooling agents. In experimental neuropathic pain models, an up‐regulation of this receptor in DRG and TG has been observed, suggesting a key role for TRPM8 in the development and maintenance of pain. Consistent with this hypothesis, TRPM8 knockout mice are less responsive to pain stimuli.

Conformational Change in the Mechanism of Inclusion of Ketoprofen in β-Cyclodextrin: NMR Spectroscopy, Ab Initio Calculations, Molecular Dynamics Simulations, and Photoreactivity.

Inclusion of drugs in cyclodextrins (CDs) is a recognized tool for modifying several properties such as solubility, stability, bioavailability, and so on. The photoreactive behavior of the β-CD/ketoprofen (KP) complex upon UV exposure showed a significant increase in photodecarboxylation, whereas the secondary degradation products by hydroxylation of the benzophenone moiety were inhibited. The results may account for an improvement of KP photophysical properties upon inclusion, thus better fostering its topical use. To correlate the structural details of the inclusion with these results, an NMR spectroscopic study of KP upon inclusion in β-CD was performed. Effects of the magnetically anisotropic centers of KP, changing their orientations upon inclusion and giving chemical shift variations, were specifically correlated with the results of the molecular dynamic simulations and ab initio calculations. In the large variety of papers focusing on the structural analysis of β-CD complexes, this work represents one of the few examples in which a detailed analysis of these simultaneous upfield-downfield NMR shifts of the same aromatic molecule upon inclusion is reported. Interestingly, the results demonstrate that the observed upfield and downfield shifts upon inclusion are not related to any direct magnetic role of β-CD. The conformational change of KP upon the inclusion process consists of a slight reduction in the angle between the two phenyl rings and in a remarkable reduction in the mobility of the carboxyl group, the latter being one of the main contributions to the NMR resonance shifts. These structural details help in understanding the features of the inclusion complex and, eventually, the driving force for its formation.

Differential protein modulation by ketoprofen and ibuprofen underlines different cellular response by gastric epithelium

Ketoprofen L‐lysine salt (KLS), is widely used due to its analgesic efficacy and tolerability, and L‐lysine was reported to increase the solubility and the gastric tolerance of ketoprofen. In a recent report, L‐lysine salification has been shown to exert a gastroprotective effect due to its specific ability to counteract the NSAIDs‐induced oxidative stress and up‐regulate gastroprotective proteins. In order to derive further insights into the safety and efficacy profile of KLS, in this study we additionally compared the effect of lysine and arginine, another amino acid counterion commonly used for NSAIDs salification, in control and in ethanol challenged human gastric mucosa model. KLS is widely used for the control of post‐surgical pain and for the management of pain and fever in inflammatory conditions in children and adults. It is generally well tolerated in pediatric patients, and data from three studies in >900 children indicate that oral administration is well tolerated when administered for up to 3 weeks after surgery. Since only few studies have so far investigated the effect of ketoprofen on gastric mucosa maintenance and adaptive mechanisms, in the second part of the study we applied the cMap approach to compare ketoprofen‐induced and ibuprofen‐induced gene expression profiles in order to explore compound‐specific targeted biological pathways. Among the several genes exclusively modulated by ketoprofen, our attention was particularly focused on genes involved in the maintenance of gastric mucosa barrier integrity (cell junctions, morphology, and viability). The hypothesis was further validated by Real‐time PCR.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor: Therapeutic potential of a CXCR1/CXCR2 inhibitor

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL‐8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential.