ACT1 Is Required for Murine IL-23-Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity.

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases.

Foxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed RORγt and produced IL-17A. Genesis of this population was attenuated by a RORγt inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+RORγt+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+RORγt+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.

Inhibition of Interleukin-23-Mediated Inflammation with a Novel Small Molecule Inverse Agonist of RORγt.

Blockade of interleukin (IL)-23 or IL-17 with biologics is clinically validated as a treatment of psoriasis. However, the clinical impact of targeting other nodes within the IL-23/IL-17 pathway, especially with small molecules, is less defined. We report on a novel small molecule inverse agonist of retinoid acid-related orphan receptor (ROR) γt and its efficacy in preclinical models of psoriasis and arthritis. 1-(2,4-Dichloro-3-((1,4-dimethyl-6-(trifluoromethyl)-1H-indol-2-yl)methyl)benzoyl)piperidine-4-carboxylic acid (A-9758) was optimized from material identified from a high-throughput screening campaign. A-9758 is selective for RORγt and exhibits robust potency against IL-17A release both in vitro and in vivo. In vivo, we also show that IL-23 is sufficient to drive the accumulation of RORγt+ cells, and inhibition of RORγt significantly attenuates IL-23-driven psoriasiform dermatitis. Therapeutic treatment with A-9758 (i.e., delivered during active disease) was also effective in blocking skin and joint inflammation. Finally, A-9758 exhibited efficacy in an ex vivo human whole blood assay, suggesting small molecule inverse agonists of RORγt could be efficacious in human IL-17-related diseases. SIGNIFICANCE STATEMENT: Using a novel small molecule inverse agonist, and preclinical assays, we show that RORγt is a viable target for the inhibition of RORγt/Th17-driven diseases such as psoriasis. Preclinical models of psoriasis show that inhibition of RORγt blocks both the accumulation and effector function of IL-17-producing T cells.

Characterization of psoriasiform dermatitis induced by systemic injection of interleukin-23 minicircles in mice.

The interleukin (IL)-23/IL-17 axis plays a central role in the pathogenesis of psoriasis and is elevated in lesional psoriatic skin. Different murine models have been developed to mimic this pathophysiology each carrying specific merits and limitations. In an attempt to address some of these limitations, B10.RIII mice received a single hydrodynamic injection of IL-23 minicircles (MC) to induce hepatic transcription and the endogenous production of IL-23. Plasma and ear IL-23 levels were dose-dependently (0.3-3 µg) increased in MC injected mice and were sustained over the 14-day study duration. Beginning on day 7 post-injection, mice developed dose-related ear inflammation, histologically confirmed increases in epidermal and dermal area, as well as enhanced neutrophil and macrophage content. Flow cytometry demonstrated increased levels of granulocytes, T cells and monocytes/macrophages in the ear skin, with T cells identified as the main cellular source of IL-17A. Evaluation of mRNA and protein showed time-dependent, increased levels of the IL-23/IL-17 pathway and inflammatory/microbial cytokines/chemokines in the ear which differed kinetically from circulating levels. An anti-IL-23p40 antibody was assessed following both prophylactic administration and administration once the disease was established. Prophylactic dosing completely prevented the development of the ear phenotype across endpoints. Treatment administration showed a dose-related response, with a maximum inhibition of 64-94%, depending on endpoint. These data demonstrate that the IL-23 MC model is a useful approach to study IL-23/IL-17-driven skin inflammation and may facilitate preclinical assessment of novel therapies.

Monocytes/Macrophages play a pathogenic role in IL-23 mediated psoriasis-like skin inflammation.

Psoriasis is an immune-mediated inflammatory skin disease that affects millions worldwide. Studying immune cells involved in psoriasis pathogenesis is essential to identify effective and safe therapeutics for the disease. Using human psoriasis skin, activated macrophages were observed in both lesional and non-lesional skin, but were elevated in lesional skin. Activation of the IL-23/IL-17 pathway is integral to the development of psoriasis. To further characterize the monocyte/macrophage (Mon/Mac) population when the IL-23 pathway is activated, a murine model of intradermal injection of IL-23 was used. Flow cytometry revealed that Mon/Mac cells were the dominant immune population, particularly late in the model, highlighted by strong presence of Ly6ChiMHC IIhi cells. The Mon/Mac cells were also shown to have high expression for TNFα but not IL-17A. Prophylactic dosing of a CSF-1R inhibitor to deplete Mon/Mac cells significantly reduced several inflammatory mediators from the skin tissue suggesting a pathogenic role for Mon/Mac. Treatment dosing of the inhibitor produced a less robust effect. Mon/Mac cells were also differentiated by levels of Ki67 and TNFα expression. These data point to an important contribution of Mon/Mac cells in IL-23 related skin inflammation and suggest that these cells are a significant player in the underlying pathophysiology of psoriasis.

Mechanistic and pharmacological assessment of murine IL-23 mediated psoriasiform dermatitis; implications for drug discovery.

BACKGROUND: Animal models of Psoriasis (PsO) are important for our understanding of the pathophysiology of human disease but rarely manifest all features of the disease. In order to facilitate greater understanding of the underlying biology of PsO it is key that we understand the strengths and limitations of models used. OBJECTIVE: While humanized mouse models are available for PsO they remain technically challenging, expensive, require prolonged timelines and require a continued source of human tissue. Another approach is to focus on developing mechanistic models which recapitulate key features of human PsO. The role of the IL-23/IL-17 pathway as a key driver of human PsO is both well characterized and clinically validated. The goal of this manuscript is to provide a comprehensive disease and pharmacological assessment of IL-23 driven skin inflammation and its similarity to human psoriatic skin. METHODS: Intradermal injection of IL-23 has been used to study the IL-23 pathway in rodents, and this current study further characterizes pathology, cellular infiltrate, and gene signature kinetics, as well as the modulation of disease features by clinically relevant agents. RESULTS: Our results indicate that IL-23 triggers an early and robust activation of the immune system resulting in accumulation of T cell and monocyte/macrophage populations. It also supports changes in gene expression that parallel those observed in human PsO samples and is responsive to biologics commonly used to treat PsO in the clinic. CONCLUSIONS: Collectively, our studies indicate that a 5 day model of IL-23 psoriasiform dermatitis can be used to assess the pharmacology of novel small molecules/biologics in the treatment of PsO.

Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Discovery of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442): a temperature-neutral transient receptor potential vanilloid-1 (TRPV1) antagonist with analgesic efficacy.

The synthesis and characterization of a series of selective, orally bioavailable 1-(chroman-4-yl)urea TRPV1 antagonists is described. Whereas first-generation antagonists that inhibit all modes of TRPV1 activation can elicit hyperthermia, the compounds disclosed herein do not elevate core body temperature in preclinical models and only partially block acid activation of TRPV1. Advancing the SAR of this series led to the eventual identification of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442, 52), an analogue that possesses excellent pharmacological selectivity, has a favorable pharmacokinetic profile, and demonstrates good efficacy against osteoarthritis pain in rodents.

Mechanistic insights into the analgesic efficacy of A-1264087, a novel neuronal Ca(2+) channel blocker that reduces nociception in rat preclinical pain models.

UNLABELLED: Voltage-gated Ca(2+) channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca(2+) channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca(2+) channels with similar potency (IC50 = 1-2 µM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene-related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant-induced inflammatory, and chronic constrictive injury of sciatic nerve-induced, neuropathic pain models with ED50 values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval = 2.2-3.5, 3.7-10, and 5.5-12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal-regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant-inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations. PERSPECTIVE: The present results demonstrate that the neuronal Ca(2+) channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.

PARP inhibitors attenuate chemotherapy-induced painful neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major toxicity of chemotherapy treatment for which no therapy is approved. Poly(ADP-ribose) polymerase (PARP)1/2 are nuclear enzymes activated upon DNA damage, and PARP1/2 inhibition provides resistance against DNA damage. A role for PARP inhibition in sensory neurotransmission has also been established. PARP inhibitors attenuate pain-like behaviors and neuropathy-associated decreased peripheral nerve function in diabetic models. The hypothesis tested was that PARP inhibition protects against painful neuropathy. The objective of this study was to investigate whether the novel, selective PARP1/2 inhibitors (ABT-888 and related analogues) would attenuate development of mechanical allodynia in vincristine-treated rats. PARP inhibitors were dosed for 2 days, and then co-administered with vincristine for 12 days. Mechanical allodynia was observed in rats treated with vincristine. PARP1/2 inhibition significantly attenuated development of mechanical allodynia and reduced poly ADP-ribose (PAR) activation in rat skin. The data presented here show that PARP inhibition attenuates vincristine-induced mechanical allodynia in rats, and supports that PARP inhibition may represent a novel therapeutic approach for CIPN.

Potentiation of analgesic efficacy but not side effects: co-administration of an α4ß2 neuronal nicotinic acetylcholine receptor agonist and its positive allosteric modulator in experimental models of pain in rats.

Positive modulation of the neuronal nicotinic acetylcholine receptor (nAChR) α4ß2 subtype by selective positive allosteric modulator NS-9283 has shown to potentiate the nAChR agonist ABT-594-induced anti-allodynic activity in preclinical neuropathic pain. To determine whether this benefit can be extended beyond neuropathic pain, the present study examined the analgesic activity and adverse effect profile of co-administered NS-9283 and ABT-594 in a variety of preclinical models in rats. The effect of the combined therapy on drug-induced brain activities was also determined using pharmacological magnetic resonance imaging. In carrageenan-induced thermal hyperalgesia, co-administration of NS-9283 (3.5 µmol/kg, i.p.) induced a 6-fold leftward shift of the dose-response of ABT-594 (ED(50)=26 vs. 160 nmol/kg, i.p.). In the paw skin incision model of post-operative pain, co-administration of NS-9283 similarly induced a 6-fold leftward shift of ABT-594 (ED(50)=26 vs. 153 nmol/kg). In monoiodo-acetate induced knee joint pain, co-administration of NS-9283 enhanced the potency of ABT-594 by 5-fold (ED(50)=1.0 vs. 4.6 nmol/kg). In pharmacological MRI, co-administration of NS-9283 was shown to lead to a leftward shift of ABT-594 dose-response for cortical activation. ABT-594 induced CNS-related adverse effects were not exacerbated in presence of an efficacious dose of NS-9283 (3.5 µmol/kg). Acute challenge of NS-9283 produced no cross sensitization in nicotine-conditioned animals. These results demonstrate that selective positive allosteric modulation at the α4ß2 nAChR potentiates nAChR agonist-induced analgesic activity across neuropathic and nociceptive preclinical pain models without potentiating ABT-594-mediated adverse effects, suggesting that selective positive modulation of α4ß2 nAChR by PAM may represent a novel analgesic approach.

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model.

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7+/-7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 micromol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 micromol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

Synthesis and biological evaluation of 5-substituted and 4,5-disubstituted-2-arylamino oxazole TRPV1 antagonists.

The synthesis and structure-activity relationships of a series of 5-monosubstituted and 4,5-disubstituted 2-arylaminooxazoles as novel antagonists of the transient receptor potential vanilloid 1 (TRPV1) receptor are described. The 7-hydroxy group of the tetrahydronaphthyl moiety on the 2-amino substituent of the oxazole ring was important for obtaining excellent in vitro potency at the human TRPV1 receptor, while a variety of alkyl and phenyl substituents at the 4- and 5-positions of the oxazole ring were well tolerated and yielded potent TRPV1 antagonists. Despite excellent in vitro potency, the 5-monosubstituted compounds suffered from poor pharmacokinetics. It was found that 4,5-disubstitution on the oxazole ring was critical to the improvement of the overall pharmacokinetic profile of these analogues, which led to the discovery of compound (R)-27, a novel TRPV1 antagonist with good oral activity in preclinical animal models of pain.

Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT-102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post-operative, osteoarthritic, and bone cancer pain. ABT-102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT-102 for 5-12 days increased its analgesic activity in models of post-operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT-102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A-993610. Although a single dose of ABT-102 produced a self-limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT-102 effectively tolerated following twice-daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT-102 supports its advancement into clinical studies.

Discovery of potent furan piperazine sodium channel blockers for treatment of neuropathic pain.

The synthesis and pharmacological characterization of a novel furan-based class of voltage-gated sodium channel blockers is reported. Compounds were evaluated for their ability to block the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3) as well as the Na(v)1.2 and Na(v)1.5 subtypes. Benchmark compounds from this series possessed enhanced potency, oral bioavailability, and robust efficacy in a rodent model of neuropathic pain, together with improved CNS and cardiovascular safety profiles compared to the clinically used sodium channel blockers mexiletine and lamotrigine.

Analgesic activity of metabotropic glutamate receptor 1 antagonists on spontaneous post-operative pain in rats.

Activation of metabotropic glutamate (mGlu) receptors has previously been shown to play a role in inflammatory or neuropathic pain states. However, the role of mGlu type 1 receptors in post-operative pain remains to be investigated. In the present study, effects of potent and selective mGlu1 receptor antagonists A-841720, A-794282, A-794278, and A-850002 were evaluated in a skin incision-induced post-operative pain model in rats. Post-operative pain was examined 2 h following surgery using weight-bearing difference between injured and uninjured paws as a measure of spontaneous pain. In this model, A-841720, A-794282, A-794278, and A-850002 induced significant attenuation of spontaneous post-operative pain behavior, with ED50s of 10, 50, 50, and 65 micromol/kg i.p., respectively. Depending on the compound, significant motor side effects were also observed at 3 to 10 fold higher doses. These results support the notion that mGlu1 receptor activation plays a significant role in nociceptive transmission in post-operative pain, though motor impairment may be a limiting factor in developing mGlu1 receptor antagonists as novel analgesics.

A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na(v)1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na(v)1.8 (IC(50) = 8 nM) and was >100-fold selective vs. human Na(v)1.2, Na(v)1.3, Na(v)1.5, and Na(v)1.7 (IC(50) values >or=1 microM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Zotarolimus, a novel sirolimus analogue with potent anti-proliferative activity on coronary smooth muscle cells and reduced potential for systemic immunosuppression.

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2 values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

A-740003 [N-(1-{[(cyanoimino)(5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a novel and selective P2X7 receptor antagonist, dose-dependently reduces neuropathic pain in the rat.

ATP-sensitive P2X(7) receptors are localized on cells of immunological origin including glial cells in the central nervous system. Activation of P2X(7) receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1beta (IL-1beta), and following prolonged agonist exposure, cytolytic plasma membrane pore formation. P2X(7) knockout mice show reduced inflammation as well as decreased nociceptive sensitivity following peripheral nerve injury. A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel competitive antagonist of P2X(7) receptors (IC(50) values = 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC(50) > 10 muM) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes. A-740003 potently blocked agonist-evoked IL-1beta release (IC(50) = 156 nM) and pore formation (IC(50) = 92 nM) in differentiated human THP-1 cells. Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation model (ED(50) = 19 mg/kg i.p.) in the rat. A-740003 also attenuated tactile allodynia in two other models of neuropathic pain, chronic constriction injury of the sciatic nerve and vincristine-induced neuropathy. In addition, A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan or complete Freund's adjuvant (ED(50) = 38-54 mg/kg i.p.). A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. These data demonstrate that selective blockade of P2X(7) receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.