Optimized protein kinase Cθ (PKCθ) inhibitors reveal only modest anti-inflammatory efficacy in a rodent model of arthritis.

We previously demonstrated that selective inhibition of protein kinase Cθ (PKCθ) with triazinone 1 resulted in dose-dependent reduction of paw swelling in a mouse model of arthritis.1,2 However, a high concentration was required for efficacy, thus providing only a minimal safety window. Herein we describe a strategy to deliver safer compounds based on the hypothesis that optimization of potency in concert with good oral pharmacokinetic (PK) properties would enable in vivo efficacy at reduced exposures, resulting in an improved safety window. Ultimately, transformation of 1 yielded analogues that demonstrated excellent potency and PK properties and fully inhibited IL-2 production in an acute model. In spite of good exposure, twice-a-day treatment with 17l in the glucose-6-phosphate isomerase chronic in vivo mouse model of arthritis yielded only moderate efficacy. On the basis of the exposure achieved, we conclude that PKCθ inhibition alone is insufficient for complete efficacy in this rodent arthritis model.

Discovery of selective and orally bioavailable protein kinase Cθ (PKCθ) inhibitors from a fragment hit.

Protein kinase Cθ (PKCθ) regulates a key step in the activation of T cells. On the basis of its mechanism of action, inhibition of this kinase is hypothesized to serve as an effective therapy for autoimmune diseases such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis. Herein, the discovery of a small molecule PKCθ inhibitor is described, starting from a fragment hit 1 and advancing to compound 41 through the use of structure-based drug design. Compound 41 demonstrates excellent in vitro activity, good oral pharmacokinetics, and efficacy in both an acute in vivo mechanistic model and a chronic in vivo disease model but suffers from tolerability issues upon chronic dosing.

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Na(v)1.8 sodium channel with efficacy in a model of neuropathic pain.

Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.

A-887826 is a structurally novel, potent and voltage-dependent Na(v)1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats.

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

Comparison of mechanical allodynia and the affective component of inflammatory pain in rats.

Most animal models of pain cannot separate the sensory and affective components of pain. One model that has been used to assess affective pain is the place escape avoidance paradigm (PEAP). The aim of the current study is two-fold. First, validate PEAP with Complete Freund's Adjuvant (CFA)-induced inflammation for the assessment of the affective component of pain using the reference analgesics celecoxib, diclofenac and duloxetine; fluoxetine and scopolamine were tested as negative controls. Secondly, determine if there is a difference in efficacy in PEAP in comparison to the effects of the same compounds on von Frey-evoked mechanical allodynia in CFA animals. All compounds were tested in mechanical allodynia, place escape/avoidance, and for potentially confounding side effects in locomotor activity. Results show that celecoxib, diclofenac, and duloxetine significantly increased the time spent on the side associated with stimulation of the injured paw, whereas fluoxetine and scopolamine had no effect. Higher doses of celecoxib, diclofenac, duloxetine, and fluoxetine were required to attenuate von Frey-evoked mechanical allodynia. In the side effect assays, only fluoxetine decreased locomotor activity at doses used in PEAP. These results show that in inflammatory pain induced by CFA injection, PEAP is more sensitive to the effects of pain relieving compounds than mechanical allodynia. Fluoxetine showed efficacy in the mechanical allodynia test, but not PEAP, whereas duloxetine showed efficacy in mechanical allodynia and PEAP. These studies show that methods other than reflex based measures of pain such as affective pain models could be more predictive of efficacy/potency in the clinic.

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1alphabeta knockout mice.

The pro-inflammatory cytokine interleukin-1beta (IL-1beta) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1beta from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC50=20-150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1beta release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED50=100 micromol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED50=40 micromol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1alphabeta knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1beta.

cis-4-(Piperazin-1-yl)-5,6,7a,8,9,10,11,11a-octahydrobenzofuro[2,3-h]quinazolin-2-amine (A-987306), a new histamine H4R antagonist that blocks pain responses against carrageenan-induced hyperalgesia.

cis-4-(Piperazin-1-yl)-5,6,7a,8,9,10,11,11a-octahydrobenzofuro[2,3-h]quinazolin-2-amine, 4 (A-987306) is a new histamine H(4) antagonist. The compound is potent in H(4) receptor binding assays (rat H(4), K(i) = 3.4 nM, human H(4) K(i) = 5.8 nM) and demonstrated potent functional antagonism in vitro at human, rat, and mouse H(4) receptors in cell-based FLIPR assays. Compound 4 also demonstrated H(4) antagonism in vivo in mice, blocking H(4)-agonist induced scratch responses, and showed anti-inflammatory activity in mice in a peritonitis model. Most interesting was the high potency and efficacy of this compound in blocking pain responses, where it showed an ED(50) of 42 mumol/kg (ip) in a rat post-carrageenan thermal hyperalgesia model of inflammatory pain.

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists.

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.

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.

Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states.

Antisense (AS) oligodeoxynucleotides (ODNs) targeting the Nav 1.8 sodium channel have been reported to decrease inflammatory hyperalgesia and L5/L6 spinal nerve ligation-induced mechanical allodynia in rats. The present studies were conducted to further characterize Nav 1.8 AS antinociceptive profile in rats to better understand the role of Nav 1.8 in different pain states. Consistent with earlier reports, chronic intrathecal Nav 1.8 AS, but not mismatch (MM), ODN decreased TTX-resistant sodium current density (by 60.5+/-10.2% relative to MM; p<0.05) in neurons from L4 to L5 dorsal root ganglia and significantly attenuated mechanical allodynia following intraplantar complete Freund's adjuvant. In addition, 10 days following chronic constriction injury of the sciatic nerve, Nav 1.8 AS, but not MM, ODN also attenuated mechanical allodynia (54.3+/-8.2% effect, p<0.05 vs. MM) 2 days after initiation of ODN treatment. The anti-allodynic effects remained for the duration of the AS treatment, and CCI rats returned to an allodynic state 4 days after discontinuing AS. In contrast, Nav 1.8 AS ODN failed to reduce mechanical allodynia in the vincristine chemotherapy-induced neuropathic pain model or a skin-incision model of post-operative pain. Finally, Nav 1.8 AS, but not MM, ODN treatment produced a small but significant attenuation of acute noxious mechanical sensitivity in naïve animals (17.6+/-6.2% effect, p<0.05 vs. MM). These data demonstrate a greater involvement of Nav 1.8 in frank nerve injury and inflammatory pain as compared to acute, post-operative or chemotherapy-induced neuropathic pain states.

A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel and selective transient receptor potential type V1 receptor antagonist, blocks channel activation by vanilloids, heat, and acid.

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC50 = 5 nM). A-425619 showed similar potency (IC50 = 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC50 = 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol ester-sensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA2 = 2.5 nM) of capsaicin-evoked calcium flux. Moreover, A-425619 was 25- to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.

ABT-594 (a nicotinic acetylcholine agonist): anti-allodynia in a rat chemotherapy-induced pain model.

ABT-594 ((R)-5-(2-azetidinylmethoxy)-2-chloropyridine) represents a novel class of broad-spectrum analgesics whose primary mechanism of action is activation of the neuronal nicotinic acetylcholine receptors. The present study characterized the effects of ABT-594 in a rat chemotherapy-induced neuropathic pain model, where it attenuated mechanical allodynia with an ED50 = 40 nmol/kg (i.p.). This anti-allodynic effect was not blocked by systemic (i.p.) pretreatment with naloxone but was blocked completely with mecamylamine. Pretreatment with chlorisondamine (0.2-5 micromol/kg, i.p.) only partially blocked the effects of ABT-594 at the higher doses tested. In contrast, central (i.c.v.) pretreatment with chlorisondamine completely blocked ABT-594's anti-allodynic effect. Taken together, the data demonstrate that ABT-594 has a potent anti-allodynic effect in the rat vincristine model and that, in addition to its strong central site of action, ABT-594's effects are partially mediated by peripheral nicotinic acetylcholine receptors in this animal model of chemotherapy-induced neuropathic pain.

A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat.

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22-92 nM) and was highly selective (IC50 >10 microM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 micromolkg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10-15 micromolkg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 micromolkg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats.

Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X(3) receptor, as it is preferentially expressed on nociceptive C-fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X(3) knock-out mice. In order to further characterize the role of P2X(3) receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X(3) antisense oligonucleotides for 7 days in the rat. P2X(3) receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freund's adjuvant (CFA), formalin or alphabeta-methylene ATP into the rat's hind paw. The anti-hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X(3) antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X(3) receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X(3) receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X(3) receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X(3) receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X(3 )receptor expression or activation.