Pyrido[2,3-b]pyrazines, discovery of TRPV1 antagonists with reduced potential for the formation of reactive metabolites.

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a non-selective cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to identify novel agents for pain treatment. During pre-clinical development, the 1,8-naphthyridine 2 demonstrated unacceptably high levels of irreversible covalent binding. Replacement of the 1,8-naphthyridine core by a pyrido[2,3-b]pyrazine led to the discovery of compound 26 which was shown to have significantly lower potential for the formation of reactive metabolites. Compound 26 was characterized as an orally bioavailable TRPV1 antagonist with moderate brain penetration. In vivo, 26 significantly attenuated carrageenan-induced thermal hyperalgesia (CITH) and dose-dependently reduced complete Freund's adjuvant (CFA)-induced chronic inflammatory pain after oral administration.

Locomotor activity in a novel environment as a test of inflammatory pain in rats.

Creating a robust and unbiased assay for the study of current and novel analgesics has been a daunting task. Traditional rodent models of pain and inflammation typically rely on a negative reaction to various forms of evoked stimuli to elicit a pain response and are subject to rater interpretation. Recently, models such as weight bearing and gait analysis have been developed to address these drawbacks while detecting a drug's analgesic properties. We have recently developed the Reduction of Spontaneous Activity by Adjuvant (RSAA) model as a quick, unbiased method for the testing of potential analgesics. Rats, following prior administration of an activity-decreasing inflammatory insult, will positively increase spontaneous locomotor exploration when given single doses of known analgesics. The RSAA model capitalizes on a rat's spontaneous exploratory behavior in a novel environment with the aid of computer tracking software to quantify movement and eliminate rater bias.

Discovery of novel 6,6-heterocycles as transient receptor potential vanilloid (TRPV1) antagonists.

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a nonselective cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to identify novel agents for pain treatment. The design and synthesis of a series of novel TRPV1 antagonists with a variety of different 6,6-heterocyclic cores is described, and an extensive evaluation of the pharmacological and pharmacokinetic properties of a number of these compounds is reported. For example, the 1,8-naphthyridine 52 was characterized as an orally bioavailable and brain penetrant TRPV1 antagonist. In vivo, 52 fully reversed carrageenan-induced thermal hyperalgesia (CITH) in rats and dose-dependently potently reduced complete Freund's adjuvant (CFA) induced chronic inflammatory pain after oral administration.

TRP channels and pain.

Preclinical research has identified an array of ion channels in sensory neurons involved in the generation and transduction of pain as potential targets for pharmacological intervention. Paramount among these new targets is the family of thermosensitive transient receptor potential channels, referred to as "thermoTRPs". We detect a wide range of noxious stimuli via a limited number (as of today, six) of thermoTRP channels, four of which (TRPV1-TRPV4) respond to heat and two (TRPA1 and TRPM8) are sensitive to cold. Targeting these thermoTRP channels represents a new and logical strategy in pain relief. Unlike traditional analgesic drugs that either suppress inflammation (e.g. NSAIDs and COX-2 inhibitors) or block pain transmission (e.g. opiates), TRP channel inhibitors aim to prevent pain by blocking a receptor where pain is generated. The archetypal thermoTRP is the vanilloid (capsaicin) receptor TRPV1. TRPV1 has a dynamic threshold of activation. Agents in inflammatory soup, including endogenous TRPV1 agonists (so-called "endovanilloids"), act in concert to reduce the heat activation threshold of TRPV1. In patients, the expression of TRPV1 is up-regulated in a number of painful inflammatory disorders. TRPV1 as a pain target has been validated by genetic deletion and pharmacological inhibition experiments. This area of drug development has been moving rapidly. It took less than a decade from the cloning of TRPV1 to clinical trials with potent small molecule TRPV1 antagonists. This review evaluates current evidence that supports particular TRP channels as targets for novel analgesic drugs, along with potential adverse effects that may limit drug development.

C5a, but not C3a, increases VEGF secretion in ARPE-19 human retinal pigment epithelial cells.

PURPOSE: We examined the potential for the pro-inflammatory complement proteins C5a and C3a to increase VEGF expression in ARPE-19 cells. MATERIALS AND METHODS: Expression of complement receptors in ARPE-19 cells was evaluated by RT-PCR. VEGF secretion from ARPE-19 cells treated with C5a or C3a was determined by ELISA. RESULTS: C5a and C3a receptor, but not C5L2, were detected in human eye tissue and ARPE-19 cells. C5a, but not C3a, treatment increased VEGF secretion from ARPE-19 cells, an effect inhibited by the C5aR antagonist, NDT 9513727. CONCLUSIONS: C5a receptor mediates increased VEGF secretion from ARPE-19 cells, suggesting a role for the C5a receptor in the pathogenesis of macular degeneration.

Characterization of N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 antagonist in animal models of pain and inflammation.

Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

1-Benzylbenzimidazoles: the discovery of a novel series of bradykinin B(1) receptor antagonists.

The design, synthesis, and structure-activity studies of a novel series of BK B(1) receptor antagonists based on a 1-benzylbenzimidazole chemotype are described. A number of compounds, for example, 38g, with excellent affinity for the cynomolgus macaque and rat bradykinin B(1) receptor were discovered.

Identification and characterization of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a novel, orally bioavailable C5a receptor inverse agonist.

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca(2+) mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC(50)s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC(50) of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.

Aminoquinazolines as TRPV1 antagonists: modulation of drug-like properties through the exploration of 2-position substitution.

A focused SAR exploration of the lead 4-aminoquinazoline TRPV1 antagonist 2 led to the discovery of compound 18. In rats, compound 18 is readily absorbed following oral dosing and demonstrates excellent in vivo potency and efficacy in an acute inflammatory pain model.

New frontiers in assessing pain and analgesia in laboratory animals.

BACKGROUND: Translating promising analgesic compounds into reliable pain therapeutics in humans is made particularly challenging by the difficulty in measuring the pain quantitatively. This problem is manifest not only in clinical settings in which patient pain assessments involve mostly subjective measures but also in preclinical settings wherein laboratory animals, most commonly rodents, are typically evaluated in stimulus-evoked response tests. OBJECTIVE: Given the limitations of traditional pain tests, we sought out new approaches to measure pain, and analgesia, in laboratory animals. METHODS: We reviewed the peer reviewed literature to identify pain tests that could be utilized in preclinical settings to understand the effects of new and established analgesics. RESULTS/CONCLUSIONS: The tests identified include weight bearing differential, suppression of feeding, reduction in locomotor activity, gait analysis, conditioning models and functional MRI. Although the pharmacology of known and new analgesics has not been broadly established in these models, they hold the promise of better predictive utility for the discovery of pain relievers.

TRP channels and pain.

Since the molecular identification of the capsaicin receptor, now known as TRPV1, transient receptor potential (TRP) channels have occupied an important place in the understanding of sensory nerve function in the context of pain. Several TRP channels exhibit sensitivity to substances previously known to cause pain or pain-like sensations; these include cinnamaldehyde, menthol, gingerol, and icillin. Many TRP channels also exhibit significant sensitivity to increases or decreases in temperature. Some TRP channels are sensitized in vitro by the activation of other receptors such that these channels may be activated by processes, such as inflammation that result in pain. TRP channels are suggested to be involved in processes as diverse as sensory neuron activation events, neurotransmitter release and action in the spinal cord, and release of inflammatory mediators. These functions strongly suggest that specific and selective inhibition of TRP channel activity will be of use in alleviating pain.

The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept.

The clinical use of TRPV1 (transient receptor potential vanilloid subfamily, member 1; also known as VR1) antagonists is based on the concept that endogenous agonists acting on TRPV1 might provide a major contribution to certain pain conditions. Indeed, a number of small-molecule TRPV1 antagonists are already undergoing Phase I/II clinical trials for the indications of chronic inflammatory pain and migraine. Moreover, animal models suggest a therapeutic value for TRPV1 antagonists in the treatment of other types of pain, including pain from cancer. We argue that TRPV1 antagonists alone or in conjunction with other analgesics will improve the quality of life of people with migraine, chronic intractable pain secondary to cancer, AIDS or diabetes. Moreover, emerging data indicate that TRPV1 antagonists could also be useful in treating disorders other than pain, such as urinary urge incontinence, chronic cough and irritable bowel syndrome. The lack of effective drugs for treating many of these conditions highlights the need for further investigation into the therapeutic potential of TRPV1 antagonists.

Inflammation-induced reduction of spontaneous activity by adjuvant: A novel model to study the effect of analgesics in rats.

The majority of rodent models used to evaluate analgesic drug effects rely on evoked measures of nociceptive thresholds as primary outcomes. These approaches are often time-consuming, requiring extensive habituation sessions and repeated presentations of eliciting stimuli, and are prone to false-positive outcomes due to sedation or tester subjectivity. Here, we describe the reduction of spontaneous activity by adjuvant (RSAA) model as an objective and quantifiable behavioral model of inflammatory pain that can predict the analgesic activity of a variety of agents following single-dose administration. In the RSAA model, activity was measured in nonhabituated rats using standard, photocell-based monitors. Bilateral inflammation of the knee joints by complete Freund's adjuvant (CFA) reduced the normal level of activity (horizontal locomotion and vertical rearing) by approximately 60% in a novel environment. This reduction in activity was dose-dependently reversed by ibuprofen, rofecoxib, celecoxib, piroxicam, and dexamethasone, whereas gabapentin and amitriptyline were inactive. Morphine significantly reversed the activity-suppressing effects of CFA, at 1 mg/kg s.c., but at higher doses locomotor activity progressively declined, coincident with the induction of sedation. In contrast to morphine and anti-inflammatory therapies, amphetamine did not affect vertical rearing, even though it increased horizontal locomotion. Thus, unlike standard measures of analgesia such as alteration in thermal or mechanical sensitivity, the RSAA model operationally defines analgesia as a drug-induced increase in spontaneous behavior (vertical rearing in a novel environment). We conclude that the RSAA model is valuable as an objective measure of analgesic efficacy that is not dependent on an evoked stimulus response.

Leukocyte effects of C5a-receptor blockade during simulated extracorporeal circulation.

BACKGROUND: Distinct pathways of leukocyte activation during simulated cardiopulmonary bypass are mediated by the complement C5a anaphylatoxin. We hypothesized that a human C5a receptor antagonist would specifically inhibit the inflammatory response of neutrophils to simulated extracorporeal circulation, while preserving the C5b-9 pathway for innate immunity. METHODS: An in vitro extracorporeal circuit recirculated fresh heparinized whole blood through a membrane oxygenator with and without addition of a small molecule human C5a receptor antagonist. Samples were periodically drawn over 90 minutes for complement and leukocyte activation studies. RESULTS: Addition of the C5a receptor antagonist to simulated extracorporeal circulation abrogated both neutrophil CD11b upregulation and interleukin 8 release (p < 0.01 for both), despite full generation of C3a and C5b-9; however, elastase release from neutrophils was unaffected. Although C5a receptor blockade only trended toward inhibiting monocyte CD11b upregulation (p = 0.09), circuit clearance of both monocytes (p = 0.04) and neutrophils (p = 0.01) was significantly decreased. In addition, the C5a receptor antagonist completely blocked both neutrophil-platelet and monocyte-platelet conjugate formation (p < 0.001 for both), without affecting platelet P-selectin expression. CONCLUSIONS: C5a receptor blockade during simulated extracorporeal circulation completely blocked neutrophil beta2 integrin upregulation and induction of plasma interleukin 8, suggesting an acute downregulatory effect on neutrophil chemotaxis-related pathways, while preserving terminal complement generation and neutrophil elastase release. Inhibition of leukocyte-platelet conjugate formation suggests a novel function for leukocyte adhesive receptors, possibly related to preservation of elastase generation.

From arylureas to biarylamides to aminoquinazolines: discovery of a novel, potent TRPV1 antagonist.

Bioisosteric replacement of piperazine with an aryl ring in lead VR1 antagonist 1 led to the biarylamide series. The development of B-ring SAR led to the conformationally constrained analog 70. The resulting aminoquinazoline 70 represents a novel VR1 antagonist with improved in vitro potency and oral bioavailability vs the analogous compounds from the lead series.

Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion.

Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the beta chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3/L6) with compressed lumbar DRG (L4/L5) for changes in the gene expression of CCR2. In situ hybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4/L5 and ipsilateral noncompressed L3/L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 +/- 3.5%, 49 +/- 6.2%, and 41 +/- 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4/L5 and noncompressed L3/L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRG in vitro produced potent excitatory effects not observed in control ganglia. MCP-1/CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.

Transient receptor potential ion channels as targets for the discovery of pain therapeutics.

A subset of transient receptor potential (TRP) channels exhibits activity that is highly sensitive to temperature changes and is expressed in sensory tissues, such as nociceptors and skin. Some of these thermosensitive TRP channels, such as TRPV1, TRPV4 and TRPA1, are activated or sensitized by molecules generated by inflammation and/or cell damage. TRPV1, also known as the capsaicin receptor, is particularly important in mediating hyperalgesic responses in inflammatory pain states, as demonstrated by research in knockout animals and with small-molecule antagonists. It is anticipated that TRPV1 antagonists, and perhaps antagonists at other thermosensitive TRP channels, will provide new therapeutic options with which to treat clinical pain.

Biochemical pharmacology of the vanilloid receptor TRPV1. An update.

There is mounting evidence that the vanilloid (capsaicin) receptor; transient receptor potential channel, vanilloid subfamily member 1 (TRPV1), is subjected to multiple interacting levels of control. The first level is by reversible phosphorylation catalyzed by intrinsic kinases (e.g. protein kinase A and C) and phosphatases (e.g. calcineurin), which plays a pivotal role in receptor sensitization vs. tachyphylaxis. In addition, this mechanism links TRPV1 to intracellular signaling by various important endogenous as well as exogenous substances such as bradykinin, ethanol, nicotin and insulin. It is not clear, however, whether phosphorylation per se is sufficient to liberate TRPV1 under the inhibitory control of phosphatydylinositol-4,5-bisphosphate. The second level of control is by forming TRPV1 heteromers and their association with putative regulatory proteins. The next level of regulation is by subcellular compartmentalization. The membrane form of TRPV1 functions as a nonselective cation channel. On the endoplasmic reticulum, TRPV1 is present in two differentially regulated forms, one of which is inositol triphosphate-dependent whereas the other is not. These three TRPV1 compartments provide a versatile regulation of intracellular Ca(2+) levels. Last, there is a complex and poorly understood regulation of TRPV1 activity via control of gene expression. Factors that downregulate TRPV1 expression include vanilloid treatment and growth factor (notably, nerve growth factor) deprivation. By contrast, TRPV1 appears to be upregulated during inflammatory conditions. Interestingly, following experimental nerve injury and in animal models of diabetic neuropathy TRPV1 is present on neurons that do not normally express TRPV1. Combined, these findings imply an important role for aberrant TRPV1 expression in the development of neuropathic pain and hyperalgesia. In humans, disease-related changes in TRPV1 expression have already been described (e.g. inflammatory bowel disease and irritable bowel syndrome). The mechanisms that regulate TRPV1 gene expression under pathological conditions are unknown but a better understanding of these pathways has obvious implications for rational drug development.

Formalin- or adjuvant-induced peripheral inflammation increases neurokinin-1 receptor gene expression in the mouse.

Substance P (SP) has been widely studied as a mediator of nociception. The release of SP from primary afferent neurons is increased during nociception, and SP activates neurokinin-1 (NK-1) receptors in the spinal cord and periphery. Nociception-evoked alterations in NK-1 receptor gene expression have been studied in rat models of persistent pain but have not been characterized in any murine models of peripheral inflammation. This study assessed behavioral responses and NK-1 receptor mRNA gene expression in mice receiving formalin or Freund's complete adjuvant (CFA) as an inflammatory stimulus. Mechanical withdrawal thresholds were measured before injection of formalin or CFA and hind paw licking/biting timed during the late-phase of the formalin response. Two and 24 hours after formalin or CFA injection, mechanical withdrawal thresholds were measured and the mice euthanized. Solution hybridization-nuclease protection assays were used to quantify NK-1 receptor mRNA levels. Results demonstrated that inflamed hind paws were edematous, and the withdrawal thresholds of the inflamed hind paws were significantly lower after formalin or CFA injection. Neurokinin-1 receptor mRNA levels in the ipsilateral dorsal spinal cords of mice were higher at 24 h after formalin injection or 4 days after CFA injection. These results confirm that mice are hyperalgesic at late time points after formalin or adjuvant injection when NK-1 receptor gene expression is elevated in the dorsal spinal cord. This supports the hypothesis that increased NK-1 receptor gene expression contributes to the development and maintenance of a hyperalgesic state.