Sulfonamido-aryl ethers as bradykinin B1 receptor antagonists.

The synthesis and identification of sulfonamido-aryl ethers as potent bradykinin B1 receptor antagonists from a approximately 60,000 member encoded combinatorial library are reported. Two distinct series of compounds exhibiting different structure-activity relationships were identified in a bradykinin B1 whole-cell receptor-binding assay. Specific examples exhibit K(i) values of approximately 10nM.

Role of the cysteine protease cathepsin S in neuropathic hyperalgesia.

Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up-regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naïve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 microg/rat) induced a mechanical hyperalgesia that developed within half-an-hour, diminished by 3h and was absent after 24h. Activated rrCathepsin B (CatB) and non-activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve-injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3h by systemic administration of a non-brain penetrant, irreversible CatS inhibitor (LHVS, 3-30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti-hyperalgesic effect of LHVS. Our results demonstrate a pro-nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury.

Potent and orally bioavailable non-peptide antagonists at the human bradykinin B(1) receptor based on a 2-alkylamino-5-sulfamoylbenzamide core.

The bradykinin B(1) receptor is rapidly induced after inflammation or tissue trauma and appears to play an important role in the maintenance of hyperalgesia in inflammatory conditions. Here, we describe the optimization process to identify novel, potent non-peptide human B(1) receptor antagonists based on a 2-alkylamino-5-sulfamoylbenzamide core. Optimized derivatives are selective, functional B(1) antagonists with low nanomolar affinity and exhibit oral bioavailability in animals.

Potential applications of siRNA for pain therapy.

Chronic pain is a major socio-economic burden which remains poorly treated owing to either the lack of efficacy or the dose-limiting, adverse effects of current clinical therapies. It is currently a subject of intense pharmaceutical activity, focused on developing more efficacious therapies. A recent development involving RNA interference (RNAi) in a pathophysiological model of chronic pain highlighted a novel approach to potential treatment in humans. Delivery of oligonucleotides led to a potent and highly selective degradation of a target mRNA and produced a measurable abrogation in pain-related behaviour. This review will discuss the potential of small interfering RNA (siRNA) in the treatment of chronic pain by evaluating points of intervention in chronic pain pathways that may be amenable to siRNA application. Selected studies using RNAi in vivo in various model systems which allow insight into its application in humans are reviewed. Chemical formats and routes of administration that may suit chronic pain siRNA therapies are considered, and some potential clinical hurdles to siRNA delivery in humans are discussed.

siRNA relieves chronic neuropathic pain.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

Fluorescent peptide probes for high-throughput measurement of protein phosphatases.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.

Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain.

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.