P2X7-related modulation of pathological nociception in rats.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

Pharmacological characterization of P2X7 receptors in rat peritoneal cells.

OBJECTIVE AND DESIGN: P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). MATERIALS AND METHODS: RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RESULTS: RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. CONCLUSIONS: RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana).

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.

Symmetrical bis(heteroarylmethoxyphenyl)alkylcarboxylic acids as inhibitors of leukotriene biosynthesis.

Symmetrical bis(quinolylmethoxyphenyl)alkylcarboxylic acids were investigated as inhibitors of leukotriene biosynthesis and 4, 4-bis(4-(2-quinolylmethoxy)phenyl)pentanoic acid sodium salt (47.Na) met our design parameters for a drug candidate (ABT-080). This compound was readily synthesized in three steps from commercially available diphenolic acid. Against intact human neutrophils, 47.Na inhibited ionophore-stimulated LTB(4) formation with an IC(50) = 20 nM. In zymosan-stimulated mouse peritoneal macrophages producing both LTC(4) and PGE(2), 47.Na showed 9000-fold selectivity for inhibition of LTC(4) (IC(50) = 0.16 nM) over PGE(2) (IC(50) = 1500 nM). Preliminary pharmacokinetic evaluation in rat and cynomolgus monkey demonstrated good oral bioavailability and elimination half-lives of 9 and 5 h, respectively. Pharmacological evaluation of leukotriene inhibition with oral dosing was demonstrated in a rat pleural inflammation model (ED(50) = 3 mg/kg) and a rat peritoneal passive anaphylaxis model (LTB(4), ED(50) = 2.5 mg/kg; LTE(4), ED(50) = 1.0 mg/kg). In a model of airway constriction induced by antigen challenge in actively sensitized guinea pigs, 47.Na dosed orally blocked bronchoconstriction with an ED(50) = 0.4 mg/kg, the most potent activity we have observed for any leukotriene inhibitor in this model. The mode of inhibitory action of 47.Na occurs at the stage of 5-lipoxygenase biosynthesis as it blocks both leukotriene pathways leading to LTB(4) and LTC(4) but not PGH(2) biosynthesis. However, 47.Na does not inhibit 5-lipoxygenase catalysis in a broken cell enzyme assay; therefore it is likely that 47.Na acts as a FLAP inhibitor.

Heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids as leukotriene biosynthesis inhibitors.

A novel series of heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids was studied as leukotriene biosynthesis inhibitors. A hypothesis of structure-activity optimization by insertion of an oxime moiety was investigated using REV-5901 as a starting point. A systematic structure-activity optimization showed that the spatial arrangement and stereochemistry of the oxime insertion unit proved to be important for inhibitory activity. The promising lead, S-(E)-11, inhibited LTB(4) biosynthesis in the intact human neutrophil with IC(50) of 8 nM and had superior oral activity in vivo, in a rat pleurisy model (ED(50) = 0.14 mg/kg) and rat anaphylaxis model (ED(50) = 0.13 mg/kg). In a model of lung inflammation, S-(E)-11 blocked LTE(4) biosynthesis (ED(50) of 0.1 mg/kg) and eosinophil influx (ED(50) of 0.2 mg/kg). S-(E)-11 (A-93178) was selected for further preclinical evaluation.

A new spin on an old model: in vivo evaluation of disease progression by magnetic resonance imaging with respect to standard inflammatory parameters and histopathology in the adjuvant arthritic rat.

OBJECTIVE: To noninvasively examine the pathogenesis of rat adjuvant-induced arthritis (AIA) by magnetic resonance imaging (MRI), and to correlate MRI indices of disease progression with classic inflammatory parameters and histologic evaluation. METHODS: AIA was established in male Lewis rats following subcutaneous injection in the right hindpaw with 0.5 mg of heat-killed Mycobacterium butyricum suspended in light mineral oil. In vivo MRI evaluations of soft tissue and bony changes in AIA rats with matched histopathology were correlated with changes in left hindpaw volumes, circulating leukocytes, acute-phase reactants, and urinary collagen crosslinks throughout the disease process. RESULTS: MRI of arthritic tibiotarsal joints of the uninjected left hindpaws from AIA rats demonstrated 2 distinct phases of disease activity. The first phase, apparent between days 10 and 18, was characterized by periarticular inflammation with marked synovitis, synovial fibroplasia, and distension of the joint capsule into the surrounding tissue. The secondary phase, occurring between days 18 and 30, was marked by continued soft tissue inflammation, periostitis with osteolysis, and periosteal new bone formation progressing to a state of near complete ankylosis by day 30. These 2 phases of disease activity observed by MRI paralleled biochemical, cellular, and histologic markers of disease progression. CONCLUSION: MRI can be used to noninvasively detect, monitor, and quantify the chronic synovitis and progressive destruction of soft tissue and bone in live AIA rats, thereby improving the ability to evaluate disease progression in this preclinical animal model of rheumatoid arthritis.

A morphological study on posterior gills of the mangrove crab Ucides cordatus.

Morphological and histological studies on posterior gills of the mangrove crab Ucides cordatus showed that the 5th gill (of 7) has a larger surface area and a greater number of lamellae compared to the 6th gill. Regular separation of gill lamellae, important when the gill is in air, is maintained by enlargements of the marginal canals. Conical, spine-like structures along the efferent vessel of both 5th and 6th gills were also observed. In addition, pillar cells, a discontinuous lamellar septum and a hypobranchial artery were observed. The presence of valve-like structures near the efferent vessel was also indicated. These structures, together with the pillar cells, may have a role in directing the hemolymph flow towards certain gills during particular physiological states. Localization of osmoregulatory epithelia in the lamellae of both gills was inferred from dimethylaminostyrylethylpyridiniumiodine staining. Apparently gills 5 and 6 have osmoregulatory epithelial cell patches of similar area, corresponding to 43% and 38% of the total lamellae area, respectively. However, their localization is quite different. Gill number 5 osmoregulatory patches seem to be restricted to the afferent region of the lamella whereas in gill number 6, they are more dispersed over the entire lamella. These differences may be related to the particular functional characteristics of these gills.

The role of tissue mast cells in polyacrylamide gel-induced inflammation in mice.

OBJECTIVE AND DESIGN: In the present study, we investigated the role of mast cells in a model of polyacrylamide gel (PAG)-induced inflammation in mice. SUBJECTS: Balb/c mice and two strains of mast cell deficient mice (WBB6F1/J-W/Wv, WCB6F1/J-S1/S1d). TREATMENT: Various quantities of polyacrylamide gel (Bio-Gel P4) were injected subcutaneously in the backs of mice. METHODS: Five hours after the injection of PAG the animals were euthanized, the injection sites lavaged and levels of LTB4, PGE2, TNF alpha and cells were determined. RESULTS: Subcutaneous injection of PAG caused a time-dependent response characterized by the accumulation of inflammatory cells peaking at 10 h and the formation of LTB4, PGE2 and TNF alpha, peaking at 5 h. PAG injection into W/Wv or SL/SLd mice (mice lacking mast cells) resulted in an attenuated response, i.e. LTB4 levels were reduced by 60% and minimal cell influx was seen. The lack of mast cells caused about a 30% reduction in the levels of TNF alpha found. CONCLUSIONS: These data suggest that mast cells play a prominent role in the PMN influx, TNF alpha production and eicosanoid formation in the PAG-induced inflammatory response.

Effect of mast cell deficiency and leukotriene inhibition on the influx of eosinophils induced by eotaxin.

Eosinophils are believed to be important cells in the pathogenesis of asthma and allergic disease. Mast cells and leukotrienes may play a role in eosinophil recruitment. Eotaxin was recently described as a specific chemoattractant for eosinophils. Therefore we examined the effects of eotaxin on eosinophil flux in the mast cell-deficient WBB6F1/J-KitW/ KitW-v (W/Wv) mice and in mice treated with zileuton or ABT-761, specific 5-lipoxygenase inhibitors. Mice were injected intraperitoneally with eotaxin and at various times later the peritoneal cavities were lavaged and cell populations determined. Murine recombinant eotaxin induced a dose-dependent increase in eosinophils, which reached a maximum at 1-2 h and subsided at 4 h in both BALB/c and W/WV littermate control mice (no other cell population was altered). However, in eotaxin-injected W/Wv mice, the peak of eosinophil influx was delayed, peaking at 2 h, and a lower number of eosinophils was seen. The specific lipoxygenase inhibitors zileuton and ABT-761 given 30 min before eotaxin caused a 63-79% reduction in the level of eosinophils seen in the lavage fluid. These data suggest that eotaxin may either be activating eosinophils to release leukotrienes or making them more responsive to leukotrienes. In addition, mast cells may be playing a role in the amplification of the eotaxin effect.

Synthesis of indolylalkoxyiminoalkylcarboxylates as leukotriene biosynthesis inhibitors.

A series of substituted indolylalkoxyiminoalkylcarboxylates were found to be potent leukotriene biosynthesis inhibitors. The structure-activity relationships were investigated. Representative potent inhibitors identified were the quinolyl 3a (A-86885) and pyridyl 3b (A-86886) congeners with in vitro IC50s of 21 and 9 nM and in vivo leukotriene inhibition in the rat with oral ED50s of 0.9 and 1.7 mg/kg, respectively.

ABT-761 attenuates bronchoconstriction and pulmonary inflammation in rodents.

Our primary goal has been to discover leukotriene biosynthesis inhibitors with characteristics that are appropriate for use as clinical agents. The success of the use of zileuton in the treatment of asthma led us to explore further the use of the N-hydroxyurea class of 5-lipoxygenase inhibitors as longer-acting compounds with good lung penetration. A variety of in vitro and in vivo methods were used to evaluate a large number of compounds, from which ABT-761 [(R)-N-(3-(5-(4-fluorophenylmethyl)thien-2-yl)-1-methyl-2-pr opynyl)-N-hydroxyurea] was selected for study. ABT-761 exhibited potent and selective inhibition of leukotriene formation both in vitro and in vivo. More importantly, the compound potently inhibited antigen-induced bronchospasm in guinea pigs when given either prophylactically or therapeutically. In addition, ABT-761 was a potent inhibitor of eosinophil influx into the lungs of Brown Norway rats. These data provide added support for the role of leukotrienes in both bronchospasm and eosinophilic inflammation and characterize ABT-761 as a particularly potent inhibitor of leukotrienes formed in pulmonary tissues. These data combined with the excellent pharmacokinetic characteristics of the compound indicate its potential use in the treatment of leukotriene-dependent human disease.

Pharmacological modulation of eosinophil influx into the lungs of Brown Norway rats.

A model of lung inflammation was developed in Brown Norway rats. Intense lung eosinophilia was induced by a single intravenous injection of Sephadex G-200 particles. The eosinophilia observed was preceded by an increase in cysteinyl leukotrienes found in lung lavage fluids. Theophylline and albuterol were tested in the model and found to be inactive, while dexamethasone was effective. Zileuton, a specific leukotriene inhibitor, was found to effectively inhibit leukotriene formation and the influx of eosinophils into the lungs of these Sephadex-treated animals. Studies with specific leukotriene D4 antagonists of the cysLT1 type receptor indicate that this leukotriene receptor is probably not involved directly in the eosinophilic inflammation. This model appears to be useful in characterizing potential anti-inflammatory effects of inhibitors by evaluating their ability to prevent eosinophil influx into the lung.

Interleukin-1 receptor antagonist inhibits proteoglycan breakdown in antigen induced but not polycation induced arthritis in the rabbit.

OBJECTIVE: To compare the alterations in proteoglycan metabolism in antigen induced arthritis and polycation induced arthritis and to determine the involvement of interleukin-1 (IL-1) in the cartilage degradation that occurs in these models of rheumatoid arthritis (RA). METHODS: The time course for loss of proteoglycan into the synovial fluid (SF) and inhibition of proteoglycan synthesis, as well as depletion of articular cartilage proteoglycan content, was compared in rabbit antigen arthritis and polycation arthritis. The ability of recombinant IL-1 receptor antagonist IL-1ra to block the acute cartilage loss at 24 h in these models was investigated, compared to its ability to block the cartilage breakdown induced by direct administration of IL-1 in rabbits. RESULTS: Initial loss of cartilage proteoglycan was accompanied by release of high levels of glycosaminoglycan (GAG) into the SF and decrease in proteoglycan synthetic rates in both antigen and polycation induced arthritis SF GAG rapidly returned to control levels, while proteoglycan synthesis and cartilage proteoglycan content remained depressed, suggesting that the inhibition in proteoglycan synthesis prevented recovery to normal levels. GAG loss from the cartilage into the SF in response to IL-1 injection, as well as other effects of IL-1 challenge, was blocked in a dose dependent manner by IL-1ra administered either intraarticularly (ED50 = 160 ng) or intravenously (iv) (ED50 = 0.09mg/kg). In the antigen induced arthritis model, IL-1ra (20 mg/kg, iv -2h) inhibited GAG release by 40%, whereas in polycation induced arthritis no inhibition was observed even with repeated administration of high doses of inhibitor. CONCLUSION: These studies suggest that sustained depression of proteoglycan synthesis may be responsible for the chronic depletion of articular cartilage proteoglycan in the antigen and the polycation model of RA. However, while IL-1 may play a role in the initial breakdown of articular cartilage in antigen induced arthritis, it does not appear to be involved in polycation induced arthritis in the rabbit.

Leukotrienes and alpha-naphthylisothiocyanate-induced liver injury.

alpha-naphthylisothiocyanate (ANIT) administration to rats results in periportal hepatic inflammation and injury. Glutathione (GSH) appears to be necessary for the liver injury to occur. The leukotrienes (LTs) are metabolites of arachidonic acid and potent mediators of inflammation that have been implicated in certain liver injury models. Inasmuch as GSH is a cofactor for the synthesis of cysteinyl-LTs and since inflammation is a prominent component of ANIT injury, we hypothesized that LTs are involved in producing the hepatic insult that results from ANIT administration. To test this hypothesis, rats were treated with one of several inhibitors of LT biosynthesis, A63162, Zileuton or MK-886. Each of these agents prevented the formation of LTB4 in Ca++ ionophore-stimulated whole blood from rats treated with the inhibitors. A63162 attenuated the hepatic parenchymal injury caused by ANIT and resulted in a modest decrease in ANIT-induced cholestasis. In contrast, neither Zileuton nor MK-886 attenuated liver injury. AT-125 (Acivicin) inhibits gamma-glutamyl transferase (GGT), the enzyme that catalyzes the formation of LTD4 from LTC4. AT-125 pretreatment did not prevent ANIT-induced hepatic parenchymal insult. It did, however, ameliorate the cholestasis caused by ANIT. In conclusion, the partial protection afforded by A63162 and AT-125 likely results from effects unrelated to the formation of LTs, since Zileuton and MK-886 inhibited LT synthesis without affording protection. The lack of protection by Zileuton and MK-886 in the face of LT synthesis inhibition suggests that LTs are not necessary for the expression of injury after ANIT administration.

Clinical activity of leukotriene inhibitors.

Data from the emerging clinical trials with compounds such as zileuton, ICI 204,219, Bay X1005, MK571, MK679, and MK591 are demonstrating the importance of the leukotrienes as mediators of asthma and possibly other diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. One of the major questions facing the asthma community is how much improvement in the FEV1 is needed to improve the quality of life of the asthmatic patient. Comparing the various approaches to asthma treatment, there is typically 15-20% improvement in the lung function with inhaled steroids. Leukotriene interventions apparently will improve lung function to similar levels as with inhaled steroids, and thus may offer an alternative to steroids. Like the steroids, zileuton appears to also reduce the inflammatory cell influx into the antigen-challenged site, which may have the long-term effect of reversing some of the tissue alterations that occur as a result of the inflammation seen with asthma. Importantly, the reported experience to date has shown that the leukotriene modulators do not have the same side-effects as the current therapies, and thus offer the hope that both safe and effective treatment may be derived from this approach. The clinical data reported do not yet define a preferred approach to the modulation of leukotriene pathology. As more studies are published in other diseases the broad spectrum use of these inhibitors will become known.

Optimization of the potency and duration of action of N-hydroxyurea 5-lipoxygenase inhibitors.

As in vitro glucuronidation assay and several biochemical assays were utilized to discover potent new N-hydroxyurea-containing 5-lipoxygenase inhibitors with long durations of action. The best of these, A-78773, is a racemic mixture of two enantiomers. These enantiomers were purified and the R(+)-enantiomer A-79175 was found to be superior to the S(-)-enantiomer with respect to in vitro metabolism and duration of action in the monkey. A-79175 was a potent selective inhibitor of 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemic homogenates (IC50 = 54 nM) and of calcium ionophore-induced leukotriene B4 (LTB4) formation in purified human polymorphonuclear leukocytes (IC50 = 25 nM) and human whole blood (IC50 = 80 nM). The compound inhibited LT formation in the rat with oral ED50s of 1 to 2 mg/kg. It also was a potent inhibitor of edema and inflammatory cell influx in rats and mice. A-79175 was resistant to glucuronidation and had an elimination half-life of nearly 9 hr in cynomolgus monkeys. A-79175 inhibited ex vivo LTB4 formation by monkeys for extended periods. A single 0.5-mg/kg oral dose gave > 50% inhibition of calcium ionophore-induced LTB4 formation ex vivo for 12 hr. A good correlation was found between the elimination half-life for A-78773 and its enantiomers in cynomolgus monkeys and humans. These data indicate that A-79175 is a promising long-acting agent that should be useful to delineate the importance of LTs in animal and human studies.