Bone parameters are improved with intermittent dosing of vitamin D3 and calcitonin.

Intermittent combination of an anabolic agent to promote bone formation and an antiresorptive agent that would prevent further bone loss is a theoretically attractive approach for restoring bone mass. We tested the potential of intermittently dosed calcitriol and calcitonin (CT) to restore bone properties in ovariectomized (Ovx) rats. Rats had Ovx or sham surgery at 8 weeks old and 4 weeks later were assigned to experimental groups: (1) sham vehicle, (2) Ovx vehicle, (3) Ovx + parathyroid hormone (PTH, 40 microg/kg), and (4) Ovx + calcitriol (2 microg/kg) + CT (2 microg/kg). Group 3 received PTH every week throughout the study, and group 4 received calcitriol at weeks 1, 3, 5, and 7 and CT at weeks 2, 4, 6, and 8. Dosing was carried out for 8 weeks with serum, and micro-computed tomographic analysis was done at 0, 4, and 8 weeks. Femurs and tibias were used for radiological analyses and for mechanical testing. Dosing with PTH improved bone mass and structure of cancellous bone at metaphyses of tibias and femurs as well as properties of cortical bone including geometry and strength. Intermittent dosing with calcitriol and CT was less potent in correcting loss of cancellous bone relative to treatment with PTH and had no effect on cortical bone parameters. However, intermittent dosing with calcitriol and CT was robust enough to improve cancellous bone mass and structure through bone formation without causing deleterious side effects. Our data provide additional evidence that therapies can be devised to ameliorate the skeletal defects associated with established osteoporosis.

Genetic ablation of the src kinase p59fynT exacerbates pulmonary inflammation in an allergic mouse model.

p59fynT is a protein tyrosine kinase in the src family that has been associated with and believed to function in the signaling of many receptors, including the T-cell receptor. A role for the kinase in antigen-driven pulmonary inflammation was examined using mice whose p59fynT gene had been genetically ablated. FynKO mice that were sensitized to ovalbumin exhibited a marked increase in bronchoalveolar lavage eosinophils and cytokines, including interleukin (IL)-4 and IL-5, relative to wild-type mice in response to antigen aerosol exposure. Ovalbumin-stimulated IL-5 production was also increased in cultured splenocytes derived from fynKO mice relative to wild-type mice, whereas interferon-gamma levels were unchanged. Diminished concanavalin A--stimulated IL-4 levels from fynKO splenocytes were consistent with reduced serum immunoglobulin (Ig)E levels observed in sensitized/saline aerosol-challenged animals and may reflect defective natural killer 1.1(+) T cell development. Normalization of IgE levels in sensitized fynKO mice relative to wild-type mice occurred after repeat antigen challenge, which suggests a secondary source of IL-4. Overall, these data demonstrate fyn is a negative regulator of allergic airway inflammation in mice because its absence promotes a shift to a T helper-2 phenotype that may reflect the kinase's role in T-cell receptor signaling.

Characterization of chemokine CCR3 agonist-mediated eosinophil recruitment in the Brown-Norway rat.

1. The ability of various C-C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown-Norway rat. 2. Eotaxin (0.1 - 3 microg site-1) of human and murine origin produced equivalent, dose-dependent increases in eosinophil peroxidase activity in rat dermis 4 h post-injection. 3. Human eotaxin-2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP-3, RANTES and MCP-4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 microg site-1 whereas the latter did produce a small effect at 3 microg site-1. 4. Consistent with observations in vivo, human eotaxin displaced [125I]-eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP-4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 microM. 5. Human eotaxin (5 microg) administered intravenously increased circulating eosinophils approximately 3 fold whereas MCP-4 (5 microg i.v.) increased circulating monocytes approximately 3 fold without affecting eosinophil numbers. 6. Dexamethasone pretreatment inhibited eotaxin-induced dermal eosinophil influx only at a steroid dose (0.1 mg kg-1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 microg, i.v.). 7. These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP-4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin-induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment.

7-Oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridines as novel inhibitors of human eosinophil phosphodiesterase.

High-throughput file screening against inhibition of human lung PDE4 led to the discovery of 3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4, 5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to analogues up to 50-fold more potent than 11 with IC50 values of 0.03-1.6 microM. One such compound, CP-220,629 (22) (IC50 = 0.44 microM), was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 2.0 mg/kg, po) and demonstrated a significant reduction in eosinophil (55%), neutrophil (65%), and IL-1beta (82%) responses to antigen challenge in atopic monkeys (10 mg/kg, po).

Characterization of a primate model of asthma using anti-allergy/anti-asthma agents.

The following study was performed to further characterize a primate model of asthma using classes of drugs that target allergy (pyrilamine, cetirizine), are bronchodilators for the treatment of asthma (salbutamol, salmeterol) or are anti-inflammatory (dexamethasone). These drugs were examined for their ability to inhibit acute, antigen-induced bronchoconstriction, the development of airway hyperresponsiveness (AHR) and the infiltration of leukocytes into the lungs of atopic cynomolgus monkeys (Macaca facsicularis) using a 10-day, multiple antigen (Ag) challenge protocol. All compounds except dexamethasone and cetirizine significantly (p < 0.05) reduced acute, Ag-induced bronchoconstriction (salbutamol: 74.2%, salmeterol: 52.6%%, pyrilamine: 62.4% inhibition) compared to vehicle control trials. Only dexamethasone and salmeterol prevented the development of AHR to methacholine challenge by 90.4 +/- 6.81% and 85.7 +/- 5.61% respectively. Dexamethasone significantly reduced the Ag-induced increase in BAL eosinophils by 85.9 +/- 8.53%. Cetirizine reduced the eosinophil response in 5 of 6 monkeys and salmeterol demonstrated a trend towards reduced eosinophil increases after multiple Ag challenge, but neither of these were statistically significant. These results further illustrate the utility of this model in predicting compound effects against several relevant functional endpoints that are consistent with the effects of similar classes of compounds in humans.

In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma.

To test the hypothesis that leukotriene (LT) B4 antagonists may be clinically useful in the treatment of asthma, CP-105,696 was evaluated in vitro, using chemotaxis and flow cytometry assays, and in vivo, using a primate asthma model. CP-105,696 inhibited LTB4-mediated monkey neutrophil chemotaxis (isolated cells, LTB4 = 5 nM) and CD11b upregulation (whole blood, LTB4 = 100 nM) with IC50 values of 20 nM and 16.5 microM, respectively. Using a modification of a previously described in vivo protocol (Turner et al. Am. J. Respir. Crit. Care Med. 1994. 149: 1153-1159), we observed that treatment with CP-105,696 inhibited the acute increase in bronchoalveolar lavage (BAL) levels of IL-6 and IL-8 by 56.9 +/- 13.2% and 46.9 +/- 14.5%, respectively, 4 h after challenge with Ascaris suum antigen (Ag). CP-105,696 tended to reduce the increase in BAL protein levels 0.5 h after Ag challenge by 47.5 +/- 18.3%, but this was not statistically significant. In addition, CP-105,696 prevented the significant 11-fold increase in airway responsiveness to methacholine after multiple Ag challenge. These results suggest that LTB4 partially mediates acute and chronic responses to antigen in an experimental primate asthma model and support the clinical evaluation of LTB4 antagonists in human asthma.

The effect of 5-lipoxygenase inhibition on Ascaris antigen (Ag)-induced responses in atopic monkeys.

The effects of two 5-lipoxygenase (5LO) inhibitors, ZD2138 or Zileuton, on acute, inflammatory responses to aerosolized Ascaris suum (Ag) were determined in atopic Macaca fascicularis monkeys. Monkeys (n = 6 each group) were dosed with vehicle, 3 or 10 mg/kg ZD2138, or 30 mg/kg Zileuton (p.o.). Both ZD2138 or Zileuton significantly inhibited ex vivo LTB4 production in Ca2+ ionophore-stimulated whole blood from these same monkeys (n = 6 each group) by 45.5% (3 mg/kg ZD2138), 82.5% (10 mg/kg ZD2138) and 84.3% (30 mg/kg Zileuton). ZD2138 (10 mg/kg) reduced bronchoalveolar lavage (BAL) LTE4 levels (65.1% inhibition), BAL neutrophils (88.9% inhibition), and IL-6 (54.0% inhibition) 4h post Ag. Zileuton inhibited these responses and also reduced BAL levels of IL-8 (73.4% inhibition). A second study was performed to evaluate the effects of ZD2138 on chronic Ag-induced responses. Treatment with ZD2138 did not prevent pulmonary inflammation or the development of airway hyperresponsiveness (AHR). Based upon these results, 5LO inhibition significantly reduced ex vivo LTB4 and in vivo LTE4 production as well as several acute inflammatory responses to Ag in the lung. However, ZD2138 did not inhibit more chronic responses following multiple Ag exposure.

Effects of rolipram on responses to acute and chronic antigen exposure in monkeys.

The following study was performed to test the hypothesis that treatment with rolipram, a specific inhibitor of phosphodiesterase (PDE) IV, should inhibit many pulmonary responses to acute and chronic antigen challenge in atopic monkeys by elevating intracellular cAMP and subsequently inhibiting leukocyte function. Monkeys received subcutaneous injections of either vehicle (2% DMSO) or 10 mg/kg of rolipram 1 h before exposure to Ascaris suum antigen (Ag). Acute responses to Ag, including bronchoconstriction, pulmonary leukocyte infiltration, and cytokine production, were monitored before and 4 h after single Ag aerosol administration. To monitor the effects of rolipram on chronic Ag exposure, a 10-d, multiple-Ag protocol, previously demonstrated to induce airway hyperresponsiveness (AHR) to methacholine (MCh), was performed. Ag exposure increased respiratory system resistance (Rrs) 221.7 +/- 31.88% (n = 5). This increase in Rrs was not significantly altered by rolipram. Rolipram significantly (p < 0.002) increased cAMP levels in bronchoalveolar lavage (BAL) leukocytes 1 h after administration (n = 5). Ag-induced increases in BAL IL-8 and TNF were significantly reduced by rolipram, but IL-1 beta and IL-6 increases were unaffected (n = 9). Ag-induced increases in BAL eosinophils and neutrophils were significantly reduced by rolipram (n = 9). In the multiple-Ag protocol (n = 7), rolipram significantly reduced both the number of BAL eosinophils (p < 0.02) and the development of AHR (p < 0.002). Despite its inability to inhibit acute Ag-induced bronchoconstriction, rolipram was protective against acute and chronic inflammatory responses to Ag and prevented the development of AHR, suggesting that selective PDE-IV inhibition is a relevant target for asthma therapy.

Leukotriene D4 receptor antagonism reduces airway hyperresponsiveness in monkeys.

Airway hyperresponsiveness (AHR) and pulmonary inflammation are observations that are consistently associated with asthma and also occur in a well-characterized monkey model of asthma. The following study was performed to determine whether treatment with an LTD4 receptor antagonist, ICI 198,615, could attenuate antigen-induced pulmonary inflammation and AHR in monkeys using the following protocol. On day 0, the PC200 (the concentration of methacholine (MCh) that doubled respiratory system resistance, Rrs) was determined in 6 male, atopic, cynomolgus monkeys, previously characterized in historical control trials (Control #1) as airway hyperresponsive. Bronchoalveolar lavage (BAL) was then performed to determine total and differential leukocyte counts. On days 3, 5 and 7, each monkey received 10 mg/kg ICI 198,615 (im) 30 min prior to Ascaris suum (Ag) aerosol exposures which doubled Rrs. On day 10, the post-Ag PC200 to MCh was determined and BAL was repeated. Five weeks after this trial was complete, a bracketing control trial (Control #2) was performed in which the monkeys were administered vehicle prior to each Ag exposure. In comparison to the response in both control trials, treatment with the LTD4 antagonist significantly (P < 0.05) inhibited the development of AHR and also significantly reduced (P < 0.05) peripheral blood lymphocyte counts after Ag challenge. Treatment with ICI 198,615 reduced the Ag-induced increase in BAL eosinophils, but statistical significance was obtained only when treated animals were compared to Control #1, not Control #2.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.

Triglyceride, free cholesterol, and total cholesterol were quantified in lipid extracts of liver and thoracic aorta from nonhuman primates using commercially available enzymatic reagents. Lipids were solubilized in water by the addition of Triton X-100. Results of the enzymatic assays compared favorably with chemical assays of lipids separated by thin-layer chromatography. In addition to saving time, the present method has the advantage of measuring each lipid class from a single sample preparation. Furthermore, the procedure has been adapted for use with microtiter plates that conserve both sample and reagent.