Purpose: GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objective was to characterize the antiproliferative activity, pharmacokinetics, pharmacodynamics, and safety of GS-9219. Experimental Design: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non^Hodgkin's lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy. Results: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes and hematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events. Conclusion: GS-9219 may have utility for the treatment of NHL.

The beta-adrenoceptor agonist, isoprenaline, inhibited the immunoglobulin E-mediated release of histamine from human lung mast cells (HLMC). Long-term (24 h) exposure of HLMC to isoprenaline reduced the subsequent effectiveness of isoprenaline to inhibit histamine release. The extent of this functional desensitization was variable with some HLMC preparations resistant and others highly susceptible. We sought to determine whether the variability in the degree of functional desensitization was influenced by genetic polymorphisms in the beta2-adrenoceptor. HLMC preparations were genotyped at two polymorphic loci, positions 16 (arg to gly) and 27 (gln to glu), and the effect of desensitizing conditions (24 h with 10(-6) M isoprenaline) on the subsequent ability of isoprenaline (10(-7) M) to inhibit histamine release from HLMC was determined (n = 72). In HLMC preparations expressing beta2-adrenoceptors with arg (wild-type) or gly (mutant) at position 16, desensitization was 71 +/- 5% (n = 18) or 43 +/- 5%, (n = 26), respectively, whereas the desensitization was 59 +/- 6% (n = 28) for heterozygotes at this position. In HLMC preparations expressing beta2-adrenoceptors with gln (wild-type) or glu (mutant) at position 27, desensitization was 65 +/- 5% (n = 25) or 28 +/- 7% (n = 17), respectively, whereas the desensitization was 61 +/- 5% (n = 30) for heterozygotes at this position. These data suggest that mutant (gly16 and glu27) forms of the receptor are resistant to desensitization compared to wild-type (arg16 and gln27) forms. However, analyses to determine the relative contributions of positions 16 and 27 suggest that position 27 is more important in influencing the degree of functional desensitization.

CD8hi CD57 + T cells have previously been described as effector memory T cells with minimal expansion capacity and high susceptibility to activation-induced cell death.

1 The long-acting b 2 -adrenoceptor agonist formoterol (10 À10 -10 À6 M) inhibited the IgE-dependent release of histamine from human lung mast cells in a concentration-dependent manner. Formoterol was more potent and a full agonist relative to the nonselective b-adrenoceptor agonist isoprenaline. By contrast, the long-acting b 2 -adrenoceptor agonist salmeterol (10 À10 -10 À6 M) was about two-thirds less efficacious than either formoterol or isoprenaline as an inhibitor of histamine release. 2 Isoprenaline, formoterol and salmeterol (all at 10 À5 M) increased total cell cAMP levels in mast cells over basal by 361790 (Po0.05), 321789 (Po0.05) and 64724% (P40.05), respectively. 3 Long-term (24 h) incubation of mast cells with formoterol (10 À6 M) or salmeterol (10 À6 M) essentially abolished the subsequent ability of isoprenaline to inhibit histamine release. Both formoterol and salmeterol were more effective at inducing the functional desensitisation than isoprenaline (10 À6 M) or the short-acting b 2 -adrenoceptor agonist salbutamol (10 À6 M). 4 The desensitisation induced by long-term treatments with salmeterol and formoterol was specific for b 2 -adrenoceptor-mediated inhibition of histamine release as the inhibitory effects of alternative cAMP-elevating compounds, prostaglandin E 2 , a receptor-mediated activator of adenylate cyclase, and forskolin, a direct activator of adenylate cyclase, were unaffected by desensitising treatments. 5 Radioligand binding studies were performed to determine b 2 -adrenoceptor density in cell membranes after pretreatment (24 h) of cells with agonists. Isoprenaline, formoterol and salmeterol (all at 10 À6 M) reduced b 2 -adrenoceptor density by 1375 (P40.05), 49713 (Po0.05) and 35717% (P40.05), respectively. 6 These data indicate that long-term exposure of mast cells to both salmeterol and formoterol can cause substantial levels of desensitisation to b 2 -adrenoceptor-mediated responses in mast cells.

The beta adrenergic agonist isoprenaline inhibited the IgE-triggered release of the preformed mediator histamine from human lung mast cells (HLMC) in a dose-dependent fashion. After prolonged (> or = 4 h) preexposure of HLMC to isoprenaline, there was a subsequent diminution in the effectiveness of a second exposure of isoprenaline to inhibit the release of histamine from activated HLMC. This induced hyporesponsiveness to isoprenaline was both concentration and time dependent. Although maximal levels of desensitization were obtained after an initial prolonged (14-h) preincubation with a high (10(-5) M) concentration of isoprenaline, exposure of HLMC for a shorter (4-h) time period with a lower (3 x 10(-7) M) concentration of isoprenaline was also effective at inducing a functional desensitization to isoprenaline. The inhibitory activity of the beta 2 agonist fenoterol was attenuated after a prolonged (14-h) pretreatment step with isoprenaline (10(-5)M), whereas the inhibitory properties of other adenylate cyclase activators, prostaglandin E2 and forskolin, were not affected appreciably. Prolonged (12-h) exposure of HLMC to the beta agonists fenoterol, salbutamol, and terbutaline also induced hyporesponsive states of beta agonists, qualitatively similar to that obtained with isoprenaline. The beta receptor antagonist propranolol, if coincubated with isoprenaline during the prolonged pretreatment step, protected against the subsequent refractoriness of the HLMC to isoprenaline. The glucocorticoid dexamethasone failed to prevent the isoprenaline-induced functional desensitization. In total, these results indicate that prolonged exposure of HLMC to beta agonists induces a state of selective hyporesponsiveness to agonists that act at beta adrenoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

1 The principal aim of the present study was to determine whether long-term treatment of human lung mast cells (HLMC) with the clinically-relevant b 2 -adrenoceptor agonists, salbutamol and terbutaline, leads to desensitization of b 2 -adrenoceptor-mediated responses in these cells. 2 The non-selective b-adrenoceptor agonist, isoprenaline, and the selective b 2 -adrenoceptor agonists, salbutamol and terbutaline, inhibited the IgE-mediated release of histamine from HLMC. Salbutamol (pD 2 ; 7.7+0.3) and terbutaline (pD 2 ; 7.3+0.2) were roughly equipotent as inhibitors of histamine release although both agonists were less potent than isoprenaline (pD 2 ; 8.6+0.2). 3 Isoprenaline (10 75 M), salbutamol (10 75 M) and terbutaline (10 75 M) enhanced total cell cAMP levels in HLMC over basal by 361+90, 150+38 and 165+35%, respectively. 4 Long-term exposure (24 h) of HLMC to either salbutamol (10 77 M) or terbutaline (10 77 M) led to a subsequent reduction in the e ectiveness of salbutamol and terbutaline (both 10 79 ± 10 74 M) to inhibit histamine release. However, salbutamol was signi®cantly (P50.05) more e ective than terbutaline at promoting the functional desensitization. 5 Radioligand binding studies, using iodinated cyanopindolol, were performed to determine b 2 -adrenoceptor density in cell membranes after pretreatment (24 h) of cells with either salbutamol (10 76 M) or terbutaline (10 76 M). Both agonists reduced b 2 -adrenoceptor density in membranes to about the same extent (*25% reduction) but these changes in receptor density were not statistically signi®cant (P40.05). 6 These data indicate that long-term exposure of mast cells to salbutamol causes greater levels of desensitization to b 2 -adrenoceptor-mediated responses in HLMC than terbutaline. These ®ndings may have wider clinical signi®cance in the context of asthma treatment as compromised mast cell inhibition could result following long-term exposure of mast cells to short-acting bronchodilators.

She eld S10 2JF1 The e ects of the b-adrenoceptor agonists isoprenaline and salbutamol on IgE-mediated histamine release from human lung mast cells (HLMC) were evaluated. Both agonists (10 710 ± 10 75 M) inhibited histamine release in a dose-dependent manner and isoprenaline (pD 2 , 8.3+0.1, mean+s.e.mean) was more potent than salbutamol (7.3+0.1). Moreover, the mean data indicated that salbutamol was a partial agonist when compared with isoprenaline. However, there was a large degree of interexperimental variability because, in 11 of 32 experiments, salbutamol was a full agonist and, in 21 of 32 experiments, a partial agonist relative to isoprenaline. These data suggest that di erent HLMC preparations possess variable receptor reserves.2 The e ect of the irreversible b-adrenoceptor antagonist, bromoacetylalprenolol menthane (BAAM), on the inhibition of IgE-mediated histamine release by both isoprenaline and prostaglandin E 2 (PGE 2 ) was assessed. Whereas BAAM (100 nM) antagonized the isoprenaline inhibition of histamine release from activated HLMC, BAAM had no e ect on the PGE 2 inhibition. Pretreatment of HLMC with the b 2 -selective competitive antagonist, ICI 118551 (100 nM), protected against the loss in responsiveness to isoprenaline following treatment with BAAM. 3 Concentrations of 1, 10 and 100 nM of BAAM caused dose-dependent rightward shifts in the doseresponse curve for the isoprenaline inhibition of histamine release. Furthermore, there was a dosedependent reduction in the maximal inhibitory response obtained with isoprenaline following treatments with increasing concentrations of BAAM. Although the rightward shifts in the isoprenaline doseresponse curves, with a given concentration of BAAM, were similar in all experiments, there was some variability in the depression of the maximal response in individual experiments. Thus, in 6 of 16 experiments, BAAM (1 nM) did not depress the maximal response to isoprenaline, whereas in 10 of 16 experiments there was a depression (7 to 49% reduction) in the maximal response. These data suggest that di erent HLMC preparations possess variable receptor reserves. 4 Isoprenaline was more potent as an inhibitor in those HLMC preparations in which there was a larger receptor reserve (i.e. preparations in which the maximal inhibitory response to isoprenaline was una ected by pretreatment with 1 nM BAAM). 5 The in¯uence of receptor reserve on the inhibition by salbutamol of histamine release from HLMC was evaluated. There was a good correlation (r=0.77) between receptor reserve and the maximal response (relative to isoprenaline) obtained with salbutamol. Thus, HLMC preparations with larger receptor reserves were more responsive to salbutamol. 6 Receptor reserve in¯uenced the desensitization of b-adrenoceptor-mediated responses in HLMC. Cells were incubated (24 h) with isoprenaline (1 mM), washed and then the ability of a second isoprenaline (10 710 ± 10 75 M) exposure to inhibit histamine release was assessed. The pretreatment caused a reduction in the isoprenaline inhi...

1 The long-acting b 2 -adrenoceptor agonist, salmeterol (10 79 ± 10 75 M), inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent fashion. Additional badrenoceptor agonists were studied and the rank order of potency for the inhibition of histamine release from HLMC was isoprenaline4salmeterol4salbutamol. Approximate EC 50 values for the inhibition of histamine release were 10 nM for isoprenaline and 100 nM for salbutamol. An EC 50 value for salmeterol could not be calculated because maximal responses to salmeterol were not observed over the concentration range employed. 2 Both salmeterol and isoprenaline inhibited the generation of sulphopeptidoleukotrienes (sLT) more potently and more ecaciously than the release of histamine from immunologically-activated HLMC. Salmeterol (EC 50 50.1 nM) was more potent than isoprenaline (EC 50 0.4 nM) at attenuating sLT generation. 3 The b-adrenoceptor antagonist, propranolol (1 mM), and the selective b 2 -adrenoceptor antagonist, ICI 118,551 (0.1 mM), both caused rightward shifts in the dose-response curve for the inhibition of histamine release by isoprenaline. The antagonism of salmeterol eects by propranolol and ICI 118,551 was more complex. At lower concentrations (51 mM) of salmeterol, both antagonists shifted the dose-reponse curve to salmeterol rightward. At a higher concentration (10 mM) of salmeterol, neither ICI 118,551 nor propranolol was an eective antagonist of the salmeterol-mediated inhibition of histamine release. 4 Prolonged exposure (4 h) of HLMC to isoprenaline (1 mM) caused an approximately 50% reduction in the eectiveness of a second exposure to isoprenaline (10 mM) at inhibiting the release of histamine, whereas this pretreatment did not aect the salmeterol (10 mM) inhibition of histamine release. 5 Isoprenaline (10 79 ± 10 75 M) caused a dose-dependent increase in total cell cyclicAMP levels in puri®ed HLMC which paralleled the inhibition of histamine release. Salmeterol (10 79 ± 10 75 M) was considerably less potent than isoprenaline at increasing HLMC cyclicAMP levels. 6 In summary, these data indicate that salmeterol is an eective inhibitor of the stimulated release of mediators from HLMC. The present data also suggest that salmeterol may act to inhibit mediator release from HLMC by b-adrenoceptor-dependent and independent mechanisms.