Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell nonHodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.lymphoma | X-linked agammaglobulinemia

1 Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M 2 subtype whereas the remaining minority belong to the M 3 subtype. The objective of the present study was to assess the functional role of M 2 and M 3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2 In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC 50 =6.3) which were unaected by tetrodotoxin (0.1 mM). These contractions were antagonized by muscarinic antagonists with the following rank order of anity (pA 2 ) estimates: atropine (9.1) 4 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) 4 darifenacin (8.5) 4 para¯uoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) 4 pirenzepine (6.8) 4 methoctramine (5.9). These pA 2 estimates correlated most favourably (r=0.99, P50.001) with the binding anity (pK i ) estimates of these compounds at human recombinant muscarinic m 3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically de®ned M 3 receptor. 3 As M 2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M 2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M 3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 mM) to protect M 2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC 50 =5.8) but had marginal eects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA 2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M 2 and M 3 receptors and were consistent with the involvement of both of these subtypes. 4 In urethane-anaesthetized rats, the cholinergic component (*55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID 35%inh , nmol kg 71 , i.v.): 4-DAMP (8.1) 4 atropine (20.7) 4 methoctramine (119.9) 4 darifenacin (283.3) 4 pirenzepine (369.1) 4 p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pK i estimates of these compounds at human recombinant muscarinic m 2 receptors. This is consistent with a major contribution of M 2 receptors in the generation of volumeinduced bladder contractions, although the modest potency of darifenacin does not exclude a role of M 3 receptors. Pretreatment with propranolol (1 mg kg 71 , i.v.) increased the ID 35%inh of methoctramine signi®cantly from 95.9 to 404.5 nmol kg 71 but had...

Protegrin-1 (PG-1) is a cysteine-rich, 18-residue beta-sheet peptide isolated from porcine leukocytes with antimicrobial activity against a broad range of microorganisms. The MICs of PG-1 against representative gram-positive and gram-negative bacteria ranged from 0.12 to 2 microg/ml. At these levels, PG-1 was rapidly bactericidal in vitro, reducing the number of viable CFU of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa by more than three log units in less than 15 min. Resistance to PG-1 did not develop after 11 subculturings of P. aeruginosa or 18 subcultures of MRSA in Mueller-Hinton broth containing PG-1 at one-half the MIC. Under similar conditions of serial passage, the MICs of norfloxacin and gentamicin against P. aeruginosa increased 10 and 190 times, respectively. Similarly, the MIC of norfloxacin against MRSA increased 85 times. Immunocompetent mice inoculated intraperitoneally (i.p.) with P. aeruginosa or S. aureus exhibited 93 to 100% mortality in the vehicle control group compared with 0 to 27% mortality in animals that received a single i.p. injection of PG-1 (0.5 mg/kg of body weight). Mice inoculated with S. aureus by intravenous (i.v.) injection and dosed 0 to 60 min later with a single i.v. injection of PG-1 (5 mg/kg) had a mortality of 7 to 33%, compared to a mortality of 73 to 93% in the vehicle controls. In leukopenic mice inoculated i.v. with vancomycin-resistant Enterococcus faecium, mortality was 87% in the vehicle control group and 33% in animals that received a single i.v. injection of PG-1 (2.5 mg/kg). Taken together, these data indicate that PG-1 has potential for use as an antimicrobial agent in the treatment of local or systemic infections caused by clinically relevant pathogens.

1 A series of isoquinolines have been identified as 5-HT3 receptor antagonists. One of these, RS 25259-197 [(3aS)-2-[(S)-l-azabicyclo[2.2.2]oct-3-yl]-2,3,3a,4,5,6-hexahydro-l-oxo-lH-benzo [de]isoquinoline-hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259-198 (R,R), RS25233-197 (S,R) and RS25233-198 (R,S). 2 At 5-HT3 receptors mediating contraction of guinea-pig isolated ileum, RS 25259-197 antagonized contractile responses to 5-HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nM, was 8.8 ± 0.2. In this tissue, the -log KB values for the other three enantiomers were 6.7 ± 0.3 (R,R), 6.7 ± 0.1 (S,R) and 7.4 ± 0.1 (R,S), respectively. The apparent affinities of RS 25259-197 and RS 25259-198, RS 25233-197 and RS 25233-198 6 Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5-HT3 receptor sites by [3H]-RS . High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala, and a low density of sites in hippocampal CAl, parietal cortex, medium raphe and cerebellum. 7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non-radiolabelled RS 25259-197 (S,S enantiomer) established the profile of a highly potent and selective 5-HT3 receptor antagonist.

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