Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members. [Cancer Res 2007;67(24):11924-32]

Janus kinase (JAK) enzymes are involved in cell signaling pathways activated by various cytokines dysregulated in allergy. The objective of this study was to determine whether the novel JAK inhibitor oclacitinib could reduce the activity of cytokines implicated in canine allergic skin disease. Using isolated enzyme systems and in vitro human or canine cell models, potency and selectivity of oclacitinib was determined against JAK family members and cytokines that trigger JAK activation in cells. Oclacitinib inhibited JAK family members by 50% at concentrations (IC50's) ranging from 10 to 99 nm and did not inhibit a panel of 38 non-JAK kinases (IC50's > 1000 nm). Oclacitinib was most potent at inhibiting JAK1 (IC50 = 10 nm). Oclacitinib also inhibited the function of JAK1-dependent cytokines involved in allergy and inflammation (IL-2, IL-4, IL-6, and IL-13) as well as pruritus (IL-31) at IC50's ranging from 36 to 249 nm. Oclacitinib had minimal effects on cytokines that did not activate the JAK1 enzyme in cells (erythropoietin, granulocyte/macrophage colony-stimulating factor, IL-12, IL-23; IC50's > 1000 nm). These results demonstrate that oclacitinib is a targeted therapy that selectively inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus and suggests these are the mechanisms by which oclacitinib effectively controls clinical signs associated with allergic skin disease in dogs.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members.

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BackgroundPruritus is a characteristic clinical sign of allergic skin conditions including atopic dermatitis (AD) in the dog. IL‐31 is a cytokine found in the serum of some dogs with AD and can induce pruritic behaviours in laboratory beagle dogs.Hypothesis/ObjectivesThe objectives were to characterize an IL‐31‐induced pruritus model by evaluating the efficacy of prednisolone, dexamethasone and oclacitinib, and to compare the speed of anti‐pruritic effects of oclacitinib against those of prednisolone and dexamethasone.AnimalsPurpose‐bred beagle dogs were used in all studies.MethodsRandomized, blinded, placebo‐controlled studies were designed to evaluate and compare the anti‐pruritic properties of prednisolone, dexamethasone and oclacitinib following a single intravenous injection of recombinant canine IL‐31. Video surveillance was used to monitor and score pruritic behaviours in study animals.ResultsPrednisolone [0.5 mg/kg, per os (p.o.)] reduced IL‐31‐induced pruritus when given 10 h prior to observation. When the time interval between drug treatment and observation was shortened to 1 h, dexamethasone (0.2 mg/kg, intramuscular) but not prednisolone (0.25 or 0.5 mg/kg, p.o.) reduced IL‐31‐induced pruritus. Oclacitinib (0.4 mg/kg, p.o.) reduced pruritus when given 1, 6, 11 and 16 h prior to the observation period, and the anti‐pruritic activity of oclacitinib was greater when compared to prednisolone and dexamethasone at all time points assessed.Conclusion and clinical importanceThe efficacy of prednisolone, dexamethasone and oclacitinib in the IL‐31‐induced pruritus model gives confidence that this may be a relevant model for acute pruritus associated with allergic dermatitis including AD and can be used to evaluate novel compounds or formulations.

Phenytoin (pht) is an anticonvulsant drug commonly used for the prevention of seizures. A common side effect of PHT therapy is gingival hyperplasia, occasionally so severe that it requires surgical intervention. Cyclosporine A (CSA) is a drug widely used for the control of rejection phenomena following solid organ and bone marrow transplantation. A frequent side effect of CSA administration is gingival overgrowth. As yet, the molecular mechanisms of drug-induced gingival hyperplasia are unknown although it has been postulated that certain drugs increase fibroblastic activity through alterations in levels of various growth factors and cytokines. The purpose of this study was to: 1) evaluate monocyte/macrophage platelet-derived growth factor (PDGF) and interleukin (IL)-1 beta production in vitro after exposure to CSA; 2) determine the levels of PDGF-B and IL-1 beta gene expression in minimally inflamed gingival tissues of control patients and PHT-treated patients exhibiting gingival overgrowth as well as patients with severe gingival inflammation; and 3) combine characterization of macrophage phenotype with clinical presentation and expression of PDGF-B and IL-1 beta in gingival tissues from the control and PHT-treated patients. For the in vitro studies, commercial ELISA kits were used to measure PDGF-A/PDGF-B and IL-1 beta levels in conditioned media from rat and human monocyte/macrophage cell cultures. CSA caused a significant elevation of PDGF but did not cause any changes in IL-1 beta levels. For the in vivo studies, quantitative competitive reverse transcription polymerase chain reaction (QC-RTPCR) techniques were utilized to measure PDGF-B and IL-1 beta mRNA levels in experimental groups. PHT-treated patients exhibiting gingival overgrowth demonstrated a significant increase in PDGF-B mRNA compared with minimally inflamed controls. Patients with severe gingival inflammation also demonstrated a significant increase in PDGF-B mRNA however, PHT-induced PDGF-B upregulation is approximately 6 times larger than PDGF-B upregulation produced by inflammation alone. PHT-treated patients exhibiting gingival overgrowth demonstrated no significant increase in IL-1 beta mRNA; however, IL-1 beta mRNA levels in the severely inflamed gingival samples demonstrated a significant increase. Additionally, for the clinical samples, macrophage phenotype was characterized immunohistochemically in adjacent sections using specific monoclonal antibodies for inflammatory and reparative/proliferative phenotypes. There were no significant differences in the numbers of either macrophage phenotype in minimally inflamed gingival tissues; however, in the severely inflamed tissue, there was a significant increase in the inflammatory macrophage phenotype and in the hyperplastic gingival tissue, there was a significant increase in the reparative/proliferative macrophage phenotype. The results of this investigation indicate that the clinical presentation of inflamed and hyperplastic gingival tissues is associated with specific macrophages phenotyp...

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The bis(heteroaryl)piperazine U-88204E is a potent inhibitor of HIV-1 reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the RNA- and DNA-dependent DNA polymerases of RT were carried out in order to determine whether the inhibitor interacts directly with the template:primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The experimental results were analyzed using steady-state or Briggs-Haldane kinetics, by assuming that the template:primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The results of the analysis show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template:primer and the dNTP binding sites. The potency of U-88204E on the RNA-directed DNA polymerase activity depends on the base composition of the template:primer. The Ki values for the poly(rC):(dG)10-directed reactions were at least 7 times lower than the ones for reactions directed by poly(rA):(dT)10. The inhibitor did not inhibit the RNase H function of HIV-1 RT nor did it impair the RNA-directed DNA polymerase activity of HIV-2 RT. These data thus demonstrate the unique specificity of U-88204E for HIV-1 RT.