Oclacitinib (APOQUEL®) is a selective Janus kinase 1 inhibitor with efficacy in a canine model of flea allergic dermatitis.

Oclacitinib is a novel Janus kinase (JAK) inhibitor that potently inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus (IL-2, IL-4, IL-6, IL-13, and IL-31). Oclacitinib (Apoquel®, Zoetis Inc, Parsippany, NJ) is approved for the treatment/control of pruritus associated with allergic dermatitis and treatment/control of clinical manifestations of atopic dermatitis in dogs at least 12 months of age. To evaluate the effectiveness of oclacitinib in dogs with flea allergy dermatitis, the JAK1 selective inhibitor was tested in a placebo-controlled, masked, single-dose (0.4 mg/kg) or repeat-dose (0.4 mg/kg, twice daily for 2 weeks) study. Pruritic behaviors were quantitated by video recording, and erythema and skin lesions were assessed using a 10-cm visual analog scale (VAS). Results showed that oclacitinib reduced pruritus by 61% as early as 1.5 h after a single oral dose compared to placebo, with an average reduction (compared to placebo) of 85% 1-5 h after dosing (0.4 mg/kg; p < .0001). Oclacitinib also significantly reduced erythema (p < .0001) and skin lesion (p < .0005) VAS scores on Day 14 compared to placebo in a repeat dose study. No adverse events were noted during the conduct of these studies. IL-31 concentrations were elevated in the majority of dogs after flea infestation, suggesting JAK1-dependent cytokines may drive clinical signs of flea allergy dermatitis. These findings show that oclacitinib, an inhibitor of JAK1-dependent cytokines involved in allergy and inflammation can rapidly reduce clinical signs associated with flea allergic dermatitis in dogs.

Cytokine transcriptome profiling in acute experimental canine atopic dermatitis skin lesions after IL-31 inhibition with lokivetmab.

BACKGROUND: The caninised monoclonal antibody lokivetmab (LKV), directed at interleukin (IL)-31, is very effective at controlling pruritus in most dogs with atopic dermatitis (AD). However, evidence exists that IL-31 is not required for the induction of acute allergic skin inflammation, which might explain why this treatment is less efficacious in some dogs with AD. HYPOTHESIS/OBJECTIVES: To compare the comprehensive transcriptome analysis of house dust mite (HDM)-sensitised dogs with and without treatment with LKV to attest our hypothesis that LKV does not majorly affect acute cytokine/chemokine production. ANIMALS: Six HDM-sensitised atopic Maltese-beagle dogs. MATERIALS AND METHODS: In this cross-over study, the cytokine profiling of acute AD skin lesions was compared by RNA sequencing (RNA-Seq), with or without LKV-induced inhibition of IL-31. Skin biopsies were obtained from each dog at 0, 6, 12, 24, 48, and 96 h after epicutaneous HDM allergen provocation. RESULTS: Macroscopic and microscopic skin lesion scores were not significantly different between the LKV- and nontreatment groups at any time points. Likewise, the results of RNA-Seq analysis revealed no significant difference in the messenger (m)RNA expression of the major cytokines between these two groups. In LKV-treated dogs, IL6, IL9, IL13, IL33, CCL17, and CCL22 were significantly upregulated compared to their baseline expression levels, suggesting that these cytokines are unaffected by IL-31 inhibition. CONCLUSIONS AND CLINICAL RELEVANCE: IL-31 inhibition is insufficient to prevent the expression of other proinflammatory mediators in acute AD and these could be considered as other potential therapeutic targets.

Speed of onset of a new chewable formulation of oclacitinib maleate (Apoquel®) in a canine model of IL-31-induced pruritus.

Oclacitinib maleate (Apoquel®, Zoetis Inc.) is commonly used around the world for the control/treatment of pruritus associated with allergic dermatitis and the control/treatment of atopic dermatitis in dogs at least 12 months of age. A new flavored chewable formulation of oclacitinib has been developed where more than 90% of doses offered to dogs were freely accepted when tested in clinical trials. The objective of this study was to determine whether the new chewable formulation of oclacitinib has a similar onset of anti-pruritic activity as the original oclacitinib film-coated tablets (FCT). Twenty-one laboratory beagle dogs were randomized to treatment and received placebo, 0.4-0.6 mg/kg oclacitinib FCT or 0.4-0.6 mg/kg flavored chewable oclacitinib tablet (n = 7/group). Efficacy was measured by assessing reduction in pruritus 1-3 h post-administration of treatments. Pruritus was induced by injecting canine IL-31, intravenously (2.5 µg/kg), approximately 15 min prior to the pruritus observation window. Results from this study demonstrated both oclacitinib FCT and the flavored chewable oclacitinib tablet significantly reduced IL-31-induced pruritus within 1-3 h post-dosing compared to placebo (p = .0069 and .0113, respectively), suggesting the new formulation of oclacitinib chewable tablets works as quickly to reduce pruritus in dogs as the oclacitinib FCT.

Onset and duration of action of lokivetmab in a canine model of IL-31 induced pruritus.

BACKGROUND: Interleukin (IL)-31 is a cytokine involved in allergic inflammation which induces pruritus across species including dogs. Using recombinant canine IL-31 we have developed a model of pruritus in the dog to evaluate onset of action and duration of effect of therapeutic drugs. OBJECTIVE: To assess the onset of action and duration of effect of lokivetmab (Cytopoint) in the IL-31-induced pruritus model. ANIMALS: Twenty-four purpose-bred beagle dogs (neutered males, spayed and intact females) 1.5-4.7 years old and weighing between 6 and14 kg. METHODS AND MATERIALS: Randomized, blinded, placebo-controlled studies were designed to evaluate the antipruritic properties of lokivetmab. Laboratory beagle dogs were given either placebo, 0.125, 0.5 or 2.0 mg/kg lokivetmab, subcutaneously. IL-31 then was administered to evaluate pruritus 3-5 h post-placebo or -lokivetmab administration as well as one, seven, 14, 28, 42 and 56 days post-dosing. Pruritus was evaluated over a 2 h window in animals by video monitoring and scored using a categorical scoring system. RESULTS: When animals were given 2.0 mg/kg lokivetmab, a significant reduction in pruritus was observed at 3-4, 4-5 and 3-5 h post-treatment (P ≤ 0.0001). When animals were given either 0.125, 0.5 or 2 mg/kg lokivetmab, the duration of effect was dose-dependent and statistically significant for 14, 28 and 42 days, respectively (P ≤ 0.0288). CONCLUSION: These data indicate that a single subcutaneous injection of 2 mg/kg lokivetmab produces a significant suppression of pruritus starting 3 h post-treatment that can be sustained for 42 days.

Cytokine expression in feline allergic dermatitis and feline asthma.

BACKGROUND: The pathogenesis of feline allergic dermatitis (FAD) is unclear, with several differences from allergic dermatitis in dogs and humans. HYPOTHESIS/OBJECTIVES: To survey cytokine expression levels in healthy cats and cats affected with allergic dermatitis or asthma. ANIMALS: Formalin-fixed, paraffin-embedded skin biopsies from 22 cats with allergic dermatitis and 21 cats without allergic dermatitis were used for cutaneous assays. Serum was obtained from 17 healthy cats, 18 cats with allergic dermatitis, and 18 cats with a presumptive diagnosis of asthma. METHODS AND MATERIALS: Cutaneous mRNA expression was evaluated with quantitative PCR [interleukin (IL)-31 and IL-31 Receptor A] and RNA in situ hybridisation (ISH) [IL-5, IL-31, IL-31RA, IL-33 and Oncostatin M receptor (OSMR)-ß]. IL-31 protein concentrations were evaluated in serum with an enzyme-linked immunosorbent assay. Serum levels of 19 additional cytokines were evaluated using a Luminex panel. RESULTS: IL-31, IL-31RA, IL-5 and IL-33 mRNA expression were either expressed in low quantities or undetectable in most samples. By contrast, OSMR-ß expression was significantly higher in the skin of allergic versus healthy cats (P < 0.0001). Although serum IL-31 was detected in a larger number of cats with allergic dermatitis than healthy cats, and concentrations appeared to be higher in cats with allergies, this difference was not statistically significant. Cats affected by asthma also exhibited insignificantly higher concentrations of IL-31 in the serum. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that feline allergic diseases may exhibit different pathomechanisms from allergic diseases affecting other species. These findings are useful in guiding further therapeutic development toward targets that may have a role in the pathogenesis of feline allergic skin disease.

Serum concentrations of IL-31 in dogs with nonpruritic mast cell tumours or lymphoma.

BACKGROUND: The aim of this study was to compare serum interleukin (IL)-31 concentrations in dogs with lymphoma and mast cell tumours (MCT) without pruritus to those of healthy dogs. HYPOTHESIS/OBJECTIVES: To determine if IL-31 plays a role in tumour pathogenesis and if IL-31 could be a biological marker for disease progression. ANIMALS: Forty-eight healthy dogs and 36 dogs with neoplasia [multicentric lymphoma (14), MCT (15) and cutaneous lymphoma (7)] were included in the study. METHODS AND MATERIALS: Dogs with neoplasia were assigned to three different groups. Group 1 consisted of patients with multicentric lymphoma, which were diagnosed by cytological, histopathological and clonality investigations. Thoracic radiographs, ultrasound examination of the abdominal cavity, and fine-needle aspirates from liver and spleen were used to determine the lymphoma stage Patients with cutaneous lymphoma, diagnosed by cytological and histopathological findings, were included in Group 2. Patients with MCT, diagnosed by cytological and histopathological findings, were included in Group 3. Serum was frozen at -80ºC before measuring the concentration of IL-31 via a Simoa ultra-sensitive, fully automated two-step immunoassay. RESULTS: Serum concentrations of IL-31, regardless of the disease and its staging, were within the normal range in all patients; there was no difference between any of the different tumour groups and healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: IL-31 is not likely to be involved in the pathogenesis of canine MCT or lymphoma without pruritus.

IL-31-induced pruritus in dogs: a novel experimental model to evaluate anti-pruritic effects of canine therapeutics.

BACKGROUND: Pruritus 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/OBJECTIVES: The 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. ANIMALS: Purpose-bred beagle dogs were used in all studies. METHODS: Randomized, 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. RESULTS: Prednisolone [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 IMPORTANCE: The 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.

Interleukin-31: its role in canine pruritus and naturally occurring canine atopic dermatitis.

BACKGROUND: Interleukin-31 (IL-31) is a member of the gp130/interleukin-6 cytokine family that is produced by cell types such as T helper 2 lymphocytes and cutaneous lymphocyte antigen positive skin homing T cells. When overexpressed in transgenic mice, IL-31 induces severe pruritus, alopecia and skin lesions. In humans, IL-31 serum levels correlate with the severity of atopic dermatitis in adults and children. HYPOTHESIS/OBJECTIVE: To determine the role of IL-31 in canine pruritus and naturally occurring canine atopic dermatitis (AD). ANIMALS: Purpose-bred beagle dogs were used for laboratory studies. Serum samples were obtained from laboratory animals, nondiseased client-owned dogs and client-owned dogs diagnosed with naturally occurring AD. METHODS: Purpose-bred beagle dogs were administered canine interleukin-31 (cIL-31) via several routes (intravenous, subcutaneous or intradermal), and pruritic behaviour was observed/quantified via video monitoring. Quantitative immunoassay techniques were employed to measure serum levels of cIL-31 in dogs. RESULTS: Injection of cIL-31 into laboratory beagle dogs caused transient episodes of pruritic behaviour regardless of the route of administration. When evaluated over a 2 h period, dogs receiving cIL-31 exhibited a significant increase in pruritic behaviour compared with dogs that received placebo. In addition, cIL-31 levels were detectable in 57% of dogs with naturally occurring AD (≥ 13 pg/mL) but were below limits of quantification (<13 pg/mL) in normal, nondiseased laboratory or client-owned animals. CONCLUSIONS: Canine IL-31 induced pruritic behaviours in dogs. Canine IL-31 was detected in the majority of dogs with naturally occurring AD, suggesting that this cytokine may play an important role in pruritic allergic skin conditions, such as atopic dermatitis, in this species.

Lack of Interference of Oclacitinib with the results of intradermal testing or allergen-specific IgE serology in Dermatophagoides farinae-sensitized beagle dogs.

Canine atopic dermatitis (AD) is associated with increased levels of allergen-specific IgE due to hyper-sensitization to environmental allergens. Intradermal testing (IDT) and allergen-specific IgE serology testing are often used to determine the allergens which elicit an IgE response in animals with a diagnosis of AD. The objective of this study was to determine the effects of oclacitinib on IDT and allergen-specific IgE serology testing using a laboratory model of house-dust mite sensitized Beagle dogs. Twenty-four (24) normal, healthy purpose-bred Beagle dogs were sensitized to house dust mites (HDM, Dermatophagoides farinae) and randomly assigned to placebo-, oclacitinib- (0.4 mg/kg/dose PO), or prednisolone-treated (0.5 mg/kg/dose PO) groups. After 14 days of twice daily dosing, the effects of prednisolone and oclacitinib were compared to placebo using baseline and post-dose IDT and allergen-specific IgE serum measurements. Sensitized dogs had increased circulating HDM-specific IgE for at least two weeks post-sensitization. Prednisolone significantly inhibited the measurable sensitivity of IDT, while oclacitinib did not. Neither prednisolone nor oclacitinib imposed significant effects on allergen-specific IgE serum levels, suggesting oclacitinib may have potential to be used in dogs concurrently undergoing intradermal skin testing and/or allergen-specific IgE serology testing without interference with test results.

In Pursuit of an Allosteric Human Tropomyosin Kinase A (hTrkA) Inhibitor for Chronic Pain.

Human ß-nerve growth factor (ß-NGF) and its associated receptor, human tropomyosin receptor kinase A (hTrkA), have been demonstrated to be key factors in the perception of pain. However, efficacious small molecule therapies targeting the intracellularly located hTrkA kinase have not been explored thoroughly for pain management. Herein, we report the pharmacological properties of a selective hTrkA allosteric inhibitor, 1. 1 was shown to be active against the full length hTrkA, showing preferential binding for the inactive kinase, and was confirmed through the X-ray of hTrkA···1 bound complex. 1 was also found to inhibit ß-NGF induced neurite outgrowth in rat PC12 cells. Daily oral administration of 1 improved the joint compression threshold of rats injected intra-articularly with monoiodoacetate over a 14-day period. The efficacy of 1 in a relevant chronic pain model of osteoarthritis coupled with in vitro confirmation of target mediation makes allosteric hTrkA inhibitors potential candidates for modulating pain.

Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor.

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. Oral administration of PF-00299804 causes significant antitumor activity, including marked tumor regressions in a variety of human tumor xenograft models that express and/or overexpress erbB family members or contain the double mutation (L858R/T790M) in erbB1 (EGFR) associated with resistance to gefitinib and erlotinib. Furthermore, PF-00299804 shows exceptional distribution to human tumor xenografts and excellent pharmacokinetic properties across species.

Update on pathogenesis, diagnosis, and treatment of atopic dermatitis in dogs.

Improved understanding of the pathogenesis of atopic dermatitis in dogs has led to more effective treatment plans, including skin barrier repair and new targeted treatments for management of allergy-associated itch and inflammation. The intent of this review article is to provide an update on the etiologic rationale behind current recommendations that emphasize a multimodal approach for the management of atopic dermatitis in dogs. Increasing knowledge of this complex disease process will help direct future treatment options.

Tyrosine kinase inhibitors. 19. 6-Alkynamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the erbB family of tyrosine kinase receptors.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI.HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.

Tyrosine Kinase Inhibitors. 20. Optimization of Substituted Quinazoline and Pyrido[3,4-d]pyrimidine Derivatives as Orally Active, Irreversible Inhibitors of the Epidermal Growth Factor Receptor Family.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)(*).

CI-994 (acetyldinaline) is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 &mgr;M) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 &mgr;M CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 &mgr;M CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune, skin, and neuronal target cells.

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.

PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib.

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.

G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein.

The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 microM) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27(KIP1) protein when treated with PD 158780 and increased association of p27(KIP1) with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB.

Induction of apoptosis by cationic amphiphilic drugs amiodarone and imipramine.

Phospholipidosis is the excessive accumulation of intracellular phospholipids in cell lysosomes. Drugs that induce this disease often share common physiochemical properties and are collectively classified as cationic amphiphilic drugs (CADs). Although the cause of phospholipidosis and morphologic appearance of affected lysosomes have been studied extensively, less is known about the physiologic effects of the condition. In the current study, U-937 cells were incubated with the CADs amiodarone (2.5-10 microg/mL) and imipramine (2.5-20 microg/mL). Treatment of U-937 cells with these compounds for 96 h resulted in concentration-related increases in phospholipids, as assessed by flow cytometry using the fluorophore nile red. These results were verified by measuring the concentrations of choline-derived phospholipids, which were significantly increased in drug-treated cells. Cell number in amiodarone (10 microg/mL) and imipramine (20 microg/mL) cultures following the 96-h incubation period were markedly reduced compared to control cultures. These observations suggested that accumulation of cellular phospholipids could inhibit cell proliferation. Flow cytometric analysis revealed a decrease in the percentage of cells in the S-phase of the cell cycle following drug treatment, yet DNA replication still occurred in a significant portion of cells. Interestingly, amiodarone and imipramine induced apoptosis in U-937 cells as shown by annexin V-FITC staining and DNA fragmentation. Enzymatic assays demonstrated that amiodarone and imipramine induced the activity of caspases 2 and 3. These results suggest that disruption of cell lysosomes in U-937 cells following accumulation of phospholipids does not cause a cell cycle arrest but instead induces apoptosis by activation of caspase pathways.