The effects of lebrikizumab in patients with mild asthma following whole lung allergen challenge.

BACKGROUND: Interleukin 13 (IL13) is a T-helper type 2 (Th2) cytokine associated with inflammation and pathology in allergic diseases such as bronchial asthma. We have shown that treatment with lebrikizumab, an anti-IL13 monoclonal antibody, significantly improves prebronchodilator forced expiratory volume in 1 s (FEV(1)) in a subset of subjects with uncontrolled asthma. OBJECTIVE: To evaluate efficacy and safety of lebrikizumab in subjects with mild asthma who underwent bronchial allergen challenge. METHODS: Twenty-nine subjects were randomized 1 : 1-5 mg/kg lebrikizumab (n = 13) or placebo (n = 16) administered subcutaneously every 4 weeks over 12 weeks, a total of four doses. Primary efficacy outcome was late asthmatic response (LAR) at Week 13, defined as area under the curve of FEV1 measured 2-8 h following inhaled allergen challenge. Serum biomarkers were measured to verify IL13 pathway inhibition and identify patients with an increased response to lebrikizumab. RESULTS: At Week 13, the LAR in lebrikizumab subjects was reduced by 48% compared with placebo subjects, although this was not statistically significant (95% confidence interval, -19%, 90%). Exploratory analysis indicated that lebrikizumab-treated subjects with elevated baseline levels of peripheral blood eosinophils, serum IgE, or periostin exhibited a greater reduction in LAR compared with subjects with lower baseline levels of these biomarkers. Lebrikizumab exerted systemic effects on markers of Th2 inflammation, reducing serum immunoglobulin E (IgE), chemokine ligands 13 and 17 by approximately 25% (P < 0.01). Lebrikizumab was well tolerated. CONCLUSION AND CLINICAL RELEVANCE: Lebrikizumab reduced the LAR in subjects with mild asthma. Clinical trial number NCT00781443.

OX40L blockade and allergen-induced airway responses in subjects with mild asthma.

BACKGROUND: The OX40/OX40L interaction contributes to an optimal T cell response following allergic stimuli and plays an important role in the maintenance and reactivation of memory T effector cells. OBJECTIVE: We tested whether treatment with an anti-OX40L monoclonal antibody (MAb) would inhibit allergen-induced responses in subjects with asthma. METHODS: Twenty-eight mild, atopic asthmatic subjects were recruited for a double-blind, randomized, placebo-controlled, parallel-group trial (ClinicalTrials.gov identifier NCT00983658) to compare blockade of OX40L using a humanized anti-OX40L MAb to placebo-administered intravenously in 4 doses over 3 months. Allergen inhalation challenges were carried out 56 and 113 days after the first dose of study drug. The primary outcome variable was the late-phase asthmatic response. Other outcomes included the early-phase asthmatic response, airway hyperresponsiveness, serum IgE levels, blood and sputum eosinophils, safety and tolerability. RESULTS: Treatment with anti-OX40L MAb did not attenuate the early- or late-phase asthmatic responses at days 56 or 113 compared with placebo. In the anti-OX40L MAb treatment group, total IgE was reduced 17% from pre-dosing levels, and sputum eosinophils decreased 75% by day 113 (both P = 0.04). There was no effect of anti-OX40L MAb on airway hyperresponsiveness or blood eosinophils. The frequency of AEs was similar in both groups. CONCLUSION AND CLINICAL RELEVANCE: Pharmacological activity of anti-OX40L MAb was observed by decreases in serum total IgE and airway eosinophils at 16 weeks post-dosing, but there was no effect on allergen-induced airway responses. It is possible that the treatment duration or dose of antibody was insufficient to impact the airway responses.

Current Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch.

US Food and Drug Administration (FDA)-approved diagnostic assays play an increasingly common role in managing patients to prolong lifespan while also enhancing quality of life. Diagnostic assays can be essential for the safe and effective use of therapeutics (companion diagnostic), or may inform on improving the benefit/risk ratio without restricting drug access (complementary diagnostic). This tutorial reviews strategic considerations for drug and assay development resulting in FDA-approved companion or complementary diagnostic status.

Cytotoxicity of mitomycin C and adriamycin in freshly isolated rat hepatocytes: the role of cytochrome P450.

The role of cytochrome P450 (P450) in the cytotoxicity of mitomycin C (MMC) and Adriamycin (ADR) was investigated in freshly isolated hepatocytes from phenobarbital-induced rats. The loss of cell viability [measured as lactate dehydrogenase (LDH) leakage] upon MMC exposure was accompanied by a rapid and extensive intracellular glutathione (GSH) depletion and followed by minor lipid peroxidation (LPO). Coincubation of the hepatocytes with the P450 inhibitors, metyrapone and SK&F 525-A, strongly protected against MMC-induced LDH leakage, GSH depletion, and LPO. Inasmuch as the depletion of intracellular GSH by MMC, which is considered as a critical event in the development of MMC cytotoxicity, was not accompanied by a stoichiometric oxidation to oxidized GSH (GSSG), the formation of a MMC-GSH conjugate after one-electron reductive bioactivation of MMC by P450 was anticipated. In contrast to MMC, ADR was only cytotoxic to the hepatocytes upon prior depletion of intracellular GSH with diethylmaleate. Addition of metyrapone and SK&F 525-A completely protected the hepatocytes against ADR-induced LDH leakage and LPO. Moreover, the ADR-induced LDH leakage and LPO were strongly inhibited by dimethyl sulfoxide and ethanol, indicating that hydroxyl radicals were involved in the cytotoxicity of ADR. In conclusion, the present investigations indicate that P450 plays a major role in the cytotoxicity of both MMC and ADR in freshly isolated hepatocytes from phenobarbital-induced rats. The present findings lead to a better understanding of the mechanism of the cytotoxic actions of both MMC and ADR.

The involvement of sensory neuropeptides in toluene diisocyanate-induced tracheal hyperreactivity in the mouse airways.

1. Recently, we developed a murine model to investigate toluene diisocyanate (%DI)-induced occupational asthma. After skin-sensitization and intranasal challenge with TDI (1%) mice exhibited tracheal hyperreactivity 24 h after the challenge. 2. The aim of the present study was to investigate the possible role for sensory neuropeptides in the development of this tracheal hyperreactivity. 3. First, we demonstrated that direct application of TDI in vitro induced the release of tachykinins from the sensory nerves in the mouse isolated trachea. Second, capsaicin pretreatment, resulting in the depletion of sensory neuropeptides, completely abolished the TDI-induced tracheal hyperreactivity 24 h after the challenge. Third, the selective neurokinin1 (NK1)-receptor antagonist RP 67580 (0.2 mumol kg-1) also inhibited tracheal hyperreactivity when it was administered before the challenge. However, administration of RP 67580 during the sensitization phase did not result in a suppression of the TDI-induced tracheal hyperreactivity 24 after the challenge. 4. When TDI-sensitized mice were topically challenged with TDI a marked ear swelling response was observed. The cutaneous response after TDI application was not affected by capsaicin pretreatment or RP 67580 administration. 5. These results clearly show that sensory neuropeptides, particularly tachykinins, are essential for the development of TDI-induced tracheal hyperreactivity during the effector phase. The differences between the airways and skin with respect to the sensory neuropeptides is intriguing and could suggest a local action for the tachykinins in the airways.

Accelerated alveolar bone loss in mice lacking interleukin-10.

Interleukin-10 regulates pro-inflammatory cytokines, including those implicated in alveolar bone resorption. We hypothesized that lack of interleukin-10 leads to increased alveolar bone resorption. Male interleukin-10(-/-) mice, on 129/SvEv and C57BL/6J background, were compared with age-, sex-, and strain-matched interleukin-10(+/+) controls for alveolar bone loss. Immunoblotting was used for analysis of serum reactivity against bacteria associated with colitis and periodontitis. Interleukin-10(-/-) mice had significantly greater alveolar bone loss than interleukin-10(+/+) mice (p = 0.006). The 30-40% greater alveolar bone loss in interleukin-10(-/-) mice was evident in both strains, with C57BL/6J interleukin-10(-/-) mice exhibiting the most bone loss. Immunoblotting revealed distinct interleukin-10(-/-) serum reactivity against Bacteroides vulgatus, B. fragilis, Prevotella intermedia, and, to a lesser extent, against B. forsythus. The results of the present study suggest that lack of interleukin-10 leads to accelerated alveolar bone loss.

A simple and rapid in vitro test system for the screening of histamine H3 ligands.

A simple and rapid functional test system for the screening of histamine H3 ligands is described. It is based on the inhibitory effect of histamine H3 agonists on electrically-evoked contractile response of isolated guinea pig intestine. Whole jejunum segments are continuously stimulated maximally (15 V) by electrical pulses with a frequency of 0.1 Hz and a duration of 0.5 msec. The resulting twitches are recorded isotonically (1.0 g) and can be completely abolished by atropine (0.1 mcM).

Translational pharmacokinetics and pharmacodynamics of an FcRn-variant anti-CD4 monoclonal antibody from preclinical model to phase I study.

MTRX1011A is a humanized anti-CD4 antibody with an amino acid substitution (N434H) to improve its binding to the neonatal Fc receptor (FcRn). Pharmacokinetic/pharmacodynamic (PK/PD) data in baboons suggest that the increased binding to FcRn reduces the nonspecific elimination rate (K(el)) of MTRX1011A by ~50% but does not affect its PK-PD relationship. The human PK/PD data of MTRX1011A from a phase I study in patients with rheumatoid arthritis (RA) were compared with those previously reported for TRX1, its predecessor antibody, using population PK-PD modeling. The results suggest a comparable PK-PD relationship and no significant difference between the K(el) values of the two antibodies. However, the results may have been confounded by the differences in the clinical populations in which the two antibodies were studied and the presence of preexisting immunoglobulin M (IgM) antibodies in the RA sera that recognize N434H in MTRX1011A. This study highlights the challenges in translating from animal studies to human application the effects of FcRn-directed mutations on the PK of monoclonal antibodies.

Oxygen and xenobiotic reductase activities of cytochrome P450.

The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O2-.) most likely by autooxidation of a P450 ferric-dioxyanion complex. The formation of reactive oxygen species (O2-., hydrogen peroxide, and, notably, hydroxyl free radicals) presents a potential toxication pathway, particularly when effective means of detoxication are lacking. Under anaerobic conditions, P450 may also be involved in the reduction of xenobiotics. During the xenobiotic reductase activity of P450, xenobiotics are reduced by the ferrous xenobiotic complex. After xenobiotic reduction by P450, xenobiotic free radicals are formed that are often capable of reacting directly with tissue macromolecules. Unfortunately, the compounds that are reductively activated by P450 have little structural similarity. The precise molecular mechanism underlying the xenobiotic reductase activity of P450 is, therefore, not yet fully understood. Moreover, description of the molecular mechanisms of xenobiotic and oxygen reduction reactions by P450 is limited by the lack of knowledge of the three-dimensional (3D) structure of the mammalian P450 proteins.

Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10-/- mice and Rag-2-/- mice reconstituted with CD4+CD45RBhigh T cells.

We used quantitative PCR to investigate the expression of chemokines and chemokine receptors in two Th1-mediated murine models of inflammatory bowel disease (IBD). First, mRNA levels encoding the chemokines MIG, RANTES, lymphotactin, MIP-3alpha, TCA-3, TARC, MIP-3beta, LIX, MCP-1 and MIP-1beta and the receptors CCR4, CCR6 and CCR2 were significantly increased in chronically inflamed colons of IL-10-/- mice when compared with wildtype mice. Interestingly, reversal of colitis in IL-10-/- mice by anti-IL-12 mAb was accompanied by the inhibition in the expression of LIX, lymphotactin, MCP-1, MIG, MIP-3alpha, MIP-3beta, TCA-3, CCR2 and CCR4, whereas the increased mRNA levels of MIP-1beta, RANTES, TARC and CCR6 were unaffected. Second, to investigate which chemokines and receptors were up-regulated during the inductive phase of colitis, we employed the CD4+CD45RBhigh T cell transfer model. At 4 and 8 weeks after reconstitution of Rag-2-/- mice the mRNA levels of IP-10, MCP-1, MDC, MIG, TARC, RANTES, CCR4 and CCR5 were significantly increased prior to the appearance of macroscopic lesions. Other chemokines and chemokine receptors were clearly associated with the acute phase of the disease when lesions were evident. The sum of our studies with these two models identifies chemokines that are expressed at constant levels, irrespective of inflammatory responses, and those that are specifically associated with acute and/or chronic stages of Th1-driven colitis.

Toluene diisocyanate-induced in vitro tracheal hyperreactivity in the mouse.

Toluene diisocyantate (TDI) is a low-molecular-weight compound that is known to cause occupational asthma in 5% to 10% of exposed workers. These patients exhibit marked airway hyperresponsiveness and granulocyte accumulation in the airways, and 10% to 20% were also determined to have TDI-specific IgE in their serum. In this study, we developed a murine model for TDI-induced asthma. After several sensitization and challenge regimens were tested, it was decided that optimal sensitization was observed after mice (BALB/c) were skin sensitized with TDI (1%) two times on two consecutive days and challenged intranasally 8 d later with TDI (1%). Sensitized mice exhibited tracheal hyperreactivity to carbachol 24 h after challenge (69% increase in maximal contractile response). In contrast, no differences between the control and TDI-treated groups was observed 2 and 48 h after challenge with 1% TDI. There appeared to be no elevation in TDI-specific IgE antibodies in the serum at all time points measured. In addition, no influx of leukocytes could be detected histologically in the trachea and lung tissue or airway lumen 2, 24, and 48 h after the challenge. Surprisingly, the tracheal hyperreactivity was associated with a marked increase in myeloperoxidase but not eosinophil peroxidase activity in the lung tissue and in the cells of the bronchoalveolar lavage fluid at 24 h after the challenge. To investigate the role of lymphocytes in the induction of tracheal hyperreactivity, mice were passively sensitized by intravenous injection of lymphoid cells from TDI-sensitized donor mice. Similar to active sensitization, adoptive transfer of lymphocytes from sensitized donors resulted in tracheal hyperreactivity 24 h after challenge of the recipients. In conclusion, these data show that TDI is capable of inducing lymphocyte-dependent but IgE-independent tracheal hyperreactivity in the mouse that is not associated with cellular infiltration in the airways. This model can be used to further investigate the possible mechanisms involved in the development of occupational asthma induced by TDl.

Long-term topical exposure to toluene diisocyanate in mice leads to antibody production and in vivo airway hyperresponsiveness three hours after intranasal challenge.

Toluene diisocyanate (TDI) is a low-molecular-weight compound which is known to cause occupational asthma in 5 to 10% of exposed workers. Previously, we developed a murine model to investigate TDI-induced occupational asthma. Short-term exposure to TDI (skin sensitization twice daily on Day 0 and Day 1 and intranasal challenge on Day 8) led to a nonspecific tracheal hyperractivity 24 h after the challenge in TDI-sensitized mice when compared with nonsensitized mice whereas no TDI-specific IgE antibodies were found in the serum. Because 20% of subjects with TDI-induced occupational asthma exhibit an increase in serum IgE antibodies, we exposed mice for a longer period of time to investigate whether this procedure could induce TDI-specific antibody production in exposed mice. Long-term exposure (skin sensitization on 6 consecutive weeks followed by intranasal challenge on Week 7) resulted in the production of total IgE and IgG and TDI-specific IgE and IgG antibodies. Airway reactivity to various agonists was also measured in vitro and in vivo in long-term exposed mice. TDI-sensitized mice exhibited in vitro tracheal hyperreactivity to carbachol 3 h after the challenge when compared with the nonsensitized mice. Moreover, in vivo airway hyperresponsiveness to serotonin (5-hydroxytryptamine [5HT]) was found 3 h after the challenge in TDI-sensitized mice. Interestingly, in vivo airway hyperresponsiveness was not observed at any time point in the mice exposed to TDI according to the short-term protocol. In conclusion, by altering the exposure time and/or cumulative dosage of TDI different biological reactions can be elicited in exposed mice. This important finding might be a reflection of the diversity of symptoms found in patients suffering from TDI-induced asthma. Both the short-exposure and the long-exposure model will be useful to further investigate the mechanisms of action of TDI.

Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation.

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.