Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.

4th Global CRO Council for Bioanalysis: coadministered drugs stability, EMA/US FDA guidelines, 483s and carryover.

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters.

Confirmatory reanalysis of incurred bioanalytical samples.

Bioanalytical methods used to support the drug development process are validated to ensure that they function in the manner in which they are intended. "Incurred" or study samples can vary in their composition when compared with the standards and quality control samples used to validate the method and analyze these samples. During the 3rd American Association of Pharmaceutical Scientists(AAPS)/Food and Drug Administration(FDA) Bioanalytical Workshop, it was suggested that the reproducibility in the analysis of incurred samples be evaluated in addition to the usual prestudy validation activities performed.

Anti-IgE efficacy in murine asthma models is dependent on the method of allergen sensitization.

Background: Murine models used to delineate mechanisms and key mediators of asthma have yielded conflicting results and suggest that the dominant mechanism and mediators required for disease induction differ depending on the model and method of allergen sensitization used.

Objective: The goal of this study was to determine whether the mode of allergen sensitization influenced the role that IgE had in allergen-induced pulmonary eosinophilic inflammation.

Methods: Mice were exposed to dust mite extract in 2 models of allergic inflammation that differed in the method of sensitization.

Immunotherapy approach to allergic disease.

The causal role of immunoglobulin E (IgE) in triggering the cascade of biochemical events leading to allergic disease is well established. Treatments that selectively inhibit IgE activity are a logical approach in managing the allergic response. One such strategy utilizes rhuMAb-E25, a recombinant humanized IgG(1) monoclonal anti-IgE antibody, which binds to IgE.

Effects of humanized monoclonal antibody to rhesus CD11a in rhesus monkey cardiac allograft recipients.

Introduction: Leukocyte function-associated antigen-1 (LFA-1, CD11a) monoclonal antibody (mAb) affects many leukocyte functions without cell depletion. We hypothesized that the use of a humanized, anti-rhesus modified LFA-1 mAb (H2C12) in rhesus monkeys would cause: (1) prolonged heart allograft survival, (2) inhibition of primary but not secondary antibody responses, and (3) minimal drug toxicity.

Methods And Results: Control (n=5) and H2C12-treated (n=7) (8-20 mg/kg i.

Effects of administration of a single dose of a humanized monoclonal antibody to CD11a on the immunobiology and clinical activity of psoriasis.

Background: CD11a/CD18 comprise subunits of leukocyte function associated antigen (LFA-1), a T-cell surface molecule important in T-cell activation, T-cell emigration into skin, and cytotoxic T-cell function.

Objective: We explored the immunobiologic and clinical effects of treating moderate to severe psoriasis vulgaris with a single dose of humanized monoclonal antibody against CD11a (hu1124).

Methods: This was an open label study with a single dose of hu1124 at doses of 0.

IgE inhibition as a therapy for allergic disease.

Monoclonal antibodies are potentially useful therapeutic agents in a variety of immunologically mediated diseases, since they offer the theoretical advantage of selectively targeting the mediators of the immuno-pathogenesis. It has been well established that IgE antibody synthesized by the immune system plays a pivotal role in the cascade of biochemical events leading to the allergic reaction. The aim of these studies was to eliminate IgE with a monoclonal antibody as the approach for treatment of atopic disease.

Differential effects of endogenous and exogenous interferon-gamma on immunoglobulin E, cellular infiltration, and airway responsiveness in a murine model of allergic asthma.

The inflammatory response as seen in human allergic asthma is thought to be regulated by Th2 cells. It has been shown that interferon-gamma (IFN-gamma) can downregulate the proliferation of Th2 cells and therefore might be of therapeutic use. In the present study we have investigated the in vivo role of endogenous and exogenous IFN-gamma in a murine model with features reminiscent of human allergic asthma.

Anti-immunoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice.

Inhibition of antigen-specific IgE response has been shown to lead to amelioration of allergic disease symptoms. In an effort to design a therapy aimed at decreasing IgE levels, we reported previously that treatment of mice with an anti-IgE antibody coincident with the primary antigen immunization resulted in significant decreases in antigen-specific IgE synthesis, without substantially altering IgG levels. In the present study, we employed this mouse model and a surrogate antibody to investigate the capacity of anti-IgE treatment to block an established IgE response in vivo.

Therapeutic potential of anti-IgE antibodies.

Anti-IgE antibodies directed against the Fc epsilon RI-binding region on IgE inhibit binding of IgE to IgE receptors without inducing mediator release from IgE sensitized cells. In mice these antibodies selectively reduce serum IgE, inhibit antigen induced skin reactions, cytokine production by lung Th2 cells, and pulmonary eosinophil infiltration. Clinical trials in humans reveal that such antibodies are well tolerated and reduce rhinitis symptoms and early and late phase bronchoconstriction responses.

Murine CTLA4-IgG treatment inhibits airway eosinophilia and hyperresponsiveness and attenuates IgE upregulation in a murine model of allergic asthma.

Antigen-specific T-cell activation requires the engagement of the T-cell receptor (TCR) with antigen as well as the engagement of appropriate costimulatory molecules. One of the most important pathways of costimulation is the interaction of CD28 on the T cell with B7-1/B7-2 on antigen-presenting cells. In the present study, we have examined the in vivo effects of blocking the CD28:B7 T-cell costimulatory pathway by administration of mCTLA4-IgG in a murine model of allergic asthma.

Down-regulation of Fc(epsilon)RI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody.

Treatment of allergic disease by decreasing circulating IgE with anti-IgE Abs is currently under clinical study. Based on previous unrelated studies, it appeared likely that Fc(epsilon)RI expression on basophils and mast cells might also be regulated by levels of circulating IgE Ab. Therefore, the expression of IgE and Fc(epsilon)RI on human basophils was examined in 15 subjects receiving humanized anti-IgE mAb intravenously.

Human anti-IgE monoclonal antibody blocks passive sensitization of human and rhesus monkey bladder.

IgE antibodies may play an important role in noninfectious urinary inflammation, including interstitial cystitis (IC). In this study, urinary bladder strips were passively sensitized for 20 hours with serum from a ragweed-sensitive patient in the absence or presence of an anti-human IgE monoclonal antibody (MaE11) at 0, 1-, or 5-fold IgE concentration. The urinary bladder strips then were suspended in a superfusion apparatus for measurement of contraction and histamine release in response to antigen E (AgE) challenge.

Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1.

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation. mAbs against LFA-1 have been shown to inhibit several T cell-dependent immune functions in vitro and prevent graft failure after bone marrow transplantation in vivo. A murine anti-human CD11a mAb, MHM24, has been humanized and shown to prevent adhesion of human T cells to human keratinocytes and the proliferation of T cells in response to nonautologous leukocytes in the mixed lymphocyte response assay.

Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal antibody monotherapy.

Strategies targeting lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1) have previously been shown to produce long-term survival of solid organ allografts in animals only when both CD11a and ICAM-1 are targeted for a brief (6-7 days) time or when extended (14 weeks) treatment with anti-CD11a monoclonal antibody (mAb) is administered. We show that recipient pretreatment followed by a brief (13 days) treatment course with high-dose anti-CD11a mAb alone produces long-term survival of cardiac allografts in the rigorous, nonprimarily vascularized heart allograft model in mice. This treatment regimen induces specific unresponsiveness in our model.

Anti-IgE therapy.

Controlling the IgE response at either the synthesis level or the effector phase should have a profound impact on the allergic cascade. For more than a decade, researchers have focused on ways of interfering with the binding of IgE to its high-affinity receptor on proinflammatory cells. Several approaches have also been taken to antagonize the complex interplay of cytokines and cell-associated molecules (CD40, CD23) that are implicated in IgE synthesis.

Allergen-induced histamine release in rat mast cells transfected with the alpha subunits of Fc epsilon RI.

A rat mast cell histamine assay (RMCHA) has been developed to quantitate the biological activity of a recombinant humanized, monoclonal anti-IgE antibody (rhuMAbE25). Rat mast cells (RBL 48), transfected with the alpha subunit of the high affinity human IgE receptor (Fc epsilon RI), were presensitized for 2 h with human plasma containing IgE specific for ragweed and challenged with ragweed allergen in the presence of 50% D2O. Histamine release plateaus at 0.

Inhibition of allergic reactions with antibodies to IgE.

Numerous clinical studies show that direct interference with the IgE response leads to a decrease or elimination of allergic symptoms. The aim of these studies was to design a therapy aimed at decreasing IgE levels in order to ameliorate atopic disease. To this end, a murine monoclonal antibody, MAE11, directed against IgE was identified, which had all the properties necessary to interfere with IgE responses, but lacked the harmful side effects of inducing receptor cross-linking.

IL-12 exacerbates rather than suppresses T helper 2-dependent pathology in the absence of endogenous IFN-gamma.

To assess the role of IFN-gamma in the in vivo regulation of Th subset differentiation by IL-12, schistosome egg-induced Th2 responses and granuloma formation were studied in IFN-gamma knock-out (gamma KO) mice in which the absence of endogenous IFN-gamma is assured. Rather than suppressing pathology and eosinophilia as observed in wild-type animals, exogenous IL-12 in egg-injected gamma KO mice exacerbated Th2-dependent granuloma formation while failing to reduce peak tissue eosinophilia. Similarly, instead of inhibiting its production, IL-12 caused a dramatic increase in serum IgE levels in gamma KO animals after egg injection.