Thomas T. Kawabata

Immunogenicity of biologically-derived therapeutics: Assessment and interpretation of nonclinical safety studies

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

Herpes Zoster and Tofacitinib Therapy in Patients With Rheumatoid Arthritis

Patients with rheumatoid arthritis (RA) are at increased risk for herpes zoster (HZ) (i.e., shingles). The aim of this study was to determine whether treatment with tofacitinib increases the risk of HZ in patients with RA.

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug‐induced skin diseases

To cite this article: Zawodniak A, Lochmatter P, Yerly D, Kawabata T, Lerch M, Yawalkar N, Pichler WJ. In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug‐induced skin diseases. Allergy 2010; 65: 376–384.

Covalent binding of the nitroso metabolite of sulfamethoxazole is important in induction of drug-specific T-cell responses in vivo.

Immune-mediated drug hypersensitivity reactions (IDHRs) represent a significant problem due to their unpredictable and severe nature, as well as the lack of understanding of the pathogenesis. Sulfamethoxazole (SMX), a widely used antibiotic, has been used as a model compound to investigate the underlying mechanism of IDHRs because it has been associated with a relatively high incidence of hypersensitivity. Previous studies by others showed that administration of 4-(nitroso)-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide (SMX-NO), the reactive metabolite of SMX, to rats resulted in the generation of SMX-specific antibodies and ex vivo splenocyte proliferative responses, as well as haptenation of skin keratinocytes, circulating peripheral blood mononuclear cells, and splenocytes. The objective of the present study was to further investigate SMX-NO-protein binding in relationship to its immunogenicity. In female DBA/1 mice treated with SMX-NO, varying degrees of SMX-NO-dependent T-cell responses and SMX-NO-protein adduct formation were observed in the spleen and in inguinal, brachial, and axillary lymph nodes. The data suggested a tissue-specific threshold of SMX-NO dosage that triggers the detection of adducts and immune response. Furthermore, serum albumin and immunoglobulin were identified as protein targets for SMX-NO modification. It seemed that these adducts were formed in the blood, circulated to lymphoid tissues, and initiated SMX-NO-dependent immune responses. Collectively, these data revealed a causal link between the deposition of SMX-NO-protein adducts in a lymphoid tissue and the induction of immune response in that tissue. Our findings also suggest that the immunogenicity of SMX-NO is determined by the immunogenic nature of the hapten, rather than special characteristics of the adducted protein.

Pathology considerations for, and subsequent risk assessment of, chemicals identified as immunosuppressive in routine toxicology

Several proposals have been made with the aim of assisting in the early identification of chemicals with immunotoxic potential. The Organisation for Economic Cooperation and Development is now likely to incorporate enhanced immunopathology into the test guideline for the 28-day rat study, which may be regarded as a Tier I investigation. However, no guidelines have yet been proposed either for how the new data generated will be evaluated, or for how a subsequent risk assessment will be made. In this paper, considerations for the immunopathological assessment of the thymus, spleen, lymph nodes and bone marrow are described, together with comments on haematological and organ weight changes that may be associated with immunotoxicity. Their interpretation will depend on the doses at which changes are manifest, the quantity and quality of the effects observed and the presence and severity of other forms of toxicity. Lastly, risk assessment and the approach to Tier II testing in immunotoxicity is discussed. It is concluded that much of this work must be on a case-by-case basis, but should not in principle differ from the approach adopted for any other type of toxicity identified ina 28-day study.

Synthesis and Characterization of Rhenium and Technetium-99m Labeled Insulin

A (99m)Tc-labeled insulin analogue was synthesized through a direct labeling method in which the [(99m)Tc(CO)(3)](+) core was combined with a protected insulin derivative (9) bearing a M(I) chelate linked to the first amino acid of the B-chain (B1). Regioselective labeling was achieved by careful control over the pH and the reaction time. Following a TFA-anisole mediated deprotection step (decay-corrected yield of 30 +/- 11%, n = 4), the identity of the final (99m)Tc-labeled product was confirmed by HPLC. Displacement of (125)I-insulin from the insulin receptor (IR) by the Re analogue 6 was similar to that of native insulin (17.8 nM vs 11.7 nM, respectively). The extent of autophosphorylation and Akt activation, as indicated by production of phospho-Akt (pAkt), showed no statistical difference between 6 and native insulin in both assays. These results support the use of the reported (99m)Tc-insulin derivative as a tracer for studying insulin biochemistry in vivo.

Developmental immunotoxicity (DIT) testing of pharmaceuticals: current practices, state of the science, knowledge gaps, and recommendations.

The development and regulatory approval of immunomodulatory pharmaceuticals to treat many human diseases has increased significantly over the last two decades. As discussed by FDA and ICH guidelines, all human pharmaceuticals in development should be evaluated for potential adverse effects on the immune system. Developmental immunotoxicology (DIT) focuses on the concern that early-life (during pre-/post-natal development) exposure to agents which target the immune system may result in enhanced susceptibility to immune-related disease (e.g., infection, autoimmunity, and cancer, particularly leukemia) compared to adults, unique effects not observed in adults, or more persistent effects in comparison to those following adult exposure. This article provides a substantive review of the literature and presents detailed considerations for DIT testing strategies with a specific focus on pharmaceuticals and biopharmaceuticals. In this regard, differences between small molecule and large molecule therapeutics will be considered, along with recommendations for best practices in the assessment of DIT during drug development. In addition, gaps in the DIT knowledge base and current testing strategies are identified. Finally, a summary of an ILSI-HESI-ITC sponsored Workshop conducted in 2010, entitled ‘Developmental Immunotoxicity Testing of Pharmaceuticals’ will be presented. This Workshop consisted of participants from the pharmaceutical, biotechnology, academic, and regulatory sectors, where many of the issues relating to DIT outlined in this review were discussed, key points of consensus reached, and current gaps in the science identified.

The identification of chemicals with sensitizing or immunosuppressive properties in routine toxicology

In the context of this paper, immunotoxicity is taken to encompass immunosuppression/immunopotentiation and allergy. Over the last 10 to 15 years, well characterized methods for the assessment of altered immune competence have been reported. This has led to proposals for tiered testing schemes. This review examines the suitability of immunotoxicity parameters for inclusion in routine 28-day studies and comments on methods that have been proposed for incorporation within the guidelines issued by the US FDA and US EPA and OECD. It is recommended that the existing OECD Guideline 407 is modified to incorporate total and differential blood cell counts, spleen and thymus weight and histopathology, and draining and distal lymph node histopathology for Tier I level testing. Data so generated will provide a reliable and accurate means of identifying at an early stage potential immunotoxic effects. Tier II testing should be carried out on a case by case basis and only assuming positive results are obtained at Tier I. An increasingly sophisticated understanding of the nature of immune responses to chemical allergens has facilitated the design of novel predictive methods for the identification of sensitizing activity. Opportunities which arise from these new developments in allergy testing such as the local lymph node assay, mouse ear swelling test, and the mouse IgE test should be monitored closely.

Characterization of the Action of Drug-Induced Stress Responses on the Immune System: Evaluation of Biomarkers for Drug-Induced Stress in Rats

Toxicological testing of compounds often is conducted at the maximum tolerated dose to identify potential target organs. Toxicities observed at these high doses may result in decreased body weight gain, food consumption and activity. These clinical signs are often associated with a generalized stress response. It has been known that stress may cause increased levels of corticosterone, which causes changes in circulating leukocyte profiles, decreases in thymus and spleen weights and changes in the microscopic structure of lymphoid organs. This makes it difficult to differentiate between stress-related changes and direct toxicity to the immune system in standard non-clinical toxicity testing in rats. In mice, MHC Class II expression was found to be a very sensitive biomarker of stress and maybe useful for the rat. Therefore, the objective of studies presented was to further characterize the effects of corticosterone and stressors on the immune system and identify potential biomarkers of stress in rats. Rats were treated with exogenous corticosterone (20 or 30 mg/kg BID) or ethanol (5 g/kg) for either 1 or 4 days. Restraint stress was also evaluated for a 3-day period. Blood and urine samples were collected during the treatment period for corticosterone measurements. At necropsy, blood samples for leukocyte differentials were collected. Spleen and thymus weights, cellularity, lymphocyte subpopulations and histopathology were also evaluated. Urine corticosterone levels were also investigated as a surrogate to measuring serum corticosterone. The results demonstrate that the pattern of responses to corticosterone or the stressors is different in mice and rats. Although, decreases in MHC Class II were found to be a sensitive indicator of stress in mice, only slight decreases were observed in rats with similar serum corticosterone AUC levels. Decreases in thymus weight were greater than spleen weight with corticosterone or ethanol or restraint stressor. No other single parameter or combination of parameters tested were obvious candidates as sensitive biomarkers of stress in rats. However, the good correlation between urine and serum corticosterone levels suggest that urine corticosterone may be a potential biomarker of stress induced changes to the immune response.

Development and Validation of a Canine T-Cell-Dependent Antibody Response Model for Immunotoxicity Evaluation

A T-cell dependent antibody response (TDAR) model to evaluate compounds for potential immunotoxicity in dogs has not been reported. The objective of these studies was to develop and validate a dog TDAR model using the T-cell dependent antigen, keyhole limpet hemocyanin (KLH). Studies were conducted to determine the appropriate dose of KLH, immunization route and kinetics of the antibody response to KLH in the dog. To validate the sensitivity of this method, we investigated the TDAR to KLH in the dog with a known immunosuppressive drug, cyclosporine (Neoral). The results of this study demonstrate that a robust primary IgM and IgG response to KLH can be generated in dogs and the IgG response was sensitive to cyclosporine treatment.