The weight losses which occurred as a result of these reactions suggested that the products of these reactions were water soluble. Similar treatment of isobutyronitrile and pivalonitrile gave principally water soluble products, and IR and GLC analyses of the small amounts of organic products from these nitriles showed the absence of starting materials.Conversion of Phenylacetonitrile to Benzonitrile. To a solution of 1.76 g (15 mmol) of phenylacetonitrile in 30 mL of CC14 was added 2.7 g of N-bromosuccinimide (NBS) and 25 mg of benzoyl peroxide. This solution was heated at reflux in the presence of a sunlamp for 30 min, the succinimide was removed, and the solution was concentrated to a yellow oil. This oil was added dropwise over a period of ca. 5 min to a solution of 3 g of sodium azide in 50 mL of Me2SO at room temperature. The solution was stirred for 15 min, poured into 1% NaOH solution, and extracted with pentane. The organic layer was dried, concentrated, and distilled to give 1.28 g (80%) of benzonitrile which was identified by comparison with an authentic sample.Acknowledgment. P. E. Nicholas thanks the Gillette Corp. for a fellowship.
Immunogenicity assessment during early stages of nonclinical biotherapeutic development is not always warranted. It is rarely predictive for clinical studies and evidence for the presence of anti-drug antibodies (ADAs) may be inferred from the pharmacokinetic (PK) profile. However, collecting and banking samples during the course of the study are prudent for confirmation and a deeper understanding of the impact on PK and safety. Biotherapeutic-specific ADA assays commonly developed can require considerable time and resources. In addition, the ADA assay may not be ready when needed if the study of PK and safety data triggers assay development. During early stages of drug development for antibody-drug conjugates (ADCs), there is the added complication of the potential inclusion of several molecular variants in a study, differing in the linker and/or drug components. To simplify analysis of ADAs at this stage, we developed plug-and-play generic approaches for both the assay format and the data analysis steps. Firstly, the assay format uses generic reagents to detect ADAs. Secondly, we propose a cut point methodology based on animal specific baseline variability instead of a population data approach. This assay showed good sensitivity, drug tolerance, and reproducibility across a variety of antibody-derived biotherapeutics without the need for optimization across molecules.
Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab′)2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab′)2 CDR using anti-DLL4 F(ab′)2 and a control F(ab′)2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.
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