A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target.

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.

Tracheo-innominate artery fistula: two case reports and a clinical review.

Tracheo-innominate artery fistula (TIF) is a surgical emergency with high mortality rates. Reported incidence is 0.1%-1.0% after tracheostomy with peak incidence 3 days to 6 weeks post procedure. TIF is usually fatal once it bleeds. For the successful management of TIF, treatment should be initiated immediately with the special considerations kept in mind. We describe two cases of TIF, and its clinical characteristics are reviewed in accordance with relevant literature.

Efficacy and tolerance of gastrostomy feeding in Japanese muscular dystrophy patients.

Although muscular dystrophy patients often have feeding difficulty and need long-term enteral nutrition, only a few reports have described gastrostomy feeding in these patients. This study was designed to evaluate the efficacy and tolerance of gastrostomy feeding in patients with muscular dystrophy. We performed a retrospective, multicenter study on 144 patients with muscular dystrophy who received gastrostomy feeding between 2007 and 2009 in 25 neuromuscular centers in Japan. There were 77 Duchenne muscular dystrophy (median age at gastrostomy placement 26 years, range 13-47 years), 40 myotonic dystrophy (median age 54.5 years, range 13-70 years), 11 Fukuyama congenital muscular dystrophy (median age 22 years, range 13-29 years), 5 limb girdle muscular dystrophy (median age 62 years, range 43-78 years), and 5 facioscapulohumeral muscular dystrophy (median age 52 years, range 28-67 years) patients. Many benefits including amelioration of malnutrition, swallowing difficulty and respiratory status were observed after the introduction of gastrostomy feeding. Especially in patients with Duchenne muscular dystrophy, mean body weight significantly increased after gastrostomy placement. Although most complications, which are commonly observed in other populations, were tolerable, respiratory failure and peritonitis were important concerns. These findings suggest that gastrostomy placement at an appropriate time is advisable in patients with muscular dystrophy.

Quantitative prediction of mechanism-based inhibition caused by mibefradil in rats.

It was previously demonstrated that mibefradil, which shows mechanism-based inhibition in humans, also caused drug-drug interactions (DDIs) with midazolam (MDZ) in rats. In this study, we aimed to quantitatively predict the DDIs observed in rats using a physiologically based pharmacokinetic (PBPK) model from in vitro inactivation parameters. For more precise predictions, contribution ratios of cytochrome P450 (P450) isozymes involved in MDZ metabolism and inactivation parameters of mibefradil against each isozyme were incorporated in the predictive model. The evaluation of metabolic rate using recombinant P450s suggested that CYP3A2 and CYP2C11 contributed to 89 and 11% of MDZ metabolism, respectively. Inactivation studies of mibefradil against the two isozymes showed that the maximal inactivation rate constants (k(inact)) were considerable in both isozymes (0.231-0.565 min(-1)), whereas the inhibitor concentration producing half the k(inact) (K(I, app)) of CYP3A2 (0.263-0.410 µM) was a good deal lower than that for CYP2C11 (6.82-11.4 µM). As a result of predicting the DDIs using the PBPK model, predicted increases in areas under the concentration-time curve of MDZ with coadministration of mibefradil (284 and 510% at 6 and 12 mg/kg mibefradil, respectively) closely corresponded to the observed values (226 and 545%, respectively). From those results, it was thought that the construction of a predictive model for DDIs using the PBPK model in detail would enable us to quantitatively predict in vivo DDIs from in vitro data. This approach to predict DDIs on the basis of the contributing isozymes would be important for predicting clinical DDIs of drugs metabolized by multiple enzymes.

Autoantibodies and cell-mediated autoimmunity to NMDA-type GluRepsilon2 in patients with Rasmussen's encephalitis and chronic progressive epilepsia partialis continua.

PURPOSE: To evaluate antibody-mediated and cytotoxic T cell-mediated pathogenicity that has been implicated as the autoimmune pathophysiological mechanism in Rasmussen's encephalitis. METHODS: We examined autoantibodies against the N-methyl-d-aspartate glutamate receptor (NMDA-type GluR) epsilon2 subunit and its epitopes in serum and CSF samples from 20 patients [five histologically proven (definitive) Rasmussen's encephalitis with epilepsia partialis continua (EPC), four definitive Rasmussen's encephalitis without EPC, and 11 clinical Rasmussen's encephalitis with EPC]. We examined 3H-thymidine uptake into lymphocytes after stimulation by GluRs. RESULTS: All nine definitive patients (five patients with EPC and four without EPC), and 10 of 11 clinical Rasmussen's encephalitis patients had the autoantibodies. In four patients, the autoantibodies were absent in early stage when epileptic seizures had already become frequent, and appeared subsequently. In two patients, the autoantibodies persisted in the serum after frontal lobe resection or functional hemispherectomy, although epileptic seizures were completely controlled. Autoantibodies to the C2 epitope predominated, while autoantibodies to the extracellular N epitope were rare. The mean 3H-thymidine uptake ratios (stimulation by GluRepsilon2-containing homogenates/stimulation by PHA) were significantly higher in definitive and clinical Rasmussen encephalitis patients than in controls. The mean 3H-thymidine uptake ratios (relative to PHA) were significantly higher for GluRepsilon2-containing homogenate than for control homogenate or GluRdelta2-containing homogenate. CONCLUSIONS: Autoantibodies against GluRepsilon2 may be one of the diagnostic markers for Rasmussen's encephalitis with and without EPC. Patients have activated T cells stimulated by GluRepsilon2 in peripheral blood circulation. We speculate that cellular autoimmunity and the subsequent humoral autoimmunity against GluRepsilon2 may contribute to the pathophysiological processes in Rasmussen's encephalitis.

[Development of monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for lasalocid and semduramicin].

Monoclonal antibodies (MAbs) against lasalocid and semduramicin were prepared using keyhole limpet hemocyanin conjugates for the immunization of mice. With these MAbs, we developed quantitative enzyme-linked immunosorbent assay (ELISA) methods for lasalocid and semduramicin. The ELISAs were quantitative in the ranges of 0.1-50 ng/mL for lasalocid and 0.05-12.5 ng/mL for semduramicin, and showed 50% inhibition concentrations of 1.2 ng/mL for lasalocid and 0.5 ng/mL for semduramicin. The coefficient of variations (CV%) of lasalocid were 0.3-4.4% for intra-assay and 0.5-5.1% for inter-assay and those of semduramicin were 0.1-4.6% for intra-assay and 0.3-5.2% for inter-assay. The detection limits for lasalocid and semduramicin were 10 ng/g and 5 ng/g in chicken liver and muscle, respectively. Based on the immunochromatographic method, rapid test kits for lasalocid and semduramicin were also developed. With these kits, the detection limits of lasalocid were 50 ng/mL for standard solution and 125 ng/g for chicken muscle, and those of semduramicin were 10 ng/mL for standard solution and 100 ng/g for chicken muscle.

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices.

Enrofloxacin has been increasingly used in veterinary medicine to treat microbial infections. A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography (HPLC) is sensitive but labor-intensive. This paper reports an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) and the development of a rapid test kit based on immunochromatography. The detection limits using the ELISA were 10 ppb for chicken liver and muscle, and 1 ppb for cattle milk, respectively. The mean recovery values were 77.3-96.0% for chicken liver, 72.4-92.0% for chicken muscle and 84.0-99.0% for cattle milk. The detection limits using the kit were ca. 100 ppb for chicken muscle and ca. 10 ppb for cattle milk, respectively. All ELISA results for assay of chicken liver, chicken muscle and cattle milk were confirmed using HPLC which is used as the routine assay. The HPLC (x) and ELISA (y) results showed close correlation for chicken liver (y = 8.7 + 0.85x, r2 = 0.99, n = 25), chicken muscle (y = -3.9 + 0.94x, r2 = 0.98, n = 25) and cattle milk (y = 18.4 + 0.92x, r2 = 0.99, n = 25).