The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.

Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.

Stress-strength reliability estimation involving paired observation with ties using bivariate exponentiated half-logistic model.

This paper deals with the problem of maximum likelihood and Bayesian estimation of stress-strength reliability involving paired observation with ties using bivariate exponentiated half-logistic distribution. This problem is of importance because in some real applications the strength of the component is highly dependent on the stress experienced by it. A bivariate extension of exponentiated half-logistic is discussed and an expression for the stress-strength reliability is obtained. This model is also useful to analyse data having the unusual feature of having a number of pairs with tied scores, even when the scores are continuous. The maximum likelihood estimate and interval estimate of the stress-strength reliability has been developed. The Bayes estimates of the stress-strength reliability under squared error loss function are obtained using importance sampling technique. Simulation studies are conducted to evaluate the performance of maximum likelihood and Bayes estimates. Two real-life data sets are analysed to numerically illustrate the usefulness of the developed method.

Dampening of the bone formation response following repeat dosing with sclerostin antibody in mice is associated with up-regulation of Wnt antagonists.

Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5-4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.

Structural characterization of a novel derivative of myricetin from Mimosa pudica as an anti-proliferative agent for the treatment of cancer.

The study was initiated to determine the anticancer activity of a novel compound isolated from the plant Mimosa pudica. The structure of the compound was identified as a derivative of myricetin having alkyl, hydroxy alkyl and methyl substitutions on the basis of spectral evidences (UV-vis, FT-IR, 1H NMR and Mass spectra). The isolated compound was interpreted as 2-(2',6'-dimethyl-3',4',5'-alkyl or hydroxy alkyl substituted phenyl)-3-oxy-(alkyl or hydoxy alkyl)- 5,7-dihydroxy-chromen-4-one. In vitro evaluation of anticancer activity against human lung adenocarcinoma cell line (A549) and human erythroleukemic cell line (K562) were conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In vivo anticancer activity was determined against Dalton's Ascites Lymphoma (DAL) in Swiss albino mice. The mice were treated with intraperitoneal administration of the compound at 25mg/kg and 100mg/kg body weight and were compared with the normal, DAL control and standard drug cyclophosphamide treated groups. The histology revealed that the compound could protect the cellular architecture of liver and kidney. The results from the in vitro, in vivo and histological examinations confirmed the ethnopharmacological significance of the isolated compound and could be considered further for the development of an effective drug against cancer.

Safety and pharmacokinetics of olokizumab, an anti-IL-6 monoclonal antibody, administered to healthy male volunteers: A randomized phase I study.

Interleukin-6 (IL-6) is implicated in the pathophysiology of several inflammatory conditions. Olokizumab, a humanized anti-IL-6 monoclonal antibody, selectively blocks the final assembly of the IL-6 signaling complex. A randomized, double-blind, placebo-controlled, phase I dose-escalation study assessed the safety and tolerability of escalating single doses of olokizumab administered intravenously (iv) or subcutaneously (sc) to 67 healthy male volunteers. The pharmacokinetics, pharmacodynamics and immunogenicity of olokizumab were also assessed. Olokizumab was tolerated at doses up to 3.0 mg/kg sc and 10.0 mg/kg iv; the maximum tolerated dose was not reached. No serious adverse events or withdrawals as a result of treatment-emergent adverse events were reported. Pharmacokinetic analysis showed that both maximum serum concentration and area under the concentration-time curve increased linearly with increasing dose. Mean terminal half-life was 31.5 days (standard deviation 12.4 days). The bioavailability of the sc doses ranged from 84.2% to 92.5%. Rapid decreases in C-reactive protein concentrations were observed, with no dose dependency.