Clinical trial: impact of prior infliximab therapy on the clinical response to certolizumab pegol maintenance therapy for Crohn's disease.

BACKGROUND: Certolizumab pegol (CZP) is an effective therapy for Crohn's disease refractory to aminosalicylates, corticosteroids and immunosuppressants. In PRECiSE 2, patients were also eligible for enrolment if prior infliximab therapy was terminated due to loss of response. AIM: To evaluate prior infliximab therapy on sustained response and remission to CZP for Crohn's disease. METHODS: PRECiSE 2 were was analysed for predictors of sustained response and remission. Covariates included prior infliximab therapy, and baseline Crohn's Disease Activity Index (CDAI). RESULTS: Week 26 response (> or =100-point decrease from baseline CDAI) and remission (CDAI < or = 150) were greater with CZP vs. placebo in patients previously receiving infliximab (response: 44.2% vs. 25.5%, P = 0.018; remission: 32.7% vs. 13.7, P = 0.008) and infliximab-naïve patients (response: 68.7% vs. 39.6%, P < 0.001; remission: 52.8% vs. 33.3%, P < 0.001). Prior infliximab use was the only independent predictor of week 26 response and remission in both groups [response OR(CZP vs. placebo) = 3.06 (95% CI: 1.21-7.77); remission OR(CZP vs. placebo) = 4.22 (95% CI: 1.45-12.28)]. Adverse events were similar for both groups. CONCLUSIONS: Certolizumab pegol is an effective maintenance therapy in Crohn's disease regardless of prior infliximab use. Efficacy is higher in patients receiving CZP therapy as a first-line biologic, but approximately 50% of infliximab-experienced patients benefited from second-line CZP therapy.

Effect of Brazilian, Indian, Siberian, Asian, and North American ginseng on serum digoxin measurement by immunoassays and binding of digoxin-like immunoreactive components of ginseng with Fab fragment of antidigoxin antibody (Digibind).

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.

Ticlopidine enhances the platelet inhibitory capacity of abciximab in vitro.

Ticlopidine and abciximab are two antiplatelet agents that are frequently administered during percutaneous coronary interventions. Although they have different mechanisms of action and pharmacological profiles, the two agents are often concomitantly used in complicated stent placements. The purpose of the study was to evaluate the effect of ticlopidine therapy on the capacity of abciximab to inhibit platelet aggregation, in vitro. Blood samples from 13 ticlopidine-treated stent placement patients and 8 patients undergoing PTCA who did not receive ticlopidine were obtained prior to, 12-36 hours and 7-10 days after initiating ticlopidine treatment. For each patient, the minimal ADP and the thrombin receptor activating peptide (TRAP) concentrations that elicited maximal platelet aggregation responses at baseline were used to measure the extent of platelet aggregation and the abciximab concentration that gave a 50% decrease in aggregation (IC(50)) for both agonists at the three time points. The ticlopidine group baseline and 12-36 hour mean ADP aggregation responses were equivalent, but decreased by 34% (P = 0.009) at 7-10 days. The control group ADP and TRAP, as well as the ticlopidine group TRAP aggregation responses, were equivalent at all time points. The ticlopidine group baseline and 12-36 hour abciximab IC(50) values for ADP were comparable (1.58 +/- 1.1 ng/mL vs. 1.23 +/- 0.5 ng/mL; P = 0.266), but decreased to 1.00 +/- 0.6 ng/mL (36%; P = 0.004) at 7-10 days. In contrast, the abciximab IC(50) for TRAP increased from 1.48 +/- 1.0 ng/mL to 1.85 +/- 1.1 ng/mL (25%; P = 0.033) at 12-36 hours, but returned to baseline at 7-10 days (1.40 +/- 0.8; P = 0.975). The control group IC(50) abciximab values for ADP and TRAP were comparable throughout the monitoring period. The results demonstrate that ticlopidine elicits subtle potentiation of the platelet-inhibitory capacity of abciximab to the agonist ADP, but not TRAP, at 1 week after initiation of treatment.

Characterization of ethanol-sensitive and insensitive fibroblast cell lines expressing human L1.

Ethanol inhibits L1-mediated cell-cell adhesion in fibroblast cell lines stably transfected with human L1. Here we show that this action of ethanol is present in only a subset of transfected NIH/3T3 and L cell clonal cell lines. All L1-expressing cell lines had higher levels of cell adhesion than cell lines transfected with empty vector. In all ethanol-sensitive cell lines, L1-mediated adhesion was inhibited by ethanol (IC50 5-10 mM), 2 mM butanol, but not 5 mM pentanol. In contrast, ethanol-insensitive cell lines were not inhibited by up to 200 mM ethanol, 2 mM butanol, or 5 mM pentanol. Ethanol sensitivity or insensitivity was a stable property of each cell line and was not associated with differences in electrophoretic mobility, abundance, or cell surface localization of L1. Fab fragments prepared from anti-L1 polyclonal antisera inhibited cell adhesion only in the ethanol-sensitive cell lines. These data suggest that L1 may exist in an alcohol-sensitive or an alcohol-insensitive state that may be governed by host cell factors.

Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.

Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.

Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.

The effects of histidine residue modification on the immune precipitating ability of rabbit IgG.

Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 microM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab')2. Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes. Carbethoxylation of two histidine residues per IgG molecule had little effect on rates of formation of insoluble immune complexes, but these rates were markedly decreased in samples of IgG with four to five histidines per molecule modified. There were parallel decreases in the protein A-binding activity and in the rates of formation of insoluble immune complexes in IgG treated with increasing concentrations of DEPC. The presence of complement protein C1q restored the rates of formation of insoluble immune complexes of DEPC-treated IgG.

A method for rapidly assessing and refining simple solvent treatments in molecular modelling. Example studies on the antigen-combining loop H2 from FAB fragment McPC603.

Different simple solvent models have been implemented in an extended simulated annealing process (ESAP), developed by Higo et al. [(1992) Biopolymers, 32, 33-43] and proven to be able to predict ab initio the conformation of the antigen-combining loop H2 from FAB McPC603. The rationale used here provides a useful new method for testing solvent models in general. The different solvent models comprise a high dielectric constant, a screened coulomb potential, a dummy water model and a statistical continuum treatment of the solvent effect in which the reaction field and the solvent accessible area is accounted for. To assess the effect of the solvent, we tested the ability of simulations to retain the experimental conformation of loop H2. We compared the different structures obtained at the end of the annealing process in terms of root mean square deviation (r.m.s.d.), for both the backbone and for all atoms, root mean square (r.m.s.) fluctuation, solvent accessible surface area (ASA), hydrogen bonding network and phi-psi plot distribution. The relationship between the r.m.s.d. and the internal energy of a structure is also evaluated in terms of precision and another possible method for obtaining the best conformation is discussed. The accuracy of modelling the coarse effect of the solvent and the similarities of the resulting structures with respect to the X-ray reference structure are examined. The possible choice of one of these solvent models in the structure determination of an unknown loop structure is discussed.