Clinical pharmacokinetic assessment of an anti-MAdCAM monoclonal antibody therapeutic by LC-MS/MS.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study [Vermeire, S. et al. Gut2011, 60 (8), 1068-1075]. The assay to measure free PF-00547,659 used immuno-enrichment with a biotinylated anti-idiotypic antibody and streptavidin magnetic beads. The total assay used enrichment by protein G magnetic beads. Following elution of PF-00547,659 from the beads, addition of an extended sequence stable isotope labeled peptide and trypsin digestion, a proteotypic peptide derived from the CDR region of the light chain of PF-00547,659 was quantified by LC-MS/MS. The free assay had a calibration range from 7.03 ng/mL to 450 ng/mL. The assay was precise and accurate with interbatch imprecision <16.5%, and interbatch inaccuracy <13.7% at all concentrations investigated during assay qualification. Results from LC-MS/MS methodologies are compared with historical immunoassay data originally acquired during the course of the clinical study. PK parameter estimates were highly correlated between the two analytical approaches. This work provides precedence that immunoaffinity LC-MS/MS can effectively be used to measure the serum concentrations of mAb therapeutics in clinical studies.

Label-free detection of differential protein expression by LC/MALDI mass spectrometry.

Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.

Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir.

Antagonists of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-1-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for 10 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 51 of whom experienced an HIV RNA reduction from baseline of >1 log(10) copies/ml, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day 11. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope (Env) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.

Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1.

We assessed the efficacy and safety of 10-d monotherapy with the orally administered CCR5 antagonist maraviroc in 63 HIV-1-positive individuals prescreened for the absence of CXCR4-using virus. Maximum reduction in viral load occurred at a median of 10-15 d, with a mean reduction of >or=1.6 log(10) copies/ml at all twice daily doses >or=100 mg. These results provide proof of concept that CCR5 antagonism is a viable antiretroviral therapeutic approach.

Increased skin collagen extractability and proportions of collagen type III are not normalized after 6 months healing of human excisional wounds.

In an attempt to identify potential staging markers of effective healing, changes in connective tissue properties were measured in a human skin excisional wound healing model in which tissue was re-excised at intervals up to 6 months after injury. The proportion of collagen III relative to collagen I increased significantly (p<0.001) up to 6 weeks after initial injury and remained elevated up to 6 months, at which time the proportion of collagen III was 70% above baseline values. Extractability of biopsy tissue collagen by pepsin increased significantly throughout the study (baseline, 32.8+/-6.8%; 6 months, 89.1+/-8.9%), with inverse changes in the mature skin cross-link, histidinohydroxylysinonorleucine (baseline, 1.18+/-0.11 mol/mol collagen; 6 months, 0.27+/-0.09 mol/mol collagen). Pyridinoline content increased over the period of the study, although remaining at relatively low concentrations (baseline, 0.037+/-0.011; 6 months, 0.063+/-0.014 mol/mol collagen), and the pyridinoline/deoxypyridinoline ratio was significantly higher (baseline, 3.5+/-0.6; 6 months, 10.3+/-2.2). Elastin content, measured as desmosine cross-links, decreased significantly in the first 3 weeks and continued to decline over the period of study. Overall, the data suggest that remodeling of the wound tissue continues at least up to 6 months after injury. The close inverse correlation between histidinohydroxylysinonorleucine concentrations and extractability by pepsin (r2=0.89, p<0.0001) suggests a causal relationship, consistent with the likely effects of a substantial network of mature, inter-helical bonds in collagen.