Pharmacokinetic interaction between mefloquine and ritonavir in healthy volunteers.

AIMS: To evaluate the pharmacokinetic interaction between ritonavir and mefloquine. METHODS: Healthy volunteers participated in two separate, nonfasted, three-treatment, three-period, longitudinal pharmacokinetic studies. Study 1 (12 completed): ritonavir 200 mg twice daily for 7 days, 7 day washout, mefloquine 250 mg once daily for 3 days then once weekly for 4 weeks, ritonavir restarted for 7 days simultaneously with the last mefloquine dose. Study 2 (11 completed): ritonavir 200 mg single dose, mefloquine 250 mg once daily for 3 days then once weekly for 2 weeks, ritonavir single dose repeated 2 days after the last mefloquine dose. Erythromycin breath test (ERMBT) was administered with and without drug treatments in study 2. RESULTS: Study 1: Ritonavir caused less than 7% changes with high precision (90% CIs: -12% to 11%) in overall plasma exposure (AUC(0,168 h)) and peak concentration (Cmax) of mefloquine, its two enantiomers, and carboxylic acid metabolite, and in the metabolite/mefloquine and enantiomeric AUC ratios. Mefloquine significantly decreased steady-state ritonavir plasma AUC(0,12 h) by 31%, Cmax by 36%, and predose levels by 43%, and did not affect ritonavir binding to plasma proteins. Study 2: Mefloquine did not alter single-dose ritonavir pharmacokinetics. Less than 8% changes in AUC and Cmax were observed with high variability (90%CIs: -26% to 45%). Mefloquine had no effect on the ERMBT whereas ritonavir decreased activity by 98%. CONCLUSIONS: Ritonavir minimally affected mefloquine pharmacokinetics despite strong inhibition of CYP3A4 activity from a single 200 mg dose. Mefloquine had variable effects on ritonavir pharmacokinetics that were not explained by hepatic CYP3A4 activity or ritonavir protein binding.

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

N-terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a possible first step in microfibril assembly.

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.

Processing of the fibrillin-1 carboxyl-terminal domain.

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.

Identification of an elastin cross-linking domain that joins three peptide chains. Possible role in nucleated assembly.

The alignment of elastin molecules in the mature elastic fiber was investigated by purifying and sequencing cross-link-containing peptides generated by proteolytic digestion incompletely cross-linked insoluble elastin. Peptides of interest were purified by reverse phase and size exclusion high performance liquid chromatography and characterized by amino acid analysis and protein sequencing. One peptide, consisting of the cross-linking domain encoded by exon 10, contained a modified lysine residue that had not condensed to form a polyfunctional cross-link. Although this domain contains the characteristic paired lysine residues found in other cross-linking domains of elastin, protein sequence analysis indicated that the first but not the second lysine had been oxidized by lysyl oxidase. This finding suggests that lysine residues in an individual cross-linking domain may not have equal susceptibility to oxidation by lysyl oxidase. In a second peptide, we found that a major cross-linking site in elastin is formed through the association of sequences encoded by exons 10, 19, and 25 and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links. Past structural studies and computer modeling predict that domains 19 and 25 are linked by a desmosine cross-link, while domain 10 bridges domains 19 and 25 through lysinonorleucine cross-links. These findings, together with the high degree of sequence conservation for these three domains, suggest an important function for these regions of the molecule, possibly nucleating the aggregation and polymerization of tropoelastin monomers in the developing elastic fiber.

The cysteine residues in the carboxy terminal domain of tropoelastin form an intrachain disulfide bond that stabilizes a loop structure and positively charged pocket.

Analysis of purified bovine tropoelastin with Ellman's reagent and [14C]iodoacetamide demonstrated that the only two cysteine residues in the molecule form an intrachain disulfide bond. Molecular modeling suggests that the cysteine residues are juxtaposed as the result of a tight turn that produces an antiparallel beta structure. Protruding from the C-terminal end of the turn is the sequence Arg-Lys-Arg-Lys which forms the floor of a positively charged pocket created by the extension of the arginine and lysine side chains on opposite sides of the peptide chain perpendicular to the plane of the turn. The side chain of a conserved lysine residue in the disulfide-bonded loop forms the top of the pocket. This positively charged pocket may define a binding site for acidic microfibrillar proteins that mediate elastic fiber assembly.