Proteolysis of Vaginally Administered Bovine Lactoferrin: Clearance, Inter-Subject Variability, and Implications for Clinical Dosing

Abstract

This report describes proteolytic fragmentation and clearance of bovine lactoferrin (bLF) upon intravaginal administration in premenopausal women. Solid dose tablet formulations (MTbLF) progressed through 3 phases, Pre-dissolution, Dissolution, and Washout over a 30-hour time course. Tablets dissolved slowly, replenishing intact 80 kDa bLF in vaginal fluid (VF) as proteolysis occurred. bLF was initially cleaved approximately in half between its N- and C-lobes, then degraded into sub-fragments and small peptides. The extent of proteolysis was approximately 10–20% and concentrations of both 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout and not proteolysis was the main clearance mechanism. Polyacrylamide gels, western blots, and HPLC analysis demonstrated the N-lobe 37 kDa fragment and C-lobe 43 kDa fragment were common to all subjects. These fragments possessed full sets of iron-ligand amino acids, providing iron sequestering activity in addition to that from intact bLF. Experiments with protease inhibitors in ex vivo VF digests suggested an acid protease was at least partially responsible for bLF cleavage. However, digestion with commercial pepsin or in vivo in the human stomach, demonstrated distinctly different patterns of fragments compared to vaginal proteolysis. Furthermore, the 3.1 kDa antimicrobial peptide lactoferricin B was not detected in VF. This suggests pepsin-like acid proteases are not responsible for vaginal proteolysis of bLF. Despite this proteolysis, these results support bLF as a nutritional-immunity treatment for bacterial vaginosis or other vaginal conditions by maintaining an iron-depleted environment favoring lactobacilli over pathogenic species.