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. Tablet formulations (MTbLF) containing 300 mg of bLF progressed through three phases: Pre-Dissolution, Dissolution, and Washout, over a 30-h 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 less than 10–20% across multiple subjects. Concentrations of both intact 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout not proteolysis was the main clearance mechanism. Concentrations of intact and/or nicked 80 kDa bLF peaked between 4 and 8 h after administration and remained above 5 mg/mL for approximately 24 h. Experiments with protease inhibitors in ex vivo VF digests suggested an aspartyl 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 aspartyl proteases are not responsible for vaginal proteolysis of bLF.

Lactoferrin, a Great Wall of host-defence?

“…The use of Lf should also be classified as accurately as possible: (a) As a nutraceutical food supplement to regulate immunity, iron storage, transport and release for special populations (neonates, infants, toddlers, children, adults, etc. ); (b) As a feed additive for livestock, poultry and fish, improving the growth performance and regulating intestinal flora (although at current prices the costs for such applications would be too high); (c) As a clinical drug candidate for the treatment of human diseases such as cancer or inflammatory and infectious diseases (Vogel 2012 ; Chen et al 2022 ; Hopp et al 2022 ; Nopia et al 2022 ). As examples of potential future clinical applications of Lf, here Chen et al ( 2022 ) discuss a role for Lf during atherosclerosis, while Nopia et al ( 2022 ) describe an interesting new albumin-hLf fusion protein that has improved pharmacokinetic properties for clinical applications, particularly in the area of cancer.…”

“…As examples of potential future clinical applications of Lf, here Chen et al ( 2022 ) discuss a role for Lf during atherosclerosis, while Nopia et al ( 2022 ) describe an interesting new albumin-hLf fusion protein that has improved pharmacokinetic properties for clinical applications, particularly in the area of cancer. Finally, Hopp et al ( 2022 ) delineate very careful proteolysis and stability studies describing degradation of bLf in vaginal fluids, a bodily location where this protein may have strong antibacterial effects, through its potent iron-binding properties.…”