3 research outputs found
In Vitro Protein Binding of Liraglutide in Human Plasma Determined by Reiterated Stepwise Equilibrium Dialysis
ABSTRACTLiraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (104pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2882–2888, 201
Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1
High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats
Background: Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective: To study the effects of VWF on
FVIII clearance in rats with endogenous VWF. Methods:
Anatomical and hepatocellular distribution studies were
performed in rats following intravenous administration of
glycoiodinated recombinant FVIII (rFVIII) and a FVIII
variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct
liver cell populations. The role of VWF binding was also
studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding
VWF fragment. Results: The liver was the predominant
organ of rFVIII distribution, and a radioactivity peak was
also observed in the intestines, suggesting FVIII secretion
to the bile by hepatocytes. In the liver, ~60% of recovered
radioactivity was associated with hepatocytes, 32% with
liver sinusoidal endothelial cells (LSECs), and 9% with
Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-
fold more radioactivity was associated with LSECs than
with hepatocytes. The importance of hepatocytes and
LSECs was confirmed by immunohistochemical staining;
strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was
observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F,
and the presence of the VWF fragment, D’D3A1, did not
change the FVIII staining pattern in intact livers. Conclusions: The present data support FVIII clearance via the
liver, with hepatocytes and LSECs playing a key role.
High-affinity VWF binding did not alter the anatomical or
hepatocellular distribution of FVIII