Electronic Supplemental Material
Modelling the Sitagliptin Effect on Dipeptidyl Peptidase-4 Activity in Adults with
Haematological
Malignancies
After
Umbilical
Cord
Blood
Haematopoietic
Cell
TransplantationAuthors:
Nieves Vélez de Mendizábal*1,2, Robert M. Strother1,3, Sherif S. Farag4, Hal E. Broxmeyer5,
Steven Messina-Graham5, Shripad D. Chitnis1,2, Robert R. Bies 1, 2
Affiliations:
1
Division of Clinical Pharmacology, Department of Medicine, Indiana University School of
Medicine, Indianapolis, IN, USA
2
Indiana Clinical and Translational Sciences Institute (CTSI), Indianapolis, IN, USA
3
Christchurch Hospital, Christchurch, New Zealand
4
Division of Hematology-Oncology, Department of Medicine, Indiana University School of
Medicine, Indianapolis, IN, USA
5
Department of Microbiology and Immunology, Indiana University School of Medicine,
Indianapolis, IN, USA
*Corresponding author:
Division of Clinical Pharmacology, Department of Medicine, Indiana University School of
Medicine, 1001 W. 10th Street W7138, Indianapolis, IN 46202, USA.
nvelezde@iu.edu
Running Title: Modelling Sitagliptin Effect on DPP4 Activity After UCB HCT
Bio-analytical assay
Sitagliptin was quantified in serum by internal standardization, liquid-liquid extraction and HPLCMS/MS (API 3200, Applied Biosystems, Toronto, Canada) using selected reaction monitoring with turboionspray interface in positive ion mode. The Q1/Q3’s for sitagliptin and fluoxetine, the internal standard,
were 408/235 and 310/148, respectively. The lower limit of quantification for sitagliptin is 0.1 ng/ml
using 0.1 mL serum. Plasma DPP4 activity was assayed using the DPPIV-Glo Protease Assay (Promega,
Madison, WI), according to the manufacturer’s instructions. The DPPIV-Glo Protease Assay
is a
luminescence based assay that measures DPPIV activity and has been shown to be the most sensitive of
the DPPIV assays [1]. Briefly, 50 μl of plasma was incubated in 96 well plates with 50 μl of a mixture
containing the DPP4 substrate Gly-Pro-aminoluciferin, luciferase, and assay buffer optimized for this
assay. Assay plates were incubated at 37 °C for 30 min, and DPP4 enzyme activity, assayed by luciferase
activity, was measured using an LMAX luminometer (Molecular Devices, Sunnyvale, CA, USA).
Quantification of DPP4 levels in patient plasma samples was not a validated method. However, the
Promega DPPIV-GLO enzyme activity assay was originally developed to detect DPP4 activity in human
serum and plasma. Thus, to detect DPP4 enzyme activity in human plasma with and without the DPP4
inhibitor, Diprotin A (DPA), six healthy human plasma samples were assayed with and without DPA.
DPA treated samples produced an average 7-fold lower enzyme activity versus non-treated plasma
samples. Therefore, the DPP-IV GLO assay was determined to be a feasible method of detecting the
inhibition of DPP4 in human plasma.
1.
Matheeussen V, Lambeir AM, Jungraithmayr W, Gomez N, Mc Entee K, Van der Veken P, et al.
Method comparison of dipeptidyl peptidase IV activity assays and their application in biological samples
containing reversible inhibitors. Clin Chim Acta. 2012 Feb 18;413(3-4):456-62.
Fig. S1 Sitagliptin (PK) model predictions. Dots are patient observations and black lines are model individual predictions.
Fig. S2 DPP4 (PD) model predictions. Dots are patient observations and black lines are model individual predictions.
Fig. S3 Changes in pharmacokinetic–pharmacodynamic descriptors, depending on the number
of doses per day. One thousand individuals were simulated per schedule. The simulated doses
were 200 mg once daily (OD), twice daily (BID), three times daily (TID) and four times daily
(QID). a Two pharmacokinetic descriptors were calculated: the maximum plasma drug
concentration from 0 to 24 h [Cmax(0–24h)] and the area under the plasma drug concentration–time
curve from 0 to 24 h [AUC0–24h]. b Two pharmacodynamic descriptors were calculated: the
Nadir0–24h and the AUC0–24h. The shaded areas correspond to the 90 % prediction intervals for
the pharmacokinetic descriptors (grey) and the pharmacodynamic descriptors (blue). The dashed
lines represent the medians. The black squares indicate the median values for the reference
dosage of 200 mg OD. The red circles correspond to twice the reference dose - 200 mg BID; the
values in red are the corresponding increments/decrements with respect to the reference dose
(black squares). RLU relative light units.