Interaction of Mycophenolic Acid and Pantoprazole: A Pharmacokinetic Crossover Study

177 Pages·2017·8.939 MB·English

by  Olesja Rissling (auth.)

Olesja Rissling
Interaction of
Mycophenolic Acid
and Pantoprazole
A Pharmacokinetic Crossover Study
Interaction of Mycophenolic Acid
and Pantoprazole
Olesja Rissling
Interaction of
Mycophenolic Acid
and Pantoprazole
A Pharmacokinetic Crossover Study
Olesja Rissling
Berlin, Germany
Dissertation Freie Universität Berlin, Germany, 2017
D188
ISBN 978-3-658-19888-6 ISBN 978-3-658-19889-3 (eBook)
https://doi.org/10.1007/978-3-658-19889-3
Library of Congress Control Number: 2017955535
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Acknowledgements

First, I would like to express my deepest appreciation to my supervisor Professor
Budde for the opportunity to do my PhD work in his research group and for his
immense support, advice and the valuable discussions. Also, I am thankful for the
research and learning opportunities he offered to me.
I wish to express my sincere thanks to Professor Melzig for accepting me as
an external PhD student at the Institute of Pharmacy and for his continuous and
great support and advice in all questions regarding my PhD thesis.
I would like to thank all members of the laboratory team (Christine, Kathrin,
Juan, Julie, Laili, Luisa, Marcel, Marco, Mareen, Michael, Petra, Pia) for the cre-
ative work atmosphere and the lunch breaks at the canteen. Especially, I would
like to thank Marco and Pia who supported me in the measurement of the samples,
especially for the clinical study. Also, I am thankful for the support and advice in
all questions regarding analytical aspects of my work and technical issues of the
HPLCs. For the later one, I also would like to thank Marcus who usually managed
to make both systems work again.
This thesis would not have been possible without the support of the people
working at the Charité transplant center: I am thankful to all people including doc-
tors, study nurses and medical students who were involved in the patient recruit-
ment, study organization or the blood collection.
I would like to thank Dr. Bauer for his advice in regard to bio-analytical ques-
tions. I am thankful for the cooperation with Dr. Shipkova and her team from the
Universitätsklinikum Stuttgart and the opportunity to perform method comparison
and the guidance in analytical related questions.
I would like to thank Marcel who introduced me to database query which
made my work a lot easier and Danilo for the additional support in data manage-
ment. Also, I thank Dr. Kastner for the proofreading of my thesis.
I would like to express my deepest gratitude towards my parents who always
believed in me and their patience and encouragement during the research period.
I thank my friends for the support and their patience with me whenever I was
too occupied with my research and had only little time for them.

Olesja Rissling
Summary

This thesis is about a pharmacokinetic (PK) interaction (IA) of pantoprazole
(PAN) and the immunosuppressive drug mycophenolic acid (MPA) in renal trans-
plant (tx) recipients. MPA is prescribed for acute rejection prevention with cal-
cineurin inhibitors (tacrolimus or cyclosporine = Cya) ± glucocorticoids. The
pharmacodynamic (PD) response is caused by inhibition of inosine-mono-phos-
phate-dehydrogenase (IMPDH) which is an important enzyme in proliferating
lymphocytes. There are two MPA formulations: the prodrug mycophenolate mo-
fetil (MMF) and the enteric-coated mycophenolate sodium (EC-MPS). Pantopra-
zole (PAN) is prescribed as prevention therapy against upper gastrointestinal side
effects.
To evaluate a potential interaction of PAN with MMF and EC-MPS, a 4-
sequence, 4-treatment, open-labeled, randomized crossover study was performed
in stable renal tx patients (pts) on maintenance therapy with Cya ± steroids. In
addition to MPA standard dose (daily dose: 2000 mg MMF), two lower doses were
allowed. Every patient took MMF±PAN and EC-MPS±PAN. PK/PD profiles were
obtained for MPA, the major metabolite Mycophenolic acid 7-glucuronide
(MPAG), which contributes to MPA exposure by enterohepatic recirculation, and
for IMPDH activity from 0-12 hours (h) after drug intake for every treatment op-
tion. Non-compartmental and bioequivalence analysis was performed. Addition-
ally, the IC (drug concentration to inhibit 50% of the enzyme activity) was cal-
50
culated.
For quantification, the samples were measured by an HPLC-UV-based iso-
cratic assay. The linearity range of MPA was 0.5-40.0 µg/ml, and that of MPAG
5.0-350.0 µg/ml. The method was adjusted to PK clinical studies, validated ac-
cording to recent bioanalytical guidelines and compared to a published reference
method.
In summary, PK/PD profiles of 19 pts were available for analysis. A decrease
in dose-normalized MPA maximum concentration (dC ) and exposure
max
(dAUC ) of up to 22.28 % and 10.86 % was observed after MMF+PAN versus
0-12h
MMF intake, respectively. After EC-MPS+PAN in comparison to EC-MPS treat-
ment, an increase in dC of +16.80 %, and an increase in dAUC of +7.52 %
max 0-12h
were found. Bioequivalence was reached for dAUC after MMF and EC-MPS
0-12h
intake but not after PAN administration with both formulations in reference to
VIII Summary
MMF/EC-MPS. T was comparable for MMF+PAN versus MMF treatment
max
(median 1 h, range: 0-6 h). After EC-MPS+PAN intake, t (2 h, 0.5-10 h) was
max
reached earlier than after EC-MPS treatment (3 h, 1.5-12 h) alone (p=0.030). No
impact of PAN on MPAG pharmacokinetics was found. PD parameters like area
under the enzyme activity curve (AEC ) and average activity (A ) were bio-
0-12h avg
equivalent. The estimated IC values ranged between 3.94-4.40 µg/ml which is
50
lower than the observed MPA C (>11.92±6.08 µg/ml), the primary PK param-
max
eter which is affected by PAN.
Our results suggest that the observed interaction of PAN with MPA does not
compromise the immunosuppressive effect to a clinical meaningful extent.
Zusammenfassung

In dieser Arbeit wurde eine mögliche pharmakokinetische (PK) Interaktion von
Pantoprazol (PAN) und Mycophenolsäure (MPA) bei nierentransplantierten Pati-
enten (Pts) untersucht. MPA ist ein Immunsuppressivum, dessen pharmakodyna-
mische Wirkung (PD) durch die Inhibierung der Inosinmonophosphatdehydro-
genase (IMPDH) in proliferierenden Lymphozyten verursacht wird. Es wird in ei-
ner Kombinationstherapie mit Calcineurininhibitoren (Tacrolimus oder Ciclospo-
rin) ± Glucocorticoiden eingesetzt. Est sind 2 Formulierungen auf dem Markt: Das
Prodrug Mycophenolat mofetil (MMF) und die magensaftresistente Form EC-
MPS. Pantoprazol wird zur Prophylaxe gegen gastrointestinale Nebenwirkungen,
die bei einer immunsuppressiven Therapie auftreten können, verschrieben.
Eine randomisierte, offene, 4-Sequenzen, 4-Behandlungsarme umfassende
klinische Studie wurde durchgeführt, um eine mögliche Einflussnahme von Pan-
toprazol in Kombination mit MMF und EC-MPS auf die PK und PD von MPA zu
untersuchen. Es waren neben der Standarddosis (tägliche Dosis: 2000 mg MMF)
2 niedrigere Dosierungen erlaubt. Die Patienten waren auf Ciclosporin ± Glu-
cocorticoiden eingestellt. Es wurde ein vollständiges PK/PD Profil von MPA, dem
Hauptmetaboliten MPAG (der auf Grund des enterohepatischen Kreislaufs zur
MPA AUC = Fläche unter der Kurve beiträgt) und IMPDH in einem Zeitraum von
0-12 Stunden nach Medikationseinnahme erstellt. Zusätzlich wurde die IC be-
50
rechnet (Konzentration benötigt, um 50% des Enzyms zu inhibieren).
Für die simultane Quantifizierung von MPA und MPAG wurde eine isokra-
tische HPLC-UV Methode verwendet. Für MPA betrug der Konzentrationsbereich
0.5-40.0 µg/ml und für MPAG 5.0-350.0 µg/ml. Die Methode wurde für pharma-
kokinetische Studien angepasst und gemäß den aktuellen bioanalytischen Richtli-
nien validiert.
PK/PD-Profile standen von 19 Patienten für eine Analyse zur Verfügung.
Nach Einnahme von MMF+PAN wurde eine Verringerung von 22.28% der Ma-
ximalkonzentration (C ) sowie von 10.86% der AUC von MPA im Vergleich
max 0-12h
zu MMF gefunden. Im Gegensatz dazu war C um 16.80% und die AUC um
max 0-12h
7.52% nach Einnahme von EC-MPS+PAN versus EC-MPS erhöht. Die dosis-nor-
mierte AUC war für MMF und EC-MPS bioäquivalent. Es konnte keine
0-12h
Bioäquivalenz für PAN mit Einnahme von MMF und EC-MPS nachgewiesen wer-
den. T betrug im Median 1 h für MMF±PAN. Für EC-MPS+PAN betrug t
max max
X Zusammenfassung
2 h und für EC-MPS 3 h (p=0.030). PAN zeigte keinen Einfluss auf die Pharma-
kokinetik von MPAG. PD-Parameter, wie mittlere Aktivität (A ) und die
avg
AEC (Fläche unter der Aktivitätskurve), waren ebenfalls bioäquivalent. Die
0-12h
berechneten IC lagen zwischen 3.94-4.40 µg/ml, was deutlich unter dem nied-
50
rigsten beobachteten (dosis-normiertem) C nach MMF+PAN Einnahme
max
(11.92±6.08 µg/ml) lag.
Da die Interaktion von Pantoprazol sich in einer Verringerung bzw. Erhöhung
von C zeigte, lässt dies die Vermutung zu, dass die beobachtete Interaktion kei-
max
nen klinisch relevanten Effekt auf die immunsuppressive Wirkung von MPA hat.
List of Contents

1  Introduction .......................................................................................... 1 
1.1  Mycophenolic Acid ................................................................................ 1 
1.1.1  Chemical Properties ................................................................................ 2 
1.2  Pharmacology of MPA ........................................................................... 3 
1.2.1  Pharmacokinetics of MPA ...................................................................... 3 
1.2.2  Pharmacokinetic Interactions of MPA .................................................... 8 
1.2.3  Clinical Aspects of MPA Pharmacokinetics ......................................... 10 
1.2.4  Pharmacodynamics of MPA ................................................................. 13 
1.3  Analytical Aspects of MPA Measurement and Its Metabolites ............ 14 
1.3.1  Sample Preparation ............................................................................... 15 
1.3.2  Chromatography ................................................................................... 15 
1.3.3  Stability ................................................................................................. 16 
1.3.4  Assays for Determination of MPA and Its Metabolites ........................ 16 
1.4  Pharmacology of Pantoprazole ............................................................. 18 
2  Research Objectives............................................................................ 21 
3  Materials .............................................................................................. 23 
3.1  Chemicals ............................................................................................. 23 
3.2  Kits ....................................................................................................... 23 
3.3  Consumable Material ............................................................................ 23 
3.4  Hardware and Software ........................................................................ 24 
3.4.1  Hardware .............................................................................................. 24 
3.4.2  Software ................................................................................................ 25 
4  Methods ............................................................................................... 27 
4.1  Quantification of MPA and MPAG in Human Plasma ......................... 27 
4.1.1  Sample Preparation ............................................................................... 27 
4.2  Measurement of IMPDH Activity ........................................................ 28 
4.3  Validation Conditions ........................................................................... 29 
4.3.1  Preparation of Calibration and Quality Control Standards ................... 30 
4.3.2  Selectivity ............................................................................................. 31 
4.3.3  Limit of Detection ................................................................................ 32 
4.3.4  LLOQ ................................................................................................... 32