1. M. Nöldner ¹, K. Appel², K. Fuchs³ and E. Koch¹
Preclinical investigations of possible drug interactions with WS® 1375, a proprietary dry extract from Rhodiola rosea roots
M. Nöldner ¹, K. Appel², K. Fuchs³ and E. Koch¹
¹ Preclinical Research, Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany
² Vivacell Biotechnologie GmbH, Denzlingen, Germany
³ GenPharmTox BioTech AG, Planegg, Germany
Abstract
Results
Fig. 4
WS® 1375 is the pharmaceutically active ingredient in Vitango, a traditional herbal medicinal product used as adaptogen for the treatment of fatigue, exhaustion, mild anxiety and other stress associated symptoms. The extract is prepared from the roots and rhizomes of Rhodiola rosea L. using 60% ethanol as extraction solvent. The aim of the present investigation was to determine potential drug interaction effects of WS® Drug interactions remain an important issue in clinical prac-tice and in vitro data are necessary to predict the magnitude of possible interfer-ences in humans during the development of new pharmaceuticals. Of the 57 cyto-chrome P450 isoenzymes encoded by the human genome, nearly 15 are involved in the metabolism of drugs and other xenobiotics but only 5 enzymes are responsible for the oxidative metabolism of 95% of all drugs (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). Although CYP450s are distributed widely in many tissues, the liver contains the highest amount of cytochrome enzymes (1). We investigated the inhibitory/stimulatory effects of WS® 1375 in microsomes, prepared from freshly isolated human hepatocytes. To get comprehensive information on the inhibitory effects of the extract, the activity of all five major CYP isoenzymes was measured whereas the possible induction potential of WS® 1375 was investigated only on the inducible isoenzymes CYP1A2 and CYP3A4 measuring the metabolism of isoenzyme-specific marker substrates. Results: The IC50 values for the inhibition of CYP isoenzymes were as follows: 1A2 = 63 µg/ml, 2C9 = 77 µg/ml, 2C19 = 75 µg/ml, 2D6 = 25 µg/ml and 3A4 = 104 µg/ml. In the induction experiments positive controls were performed with reference compounds. A more than 7-fold induction was observed confirming the functional state and the validity of the test system. The extract did not induce the catalytic activity of the cytochrome isoenzymes tested up to a concentration of 100 µg/ml. Conclusion: The IC50 values for all CYP isoenzymes in the inhibition experiments were between 25 µg/ml for CYP2D6 and 104 µg/ml for CYP3A4 and thus far away from clinical relevant plasma concen-trations. These data in combination with the lack of induction potential clearly demonstrates that WS® 1375 is devoid of a clinical relevant interaction potential.
References: 1. Johnson, W.W. (2008) Drug Metabolism Reviews, 40:
Tab. 1: Summary of induction of CYP1A2 (7-ethoxyresorufin-O-deethylation)
The induction of 7-ethoxyresorufin-O-deethylation activity by the model inducer omeprazole (PC) verified the functionality of the test system. Comparison of the test item incubations with the untreated negative control indicated no induction potential of WS® 1375 on CYP1A2 activity in human hepatocytes.
Tab. 2: Summary of induction of CYP3A4 (Testosterone 6ß-hydroxylation)
The induction of testosterone 6ß-hydroxylation activity by the model inducer rifampicin (PC) verified the functionality of the employed test system. Comparison of the test item incubations with the untreated negative control indicated no induction potential of WS® 1375 on CYP3A4 activity in human hepatocytes at any of the tested concentrations.
Tab. 3: Summary of inhibition of CYP IC 50 values
Determination of IC50 for inhibibition of CYP2D6 activity
Fig. 5
Determination of IC50 for inhibition of CYP3A4 activity
Introduction
The medicinal use of Rhodiola was already mentioned by Dioscorides in 77 B.C. in the De Materia Medica. Linnaeus named it Rhodiola rosea referring to the rose-like fragrance of the fresh cut roots.
Rhodiola rosea has been used in folk medicine for centuries in Russia, Scandinavia and other countries. It was employed to increase physical endurance, work produ-ctivity, longevity, resistance to high altitude sickness and to treat fatigue. Between the 18th and 20th century this drug appeared in the scientific literature of Sweden, Norway, France, Germany and other countries. In 1755 the drug was included in the first Swedish Pharmacopoeia.
From the early 1960s extensive investigations on this drug were performed. The main active ingredients are the cinnamoyl glycosides rosavin, rosin and rosarin (the rosavins), thought to be unique to Rhodiola rosea [Ramazanov (2006); Barnes (2007)].
Schwabe´s Rhodiola rosea extract, WS® 1375, is a herbal medicinal product for the relief of mental and physical symptoms of stress and overwork, such as fatigue, exhaustion, burn-out, irritability and tenseness.
In recent publications an induction and/or an inhibition of CYP450 isoenzymes by Rhodiola species was reported [Hellum et al, 2010; Brandin et al, 2007]. Thus, it was the aim of this study to evaluate a possible drug interaction potential of WS® 1375 in human hepatocytes and human hepatic microsomes.
Conclusion
In the present study the inhibitory effects of WS® 1375 upon the catalytic activity of 5 major human hepatic CYP isoenzymes 1A2, 2C9, 2C19, 2D6 and 3A4 was investi-gated with pooled human liver microsomes at 10 concentrations and the IC50 values were calculated.
Test item concentrations from to 250 µg/ml were tested and the IC50 values for all CYP isoenzymes in the inhibition experiments were between 25 µg/ml for CYP2D6 and 104 µg/ml for CYP3A4 and thus far away from clinical relevant plasma concentrations. These data in combination with the lack of an induction potential clearly demonstrates that WS® 1375 is devoid of a clinical relevant interaction potential.
Fig. 1
Determination of IC50 for inhibition of CYP1A2 activity
References
1. Brandin, H.; Viitanen, E.; Myrberg, O.; Arvidsson, A.K.
Effects of Herbal Medicinal Products and Food Supplements on Induction of CYP1A2, CYP3A4 and MDR1 in the Human Colon Carcinoma Cell Line LS180
Phytotherapy Research 21, (2007)
2. Barnes J, Anderson LA, Phillipson JD. Rhodiola. In: Herbal Medicines. Pharmaceutical Press (2007)
Hellum,B.H.; Tosse, A.; Hoybakk, K.; Thomsen, M.; Rohloff, J.;
Georg Nilsen, O.
Planta Medica 76(4): (2010)
Potent in vitro Inhibition of CYP3A4 and P-Glycoprotein by Rhodiola rosea
4. Ramazanov Z. Phytochemistry, Pharmacology and Standardization of Rhodiola rosea Root Extract.
National BioScience Corporation 2006 (Online publication)
Material and Methods
The CYP450 inhibition experiments were performed by BSL Bioservice (Planegg) and the the CYP450 induction experiments were performed by GenPharmTox Bio Tech AG (Martiensried) both in cooperation with Vivacell (Denzlingen).
(an 60% ethanolic extract from Rhodiola rosea roots)
Test compound: WS® 1375
I. Inhibition experiments with pooled human liver microsomes
1A2 - 2C9 – 2C19 – 2D6 – 3A4
The following CYP isoenzymes were investigated:
Experimental procedure:
Test item incubations were performed in triplicate in 96-well plates. CYP isoen-zyme specific marker reactions and the yield of specific metabolits were investi-gated in the presence of 10 different test item concentrations at 37 ± 1°C in phos-phate buffer containing human liver microsomes and the marker substrate.
II. Induction experiments with freshly isolated human hepatocytes
1A2 - 3A4
Freshly isolated, pre-plated human primary hepatocytes (2 individual donors) in collagen-coated 24-well plates were used. Omeprazole (CYP1A2) and rifampicin (CYP3A4) served as reference inducers (positive controls), and 7-ethoxyresorufin (CYP1A2) and testosterone (CYP3A4) served as marker substrates. Four concen-trations of WS® 1375 (1, 10, 50 and 100 µg/ml) were tested in 3 parallel incubations.
Exposure of test system was performed over 72 h incubation period with the test or reference items (induction phase). The hepatocytes, pre-plated in 24-well plates were equilibrated after transport for approximately 48 hours at 37°C before starting of the induction phase. The test item or reference inducers were applied in 500µl medium to the cells. After ever 24 hours working solutions containing test item or reference inducers were renewed.
Fig. 2
Determination of IC50 for inhibition of CYP2C9 activity
Fig. 3
Determination of IC50 for inhibition of CYP2C19 activity