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Treatment of rats with the Pelargonium sidoides extract EPs[R] 7630 has no effect on blood coagulation parameters or on the pharmacokinetics of warfarin.

Abstract

Umckaloabo[R] is a herbal drug for the treatment of respiratory tract infections. It contains an aqueous ethanolic extract from roots of Pelargonium sidoides DC (EPs[R] 7630) as the active ingredient. Polymeric polyphenols and coumarins have been identified as the principal ingredients of EPs[R] 7630. In view of the coumarin content, it has been suggested that the administration of Umckaloabo[R] could possibly be associated with an increased risk of bleeding. This study, therefore, investigated whether a change in blood coagulation parameters or an interaction with coumarin-type anticoagulants occurred after administration of EPs[R] 7630 to rats.

No effect on thromboplastin time (TPT), partial TPT (PTPT) or thrombin time (TT) was observed after oral administration of EPs[R] 7630 (10, 75, 500mg/kg) for 2 weeks, while treatment with warfarin (0.05 mg/kg) for the same period resulted in significant changes in TPT and PTPT. If EPs[R] 7630 (500mg/kg) and warfarin (0.05mg/kg) were given concomitantly, the anticoagulant action of warfarin was not influenced. Similarly, the pharmacokinetics of warfarin were unchanged after pre-treatment with EPs[R] 7630 for 2 weeks.

Coumarin-type anticoagulants inhibit the synthesis of vitamin K-dependent coagulation factors via an identical mechanism in rat and man, and have a similar pattern of metabolism in both species. Moreover, as the coumarins so far identified in EPs[R] 7630 do not posses the structural characteristics needed for anticoagulant activitity, it appears unlikely that an increased tendency to haemorrhage arises in patients after intake of Umckaloabo[R].

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Blood coagulation; Coumarins; Anticoagulant activity; Bleeding

Introduction

Umckaloabo[R] is a plant-based pharmaceutical for the treatment of acute and chronic infections of the respiratory tract and ear, nose and throat. Pharmacological investigations have shown that it has antimicrobial, immunomodulatory and cytoprotective properties (Kolodziej et al., 2003; Kolodziej and Schulz, 2003).

Umckaloabo[R] contains a hydroethanolic extract of Pelargonium sidoides DC roots (EPs[R] 7630) as the pharmacologically active ingredient. The constituents identified in P. sidoides roots mainly include polyphenols and coumarins, which are probably also responsible for the observed therapeutic effect of EPs[R] 7630 (Kolodziej and Schulz, 2003).

Anticoagulants of the coumarin type have been used for over 50 years as medicines with antithrombotic effects. The mechanism of action of these compounds is based on the inhibition of the vitamin K-dependent synthesis of coagulation factors, especially clotting factors II (prothrombin), VII, IX and X. The reduction in plasma concentration of these factors leads to a delay in blood coagulation and thus to an increased bleeding tendency (Majerus and Tollefsen, 2001).

Owing to the coumarin content of EPs[R] 7630, there have been discussions about an increased risk of bleeding in association with the administration of Umckaloabo[R] (Anonymous, 2003). As a consequence, an increase in bleeding tendency has been included as a contraindication in the instructions for use. Furthermore, a potential increase in anticoagulant action on concomitant administration of coumarin derivatives has been pointed out. A delayed return to physiological Quick values in individual patients after anticoagulant therapy and subsequent administration of Umckaloabo[R] has in fact been reported from the 1960s (Zimmermann, 1965). However, these investigations were performed under poorly controlled conditions and large intraindividual variability in the anticoagulant effect after administration of coumarin-type anticoagulants was not taken into account.

Thus, it was the aim of the present investigation to examine whether any change in coagulation parameters arises in rats after administration of EPs[R] 7630 under standardised conditions for a period of 2 weeks. In addition, we analysed if the anticoagulant effect as well as pharmacokinetics parameters of warfarin, a typical coumarin-type anticoagulant, were influenced by concomitant treatment with EPs[R] 7630 for 2 weeks. Since coumarin-type anticoagulants inhibit the synthesis of vitamin K-dependent coagulation factors via an identical mechanism in rat and man, and have a similar pattern of metabolism in both species, the rat is considered to be a suitable animal model for this type of interaction study.

Materials and methods

Experimental animals

Male Sprague-Dawley rats (Janvier Le Genest, St. Isle, France) were used as experimental animals. After an acclimatisation period of at least 1 week, the animals weighed approximately 170-210 g at the start of the investigations. The animals were maintained under standardised environmental conditions (temperature 21 [+ or -] 1 [degrees]C; relative humidity: 50-60%; 12 h light-dark cycle) in Macrolen cages with free access to drinking water and pelleted food (Altromin, Lage, Germany). All experiments were conducted in accordance with German Animal Protection Act regulations after approval by the local government ethics committee. Oral treatment of the experimental animals with EPs[R] 7630 and warfarin was performed by gavage. A 0.2% agar suspension was used as vehicle for both substances (10 ml/kg body weight).

Materials

One single batch of EPs[R] 7630 (PSc 0343/Ch. 001 (9/00) TN01-125, Ident.-No. 501790080), provided by the Phytochemistry Section of the Department of Preclinical Research, Dr. Willmar Schwabe GmbH (Karlsruhe, Germany), was used for all experiments. Warfarin (3-([alpha]-acetonylbenzyl)-4-hydroxy-coumarin) was obtained from Sigma (Taufkirchen, Germany).

Coagulation tests

The animals were sacrificed with C[O.sub.2] at the end of the experiments and a blood sample was taken by cardiopuncture. Blood (4.5 ml) was mixed with 0.5 ml sodium citrate (Sigma, No. S-5570) for the determination of coagulation parameters. The samples were kept in on ice and centrifuged within 30 min at 4000 r.p.m. for 10 min. The plasma was then immediately separated from the cellular components and stored at -80 [degrees]C until analysis.

The coagulation investigations were performed in a group practice for laboratory medicine. In detail, the following assays were performed:

Thromboplastin time/Quick test (TPT): After addition of human placenta thromboplastin and calcium chloride, thrombin is formed from prothrombin in the plasma via the so-called extrinsic coagulation pathway. This then initiates the formation of fibrin from fibrinogen. The start of fibrin formation is determined photometrically from the resulting turbidity. The time between addition of reagent and start of coagulation is measured. This global test detects deficiencies in factors II, VII and X, a reduction in activity of factor V and fibrinogen can also be determined but with lower sensitivity. TPT is often used as a standard test to check therapy with coumarin-type anticoagulants. Reagent used was Thromborel S (Dade Behring, Schwalbach, Germany) applying a BCS analyser (Dade Behring).

Partial thromboplastin time (PTPT): On addition of phospholipids or surface active dispersed particles and calcium ions, thrombin formation occurs via the intrinsic coagulation pathway. Thrombin then induces the conversion of fibrinogen to fibrin. As described above, the time between addition of reagent and start of coagulation is measured photometrically from the turbidity due to fibrin formation. This test primarily monitors deficiencies in factors VIII, IX, XI, XII, prekallikrein and high molecular weight kininogen (HMWK). The test is also sensitive to some extent for factors X, V, II and fibrinogen. Reagent used was Pathrombin SL (Dade Behring) applying a BCS analyser (Dade Behring).

Thrombin time (TT): In this test, fibrin formation is directly induced by the addition of thrombin. The test therefore only detects disturbances in the final stages of coagulation, especially dysfibrinogenaemia or the presence of thrombin inhibitors. The measurement principle corresponds to the photometric detection of turbidity due to fibrin formation as described above. Reagent used was BC thrombin (Dade Behring) applying a BCS analyser (Dade Behring).

Pharmacokinetic investigations

Experimental animals were treated orally with 500 mg/kg EPs[R] 7630 daily over a period of 2 weeks for these investigations. On day 15, a single oral dose of 0.2 mg/kg warfarin in 10 ml/kg 0.2% agar suspension was given. In total, 6-12 animals each were sacrificed at different times (0.5, 1, 2, 4, 8, 24, 36, 48 and 72 h) after the warfarin application, blood samples were taken in sodium citrate tubes by cardiopunction and the plasma frozen at -80 [degrees]C until analysis.

The plasma warfarin assay was performed by HPLC analysis (column: Nova-Pak C18, 150 x 3.9 [mm.sup.2]; mobile phase: 35% acetonitrile/65% buffer [20 mM N[a.sub.2]HP[O.sub.4], pH 4.5]; flow: 0.8ml/min; detection: 306 nm) with reference to a warfarin calibration curve (0.1-5 [micro]g/ml plasma). Plasma (500 [micro]l) was acidified with 50 [micro]l l N HCl and extracted with 4ml ethyl acetate. The organic phase (3 ml) was evaporated off and the residue taken up in 300 [micro]l mobile phase. In total, 50 [micro]l each were injected into the HPLC column.

Statistical analysis

As the absolute coagulation parameters in the individual experiments displayed some variability due to different batches of reagent, the values were standardised and calculated as percentage values compared to the control groups investigated in parallel (corresponding to 100%).

The means [+ or -] SD are given in each case in the tables and figures. The statistical analysis was performed with a one-way analysis of variance and subsequent comparison of the different test groups using a Tukey test (GraphPad Prism 3.0, GraphPad Software, San Diego, USA).

Results

Effect of EPs[R] 7630 on coagulation parameters

Rats were treated orally with three different doses of EPs[R] 7630 (10, 75 and 500mg/kg) for 2 weeks in order to investigate a potential effect on blood coagulation. The lowest dosage chosen was approximately equivalent to the recommended daily dose in humans, while the highest dose level corresponded to the maximum dose used in pharmacological investigations. The geometrical mean between these two dosages was selected as the third dosage.

Treatment with EPs[R] 7630 had no effect on body weight or weight development in the experimental animals (Table 1). Similarly, no detectable influence on the absolute or relative liver weights was found.

The results of the blood coagulation analysis are summarised in Table 2. Compared to the control group, the TPT in animals treated with 10 or 500 mg/kg EPs[R] 7630 was somewhat shortened. The analysis of the data using a one-way analysis of variance showed this to be marginally significant (p<0.044). However, a direct comparison between the individual test groups revealed no statistically significant influence (p>0.05; Tukey's Multiple Comparison Test). With regard to the PTPT and TT values, there was no evidence of a change in these parameters due to the treatment of the experimental animals with EPs[R] 7630.

Effect of the combination of EPs[R] 7630 and warfarin on coagulation parameters

The results of two independent experiments are shown in Fig. 1. As expected, a 2-week oral treatment of the experimental animals with warfarin (0.05 mg/kg p.o.) led to a statistically significant prolongation of both TPT and PTPT compared to controls, but had no effect on TT. If the animals were given EPs[R] 7630 orally at a dosage of 500 mg/kg in combination with warfarin (0.05 mg/kg p.o.), the coagulation parameters did not statistically significantly differ from those in the warfarin group. Again, no influence on the blood coagulation values could be detected in these experiments when the rats were treated with EPs[R] 7630 (500 mg/kg p.o.) alone.

Effect of EPs[R] 7630 on the pharmacokinetics of warfarin

Rats were treated for 14 days with 500 mg/kg EPs[R] 7630 or vehicle (10 ml/kg 0.2% agar suspension) p.o. in order to investigate a possible influence of EPs[R] 7630 treatment on the bioavailability and metabolism of coumarin-type anticoagulants. The animals were given 0.2mg/kg warfarin p.o. 1 day after the last treatment, and blood samples were taken 0.5, 1, 2, 4, 8, 24, 36, 48 and 72 h later to assay the plasma levels of warfarin.

[FIGURE 1 OMITTED]

The results of this experiment are summarised in Table 3 and shown in Fig. 2. There was no statistically significant difference in warfarin plasma levels between control and EPs[R] 7630 group at any time, and almost identical pharmacokinetic parameters were accordingly found for both experimental groups (Table 4).

Discussion

The constituents so far identified in EPs[R] 7630 include a number of hydroxylated and sulphated coumarin derivatives (Latte et al., 2000; Fig. 3). Owing to the inhibition of vitamin K-dependent synthesis of various coagulation factors by certain coumarin compounds, it was thought possible that an increased bleeding tendency could also arise on taking Umckaloabo[R].

The biochemical mechanisms responsible for the anticoagulant effect of coumarin anticoagulants are essentially identical in humans and rats. This is also the reason for using these compounds as rodenticides (van Sittert and Tuinman, 1994). Warfarin is the prototype of these compounds and is commercially available as a racemic mixture of R- and S.enantiomeres. There are clear pharmacokinetic and pharmacodynamic differences between the two compounds. S-warfarin has a 2-5-fold higher vitamin K-antagonistic action than R-warfarin and is mainly hydroxylated in positions 6 and 7 by CYP29C. In contrast, CYP1A2 (6 and 8 hydroxylation) and CYP3A4 (10 hydroxylation) are mainly involved in the metabolism of R-warfarin (Kaminsky and Zhang, 1997; Wittkowsky, 2002). Similar metabolites have been identified in man and rat (Sutcliffe et al., 1987) and comparable CYP2C9 enzyme activities (Km, Vmax) have been found for both species (Bogaards et al., 2000). The metabolisation by different CYP450 isoenzymes, the high plasma protein binding and a low therapeutic range are responsible for interactions between warfarin and numerous other drugs. Due to the close similarity between rats and humans with respect to the pharmacodynamic and pharmacokinetic actions of warfarin, this species appears to represent a suitable animal model for corresponding interaction studies.

The minimal structural requirements for anticoagulant characteristics in coumarins are a hydroxy group in position 4 and a non-polar rest in position 3 (Majerus and Tollefsen, 2001, Fig. 3). None of the coumarin compounds so far identified in EPs[R] 7630 meets these structural requirements (Latte et al., 2000). It is therefore not surprising that no anticoagulant effects were observed even with the administration of EPs[R] 7630 at a dosage as high as 500 mg/kg for a period of two weeks. Furthermore, no pharmacokinetic or pharmacodynamic interactions between EPs[R] 7630 and warfarin were observed in these investigations. Thus, there was no significant influence on coagulation parameters under concomitant administration of EPs[R] 7630 and warfarin compared to animals, which were only treated with warfarin. Moreover, the plasma levels of warfarin were not changed by a 2-week pre-treatment of rats with EPs[R] 7630. In view of these results, it does not therefore appear very probable that an increased bleeding tendency can arise in patients treated with Umckaloabo[R]. This conclusion is supported by recent findings which demonstrate that EPs[R] 7630 does not have any effect on the metabolisation of marker substrates by human recombinant CYP450 isoenzymes and does not influence their expression in human hepatocytes (results not shown).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

References

Anonymous, 2003. Was ist dran an Umckaloabo? Arznei-Telegramm 34, 28-29.

Bogaards, J.J., Bertrand, M., Jackson, P., Oudshoorn, M.J., Weaver, R.J., van Bladeren, P.J., Walther, B., 2000. Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man. Xenobiotica 30, 1131-1152.

Kaminsky, L.S., Zhang, Z.Y., 1997. Human P450 metabolism of warfarin. Pharmacol. Ther. 73, 67-74.

Kolodziej, H., Schulz, V., 2003. Umckaloabo. Dts. Apoth. Ztg. 143, 55-64.

Kolodziej, H., Kayser, O., Radtke, O.A., Kiderlen, A.F., Koch, E., 2003. Pharmacological profile of extracts of Pelargonium sidoides and their constituents. Phytomedicine 10 (Suppl. IV), 18-24.

Latte, K.P., Kayser, O., Tan, N., Kaloga, M., Kolodziej, H., 2000. Unusual coumarin pattern of Pelargonium species forming the origin of the traditional herbal medicine Umckaloabo. Z. Naturforsch. 55c, 528-533.

Majerus, P.W., Tollefsen, D.M., 2001. Anticoagulant, thrombolytic, and antiplatelet drugs. In: Hardman, J.G., Limbird, L.E. (Eds.), Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th ed. McGraw-Hill, New York, pp. 1519-1538.

Suttcliffe, F.A., MacNicoll, A.D., Gibson, G.G., 1987. Aspects of anticoagulant action: a review of the pharmacology, metabolism and toxicology of warfarin and congeners. Drug Metabol. Drug Interact. 5, 225-272.

Van Sittert, N.J., Tuinman, C.P., 1994. Coumarin derivatives (rodenticides). Toxicology 91, 71-76.

Wittkowsky A., 2002. Warfarin and its potential interactions with traditional drugs and botanical agents. <http://www.pshp.org/ce_files/adis_anticoag.html>. Accessed on 24.10.2003.

Zimmermann, W., 1965. Uber die gerinnungshemmende Wirkung der Umckaloabo[R]-Droge. Arztl. Forsch. 19, 278-280.

E. Koch (a,*), A. Biber (b)

(a) Preclinical Research, Dr. Willmar Schwabe Gmb H & Co. KG, Karlsruhe, Germany

(b) Analytical Development, Deutsche Homoopathie Union, Karlsruhe, Germany

*Corresponding author. Tel.: +49 0 721 4005 356; fax: + 49 0 721 4005 150.

E-mail address: egon.koch@schwabe.de (E. Koch).
Table 1. Effect of a 2-week oral treatment with EPs[R] 7630 on the body
and liver weight in rats

 Number of Body weight (g)
Test group animals Start of experiment End ofexperiment

Controls 10 176.7 [+ or -] 3.8 309.3 [+ or -] 9.5
EPs[R] 7630 10 171.9 [+ or -] 9.9 316.1 [+ or -] 29.0
 500 mg/kg
EPs[R] 7630 10 173.5 [+ or -] 10.9 298.4 [+ or -] 15.0
 75 mg/kg
EPs[R] 7630 10 180.5 [+ or -] 9.4 313.0 [+ or -] 16.8
 10 mg/kg

 Weight Liver weight
Test group increase (g) Liver weight (g) (g/kg body weight)

Controls 132.6 [+ or -] 6.2 15.2 [+ or -] 1.1 4.9 [+ or -] 0.4
EPs[R] 7630 144.2 [+ or -] 30.4 15.2 [+ or -] 1.5 4.8 [+ or -] 0.6
 500 mg/kg
EPs[R] 7630 124.9 [+ or -] 8.1 14.4 [+ or -] 1.1 4.8 [+ or -] 0.2
 75 mg/kg
EPs[R] 7630 132.5 [+ or -] 9.7 14.9 [+ or -] 1.1 4.8 [+ or -] 0.2
 10 mg/kg

Means [+ or -] SD are given in each case.

Table 2. Effect of a 2-week oral treatment with EPs[R] 7630 on the
thromboplastin time (TPT), the partial thromboplastin time (PTPT) and
the thrombin time (TT) in rats. Means [+ or -] SD are given in each case

 Number of
Test group animals TPT (s) PTPT (s) TT (s)

Controls 10 9.55 [+ or -] 28.05 [+ or -] 62.63 [+ or -]
 00.35 3.93 3.59
EPs[R] 7630 10 9.28 [+ or -] 28.81 [+ or -] 62.91 [+ or -]
 500 mg/kg 0.24 5.38 4.10
EPs[R] 7630 10 9.51 [+ or -] 27.77 [+ or -] 61.67 [+ or -]
 75 mg/kg 0.39 3.08 4.85
EPs[R] 7630 10 9.21 [+ or -] 28.73 [+ or -] 62.94 [+ or -]
 10 mg/kg 0.22 2.98 6.51

Table 3. Effect of a 2-week oral treatment with EPs[R] 7630 (500 mg/kg)
or vehicle (agar suspension, 0.2%, 10 ml/kg) on the warfarin plasma
level in rats at different times after treatment with a single dose of
warfarin (0.2mg/kg p.o.). Means [+ or -] SD

Time after
warfarin Number Warfarin (ng/ml)
treatment (h) of animals Controls EPs[R] 7630

 0.5 6 562.8 [+ or -] 247.0 709.2 [+ or -] 318.4
 1 6 772.2 [+ or -] 372.2 860.0 [+ or -] 211.5
 2 12 766.3 [+ or -] 214.4 885.8 [+ or -] 366.5
 4 6 995.7 [+ or -] 305.0 845.3 [+ or -] 242.2
 8 6 1096.3 [+ or -] 307.6 945.8 [+ or -] 330.6
24 6 495.2 [+ or -] 273.9 761.0 [+ or -] 106.8
36 6 250.0 [+ or -] 18.0 319.0 [+ or -] 136.1
48 6 274.8 [+ or -] 79.1 255.5 [+ or -] 121.2
72 6 175.3 [+ or -] 132.1 159.7 [+ or -] 165.6

Table 4. Effect of a 2-week oral treatment with EPs[R] 7630 (500 mg/kg)
or vehicle (agar suspension, 0.2%, 10 ml/kg) on the pharmacokinetic
parameters of warfarin (0.2 mg/kg p.o.) in rats

Parameter Controls EPs[R] 7630

Cmax (ng/ml) 1096 945
Tmax (h) 8 8
AUC (0-t) (ng/ml h) 32,938 35,321
AUC (0-[infinity]) (ng/ml h) 41,831 43,533
[t.sup.1/2] (h) 37 36
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Author:Koch, E.; Biber, A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:4EUGE
Date:Feb 1, 2007
Words:3382
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