Possible intrusive food interaction with oral dabigatran ’ s anticoagulant activity in a rat models

Purpose: To evaluate the synergistic or antagonistic interaction between oral dabigitran etexilate (Pradaxa) and some foods. Methods: Aqueous extracts of sweet orange, cabbage, ginger, green tea, garlic and pineapple were prepared and lyophilized, and total phenolic acids and flavonoid contents evaluated. The residues of the aqueous extracts were given daily to the rats (500 mg/kg dose, oral) 1 h after administration of dabigatran (1.83 mg/kg dose) for 14 days. Bleeding time, International Normalized Ratio (INR), prothrombin time (PT) as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in blood were measured. Results: Oral administration of Pradaxa with all the test food (except cabbage) significantly increased bleeding time, INR and PT. Pradaxa interaction with cabbage significantly decreased bleeding tendencies but the drug did not produce any major elevation of liver enzyme levels (ALT, AST and ALP). Conversely, significant elevation in ALP level was observed only after aqueous extract of ginger was administered with Pradaxa. Green tea and garlic significantly increased ALT and AST levels. Conclusion: Synergistic interaction occurrs between dabigatran etexilate with the selected foods, but interaction with cabbage antagonistic.


INTRODUCTION
Anticoagulant drugs are those drugs that decrease the danger of blood clots.Anticoagulants are used to treat atrial fibrillation and deep vein thrombosis.Dabigatran etexilate (Pradaxa) is a new oral anticoagulant drug with less side effects than other anticoagulant drugs (e.g., warfarin) [1].Dabigatran is a direct thrombin inhibitors which inhibits free and clotbound thrombin and thrombin platelet aggregation.The drug is present in three strengths 75, 110 and 150 mg, and it is always taken two times daily by the patient [2].The structure of the drug is shown in Figure.Herbal remedies have been used for centuries for their medical benefits.Some remedies have shown a significant role in treatment and prevention of some diseases, but they need to be used wisely when taken concomitantly with prescribed drugs to avoid serious adverse effects and interactions [3].Due to limited food drug interaction studies, several phytochemical and pharmacological studies must be conducted to balance the level of therapeutic efficacy with the risk of toxicity [4].Some herbs normally consumed as foods are known for their different coagulation effects either as anticoagulants or enhancing the coagulation time such as, garlic, ginger, green tea, pineapple, orange and cabbage [5].Ginger has been reported to inhibit cyclooxygenase activity, platelet aggregation, and thromboxane synthetize activity [6].In vitro studies of constituents of garlic revealed that garlic possesses antiplatelet effects [7].The main constituent of green tea named catechin, has anti-platelet effect as it can prolong the bleeding time [8].Furthermore, bromelain (the main constituent of pineapple) can increase the serum fibrinolysis ability, activated partial thromboplastin time (APTT) and prothrombin time (PT) [9].In addition, orange fruit extract possess anti-platelet and anticoagulant effect by increasing activated partial thromboplastin time (APTT) and prothrombin time (PT) [10].In contrast, cabbage and other green leaves such as cauliflower and spinach, contain vitamin K which is important for production of certain proteins in the liver (proteins C, S and Z) that is responsible for the clotting factors [11].To evaluate the synergistic or antagonistic interaction between oral Dabigitran etexilate (Pradaxa) and some foods in correlation to their composition.
This study was aimed at evaluating the synergistic or antagonistic interactions between oral dabigitran etexilate (Pradaxa) and some foods in relation to their composition.Dabigitran etexilate had been approved by FDA as having lower bleeding risk than other oral anticoagulants [12].

Foods, materials and extraction
Fresh sweet orange, cabbage, ginger, green tea, garlic and pineapple were purchased from the local vegetable market in Dubai, UAE (500 g each).All the selected foods were identified by Zayed Complex for Herbal Research and Traditional Medicine in Abu-Dhabi, UAE.The outer layers of oranges, ginger, pineapples and garlic were removed.The fresh plants were cut into small pieces.All plants under investigation except green tea, were separately mixed with distilled water using a blender while aqueous decoction of the green tea was prepared.The prepared aqueous extracts were filtered through gauze and then dried by lyophilization.The percentage yields of the dried extracts were determined and are presented in Table 1.

Standardization of the aqueous extracts of foods
The total contents of phenolic acids and flavonoids were evaluated colorimetrically using spectrophotometer.
All experiments were performed in triplicate.Folin-Ciocalteu reagent was used to determine total phenolic content in alkaline medium [13,14].Absorbance was measured at 750 nm against blank.Results were calculated as mg/g gallic acid on dry weight of material.Total flavonoid content was evaluated by the aluminum chloride method.Quercetin was used as standard.The absorbance was measured at 510 nm.The results are presented in Table 1.

Experimental animals
Healthy albino male rats weighing 200 -220 g were obtained from the animal house of Dubai Trop J Pharm Res, October 2018; 17 (10):2033 Pharmacy College.The animals were kept under standard hygienic conditions (relative humidity 50-60 %, temperature at 22 ± 2 ∘ C, with 12 h day/night light cycle) and fed with a wellbalanced diet and water supplied ad libitum.The animals spent one week of accommodation before conducting the experiments.Ethical standards for the proper care and use of laboratory animals were applied to all biological investigations [15], and the work was approved by Research Unit and Ethical Committee of Dubai Pharmacy College, Dubai, United Arab Emirates (no.12.10.16).

In vivo anticoagulant studies
The residues of the aqueous extracts of the six selected foods were separately suspended in 1 % CMC.The dose for each, was 500 mg/kg.The animals were randomly distributed to eight groups (n = 6): group I (Control), received 1 % of CMC; group II, administrated Dabigatran etexilate in a dose of 1.83 mg/kg; groups III -VIII: administrated dabigatran etexilate in a dose of 1.83 mg/kg and then after one hour they received the selected foods separately in a dose of 500 mg/kg suspended in 1% CMC.All the previous groups were daily received the doses orally.
After 14 days of administration, blood samples were collected from the retro-orbital vein.International Normalized Ratio (INR) and Prothrombin Time (PT) were analyzed using Mission PT Coagulation Monitoring System apparatus.

Bleeding model
A rat-tail incision bleeding model, as described by Gustafsson et al [16] was used.All animals were fasted overnight, and then received a single oral dose of Dabigatran etexilate alone (1.83 mg/kg) or dabigatran etexilate with the selected foods (500 mg/kg) via gastric gavage.Bleeding time was measured using a spring-loaded blade device (Surgicutt®; Loxo, Dossenheim, Germany).The device was applied on the surface of the tail to make incision.Blood flow was blotted using filter paper which held directly below the tail.Every 30 sec, the position of the filter paper was changed until no longer development of a red crescent (end of bleeding).

Assay of liver enzymes
Samples of the blood were obtained from the orbital sinus of each rat.Plasma was separated by centrifugation at 3500 rpm and was used for the liver enzymes analyses (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)).

Statistical analysis
One-way ANOVA followed by post hoc analysis were used to analyze the data with the aid of.SPSS software, version 25.The data are expressed as mean ± standard deviation (SD, n = 3).P < 0.05 and < 0.01 were considered statistically significant.

Composition of aqueous food extracts
The part used and the percentage yield of each food extract are represented in Table 1.Evaluation of the total phenolic content of the selected foods aqueous extracts revealed that highest percentage yield was observed in green tea followed by pineapple (19.0, and 7.8 % respectively) calculated as gallic acid equivalents.On the other hand, highest flavonoid contents percentages were observed in green tea, garlic and sweet orange (40, 30, and 25 % respectively) expressed as grams of quercetin per 100 grams of dry plant material.

In vivo anticoagulant effects
Oral administration of all tested foods (except cabbage) with Pradaxa significantly increased the bleeding time, INR and PT.On the other hand, oral administration of Pradaxa with cabbage significantly decreased the bleeding time as shown in Table 2.

Liver enzymes
No significant elevation on the ALT, AST and ALP was observed after treating the animals once daily with Pradaxa for two weeks, as shown in Table 2. On the other hand, significant elevation in ALP level was observed by ginger residue.Moreover, oral administration of green tea and garlic separately with Pradaxa significantly increased ALT and AST levels as shown in Table 2.In contrast, oral administration of Pradaxa with cabbage significantly decreased the level of ALT.

DISCUSSION
Dabigatran etexilate or Pradaxa is a new oral anticoagulant drug and the most extensively used for treating pulmonary embolism and deep vein thrombosis with little side effects.Few reports are available about food-Dabigatran interaction [2,17].Dabigatran (direct thrombin Trop J Pharm Res, October 2018; 17 (10):2034 a, all the selected food extracts were separately orally administered just after one hour of the administration of Pradaxa; b In minutes ; *significant difference (p < 0.05) compared to control group; **significant difference (p < 0.01) compared to control group inhibitor) has a rapid onset of action, a short plasma half-life, a predictable pharmacokinetic profile and it does not need any regular blood tests to see the blood-thinning level.No dietary restrictions were previously reported [18].
INR provides information about the blood tendency to clot (thin or thick) while the prothrombin time (PT) is the time for the blood to clot [19].Dose selection for cabbage, pineapple, ginger, green tea, orange, garlic and Pradaxa was based on published data as well as the average body weight of the human (70 kg) [20].A dose of 500 mg/kg b.wt in rats was used for the selected foods which equivalent to 35 g in human being (average body weight, 70 kg).This study proved that oral administration of all tested foods except cabbage after one hour of Pradaxa administration, significantly amplified the bleeding time and significantly increased both INR and PT.Higher INR and PT can propose bleeding tendencies and increase the risk of the bleeding when those selected foods are taken with Pradaxa.On the other hand, oral administration of Pradaxa with cabbage can decrease the bleeding tendencies and can promote the time of clotting.This clotting effect is due to the presence of vitamin K in cabbage, vitamin K is an important factor for producing a protein in liver which responsible for the clotting factors [21].
No significant elevation on ALT, AST and ALP was observed after treating the animals once daily with Pradaxa for two weeks as shown in Table 2 which confirmed that Pradaxa did not have hepatotoxicity on short treatment (two weeks only) [22].In contrast, significant elevation in ALP level was noticed by oral administration of the aqueous extract of ginger with Pradaxa.As a result of high ALP level, the cholestasis may occur [23].Oral administration of green tea with Pradaxa significantly increased ALT and AST levels.United States Pharmacopeia shows evidence for the green tea extract that causes hepatotoxicity which noticed by elevation of the levels of liver enzymes in blood [24].Preclinical Trop J Pharm Res, October 2018; 17 (10):2035 and human study proved that the epigallocatechin-3-gallate, a component of green tea, can induce mitochondrial toxicity and generate the reactive oxygen species, although green tea showed the highest antioxidant effect [25].Similarly, elevation of liver enzymes especially ALT and AST was observed after oral administration of Pradaxa with garlic (192.5, 284.0 IU/L respectively).It was proved that, oral administration of the fresh garlic in high dose up to 1g/kg to the rats showed significant deterioration in liver enzymes [26].In contrast, administration of cabbage with pradaxa significantly decreases the level of ALT (11.50 IU/L).

CONCLUSION
The findings of the present study indicate that some food/dabigatran interactions occur in rats and thus, may also occur in human beings.Further studies are, however, required to ascertain the nature of these interactions.Health care professionals who prescribe and dispense dabigatran etexilate are advised to counsel patients to avoid drinking or eating these selected foods with and/or shortly before/after the administration of the drug.

Table 1 :
Yield and total phenolic and flavonoid contents of selected foods

Table 2 :
Effect of some food extracts on the anticoagulant activity of oral dabigatran (Pradaxa) in a rat model