Effects of fenugreek, garden cress, and black seed on theophylline pharmacokinetics in beagle dogs

Abstract

Context: Herb–drug interactions are a serious problem especially for drugs with a narrow therapeutic index, taking into consideration that herbal medicines are commonly used in various parts of the world.

Objective: The present study investigates the effect of fenugreek, garden cress, and black seed on the pharmacokinetics of theophylline in beagle dogs.

Materials and methods: Beagle dogs received theophylline (200 mg) orally and blood samples were withdrawn at different time intervals (0.33, 0.66, 1.0, 1.5, 2, 3, 4, 6, 8, 12, 24, and 30 h). After a suitable washout period, each herb was given orally at doses of 25, 7.5, and 2.5 g, twice daily for 7 d. On the eighth day, theophylline was re-administrated orally and blood samples were collected. Plasma concentrations of theophylline were determined using HPLC and pharmacokinetic parameters were calculated using a non-compartmental analysis.

Results: Treatment with fenugreek (25 g, orally) lead to a decrease in C_max and AUC_0–t of theophylline of about 28% (p < 0.05) and 22% (p < 0.05), respectively, with no significant changes in T_1/2λ compared with the baseline values. Garden cress caused a decrease in C_max to a lesser extent and delayed T_max of theophylline (2.10 ± 0.24 h versus 3.40 ± 0.74 h), while AUC_0–∞ increased by 37.44%. No significant effect was observed for the black seed treatment on theophylline disposition as measured by C_max, T_max, AUC_0–∞, and CL/F.

Discussion and conclusion: The concurrent use of fenugreek or garden cress alters theophylline pharmacokinetic behavior in an animal model. This could represent a modulation in cytochrome P450 activity, which is responsible for theophylline metabolism in beagle dogs. Further confirmation of these results in humans will warrant changes in theophylline dosing before the co-administration of such herbs.

• Cytochrome P450
• disposition
• herb–drug interaction

Introduction

Theophylline has been used to treat asthma and chronic obstructive pulmonary disease for many years (Ohnishi, 2000; Sullivan et al., 1994). Its oral absorption is almost complete with a peak plasma concentration generally achieved 2 h after administration (Page et al., 1998). Theophylline is characterized by a narrow therapeutic window of 5–20 µg/mL (Desoky et al., 1993; Qiu et al., 2007). In humans, it is extensively metabolized by cytochrome P450 (CYP) CYP1A2-mediated hepatic oxidation, predominantly to produce the metabolites 1,3-dimethyluric acid, 1-methyluric acid, and 3-methylxanthine, and to a lesser extent by CYP2E1 to 1,3-dimethyluric acid (Sarkar et al., 1992). Hence, it could be expected that the co-administration of drugs or herbal products that inhibit CYPlA2 activity could increase theophylline plasma concentration by reducing its metabolism, which may contribute to the emergence of adverse effects. In contrast, induction of CYP isoenzymes possibly will enhance theophylline clearance, leading to sub-therapeutic concentrations (Jan et al., 2005).

The global increase in the popularity of alternative medicines has raised renewed concerns regarding herb–drug interactions (Blanc et al., 2001). These interactions are especially important for drugs with narrow therapeutic indices, and may either be pharmacodynamic or pharmacokinetic in nature (Shi & Klotz, 2012; Tarirai et al., 2010). According to the World Health Organization (WHO), a significant percentage of the population living in developing countries relies on the traditional medicine for their primary health-care needs (Al-Faris, 2000; Mukherjee & Wahile, 2006; WHO, 2003).

Nigella sativa Linn. (Ranunculaceae), commonly known as black seed, is an annual herb that grows in countries bordering the Mediterranean Sea, as well as, in Pakistan and India (Gali-Muhtasib et al., 2006). The seeds have been used as a natural remedy over thousand years to promote health and treat diseases. For example, the oil of Nigella sativa is used as a traditional medicine for the treatment of arthritis, lung diseases, and hypercholesterolemia (Alkharfy et al., 2013b; Khader et al., 2009). Some important constituents of the seeds include thymoquinone, nigellone, and isoquinoline alkaloid (nigellimine N-oxide). Thymoquinone has been reported to be an effective antimicrobial and anthelmintic agent. It may also play a role in women's health, stimulating menstruation, and increasing milk flow (Ajayi et al., 2006).

Garden cress is the dried ripe seeds of Lepidium sativum Linn. (Cruciferae). The major constituents of Lepidium sativum are glucosinolates. Other constituents include ascorbic acid, cucurbitacins, and cardenolides. It has been used for respiratory disorders, vitamin C deficiency, constipation, poor immunity, and as a diuretic. The practitioners of Indian medicine consider its seeds useful in dysenteric diarrhea as well as in febrile and catarrhal infections (Gilani et al., 2013).

Fenugreek is the dried ripe seed of Trigonella foenum-graecum Linn. (Fabaceae). The constituents of fenugreek are simple alkaloids consisting mainly of trigonelline, choline, gentianine, carpaine, and saponine. It is used traditionally as a nutrient and in treating kidney ailments, diabetes, cellulitis, and tuberculosis. Fenugreek has also been utilized to reduce plasma cholesterol level and enhance blood anticoagulant activity (Basch et al., 2003; Sowmya & Rajyalakshmi, 1999). Enhancement of male libido has also been seen with the standardized fenugreek and its mineral formulation (Steels et al., 2011).

Given the fact that self-medication is a prevalent practice in many Asian cultures, more information about possible drug interactions with local herbal products is of critical importance. Therefore, effects of garden cress, black seed, and fenugreek on the disposition of theophylline are being explored by the current study.

Materials and methods

Chemicals and materials

Theophylline and hydroxyethyl theophylline (internal standard; I.S.) were obtained from Sigma-Aldrich (St. Louis, MO). High-performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). The tested herbs were procured in dry form from the local market and authenticated at the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. All other chemicals used were of the highest available commercial purity.

Animals and study design

Healthy male beagle dogs (n = 5) weighing 15–20 kg were used in the study after it was duly approved by the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia (Reference no. C.P.R-153). Animals were maintained in accordance with the recommendations of the “Guide for the Care and Use of Laboratory Animals” approved by the center. They were allowed free water and food ad libitum during the study except that the chow was pulled 12 h prior to animals' dosing. After the overnight fasting, dogs were given dog food (30 g) alone to serve as controls. Fifteen minutes later, manually prepared 200 mg of theophylline hard gelatin capsule was administered orally to each dog. Blood samples (2 mL) were collected from a leg vein through an indwelling vein catheter into heparinized vacutainer tubes before theophylline administration and at 0.33, 0.66, 1.0, 1.5, 2, 3, 4, 6, 8, 12, 24, and 30 h after dosing. Plasma was then immediately separated by centrifugation of blood at 2500 g for 10 min and stored at −20 °C until analysis. In all experiments, equal volumes of normal saline were injected through the cannula to replace the fluid loss.

After a suitable washout period, animals were started on a regimen of the studied herb where fenugreek, garden cress, and black seed were given to various groups at doses of 25, 7.5, and 2.5 g, respectively. To achieve proper herbal treatment, seeds of the herbs were grounded and mixed with 30 g of dog food and administered twice daily for 7 d. On the morning of the last day and after an overnight fasting, the last dose of the herb was given and theophylline (200 mg orally) was re-administered 15 min later. The same sampling scheme was repeated as described above to determine theophylline plasma concentrations and evaluate the effect of herbal therapy on theophylline disposition in dogs.

Theophylline plasma assay

An integrated HPLC system (LC 2010C; Shimadzu Corporation, Kyoto, Japan) equipped with a quaternary pump (LC20AD), a degasser, an auto-sampler (CIL20A), a column oven, a UV detector, and a data analysis software was used to determine theophylline plasma concentrations. Drug separation was achieved by a C18 column (5 µm, 150 mm × 4.6 mm; Waters Corporation, Milford, MA) at 25 °C. The mobile phase was a mixture of HPLC water/acetonitrile (93:7, v/v) with a pH of 4.2 adjusted with glacial acetic acid (0.5 mL/L) and pumped at a flow rate of 1.0 mL/min. Detection was set at a wavelength of 272 nm.

Sample preparation

Plasma sample (0.2 mL) was pipetted into micro-centrifuge tubes, then 0.1 mL of I.S. working solution (250 µg/mL) was added and the mixture was vortex mixed for 30 s. Then, 700 µL of zinc sulfate solution (2%) was added for protein precipitation followed by vortex-mixing for 1 min. The sample was centrifuged at 3000 g for 15 min at 25 °C. The clear supernatant was transferred into injector vials and a 50 µL aliquot was injected into the chromatographic system.

Calibration and control samples

Standard stock solutions of theophylline (1 mg/mL) and I.S. (1 mg/mL) were prepared in methanol. The I.S. working solution (250 µg/mL) was prepared by diluting stock solution with a mobile phase. Working solutions of theophylline and I.S. (0.2 mL) were added to drug-free plasma (9.6 mL) to obtain final concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 25.0 μg/mL. The quality control samples at three different concentrations were also prepared in a similar manner as the calibration standards. Spiked plasma calibration standards and quality control samples were stored at −20 °C until analyzed.

Pharmacokinetic analysis

Non-compartmental analysis was used to determine the pharmacokinetic parameters of theophylline after oral administration in the beagle dogs before and after herbal treatment. The maximum observed plasma concentration (C_max) and the time to reach the maximum observed plasma concentration (T_max) following oral administration were determined from the observed data. The area under the concentration versus time curve (AUC_0–t) was calculated by the trapezoidal rule with extrapolation to infinity. AUC from time 0 to infinity (AUC_0–∞) was calculated as the sum of AUC_0–t and any residual area, which was computed as the concentration at the last time point divided the terminal rate constant. Volume of distribution at steady state (V_ss) as (AUMC_0–∞/AUC_0–∞) × CL and total body clearance (CL) as dose/AUC_0–∞. The theophylline apparent elimination rate constant (λ_z) was estimated by linear regression analysis of the terminal portion of the log concentration–time data. Theophylline apparent elimination half-life (T_1/2λ) was calculated as ln2/λ. Pharmacokinetic analysis was performed using WinNonlin software (version 4.1, Pharsight Corporation, Palo Alto, CA).

Statistical analysis

Data are presented as the mean ± SEM Differences in pharmacokinetic parameters (e.g., CL/F, V_ss, and AUC_0–∞) of theophylline before and after treatment with the tested herbs were assessed by a paired t-test on log-transformed data. Statistical significance were assumed when p ≤ 0.05. All calculations were performed using GraphPad Prism version 3.00 for Windows (San Diego, CA).

Results

The analytical method was developed and validated in our laboratory. The peak area ratio of theophylline to I.S. in dog plasma was linear with respect to the analyte concentration over the range 0.1–25.0 μg/mL. The mean coefficient of determination (r²) for the standard curves was >0.99. The intra- and inter-day precision values for all QC samples were less than 5.0% and accuracies of 95–105%.

The estimated model-independent pharmacokinetic parameters of theophylline before and after fenugreek treatment are listed in Figure 1 and Table 1. The calculated C_max in dogs was 16.45 ± 0.72 μg/mL and 11.92 ± 1.08 μg/mL (p = 0.0029), and AUC_0–t was 186.03 ± 5.71 μg h/mL and 144.38 ± 7.79 μg h/mL (p = 0.0035), respectively. A trend of reduction was observed in the calculated oral clearance (CL/F) and apparent oral volume of distribution at steady state (V_ss), which, however, did not reach statistical significance (Table 1; p > 0.05). These parameters have resulted in T_1/2λ of theophylline of 8.13 h and 8.05 h.

Figure 1. Plasma concentration–time curves of theophylline following oral administration of 200 mg dose to male beagle dogs (n = 5) alone (control) and after fenugreek, garden cress, and black seed treatment.

Table 1. Non-compartmental pharmacokinetic parameters of theophylline following an oral administration in beagle dogs before and after fenugreek treatment.

	Estimate (mean ± SEM)
Parameter	Before	After	p Value
Cmax (μg mL^−1)	16.45 ± 0.72	11.92 ± 1.08	*0.0029
Tmax (h)	2.10 ± 0.24	3.20 ± 0.86	0.2887
AUC0–t (μg h mL^−1)	186.03 ± 5.71	144.38 ± 7.79	*0.0035
AUC0–∞ (μg h mL^−1)	203.12 ± 7.78	161.07 ± 11.30	*0.0045
CL/F (mL h^−1)	49.26 ± 33.85	21.62 ± 2.27	0.4466
Vss (mL)	33.60 ± 22.73	17.47 ± 2.11	0.5140
λz (h^−1)	0.088 ± 0.004	0.081 ± 0.003	0.2820
T1/2λ (h)	8.13 ± 0.62	8.05 ± 0.72	0.412
MRT (h)	10.94 ± 0.24	11.51 ± 0.49	0.2718

*p < 0.05.

Plasma concentration–time curve and pharmacokinetic parameters of theophylline in dogs before and after garden cress are given in Figure 1 and Table 2. Theophylline C_max and T_max under garden cress treatment were 14.64 μg/mL and 3.40 h, respectively (p > 0.05). However, AUC_0–∞ was significantly increased with herbal treatment (279.16 μg h/mL compared with 203.12 μg h/mL; p < 0.05). Similarly, the relative bioavailability of theophylline was enhanced by ∼37.44%.

Table 2. Non-compartmental pharmacokinetic parameters of theophylline following an oral administration in beagle dogs before and after garden cress treatment .

	Estimate (mean ± SEM)
Parameter	Before	After	p Value
Cmax (μg mL^−1)	16.45 ± 0.72	14.64 ± 0.50	0.1091
Tmax (h)	2.10 ± 0.24	3.40 ± 0.74	0.1658
AUC0–t (μg h mL^−1)	186.03 ± 5.71	223.95 ± 17.81	0.0597
AUC0–∞ (μg h mL^−1)	203.12 ± 7.78	279.16 ± 28.88	*0.0309
CL/F (mL h^−1)	49.26 ± 33.85	12.01 ± 1.97	0.3198
Vss (mL)	33.60 ± 22.73	11.85 ± 1.40	0.374
λz (h^−1)	0.088 ± 0.004	0.066 ± 0.004	*0.0068
T1/2λ (h)	8.13 ± 0.66	11.68 ± 0.84	0.296
MRT (h)	10.94 ± 0.24	15.01 ± 0.51	*0.0002

*p < 0.05.

The comparison of plasma concentration–time profile and pharmacokinetic parameters of the theophylline after black seed treatment is shown in Figure 1 and Table 3. In this case, non-significant (p > 0.05) changes in C_max and T_max of theophylline were observed. T_1/2λ of theophylline in the black seed treated group and controls were similar (Table 3). The extent of theophylline absorption (measured by AUC_0–∞) was non-significantly (p > 0.05) increased when the drug was taken with black seed. In addition, there were no significant differences in the rests of the pharmacokinetic parameters including CL/F and V_ss, relative to those of the control group.

Table 3. Non-compartmental pharmacokinetic parameters of theophylline following an oral administration in beagle dogs before and after black seed treatment.

	Estimate (mean ± SEM)
Parameter	Before	After	p Value
Cmax (μg mL^−1)	16.45 ±  0.72	15.89 ± 0.50	0.6597
Tmax (h)	2.10 ± 0.24	2.70 ± 0.53	0.3239
AUC0–t (μg h mL^−1)	186.03 ± 5.71	187.65 ± 6.95	0.7572
AUC0–∞ (μg h mL^−1)	203.12 ± 7.78	216.61 ± 8.12	0.1069
CL/F (mL h^−1)	49.26 ± 33.85	15.05 ± 0.42	0.3675
Vss (mL)	33.60 ± 22.73	12.77 ± 0.65	0.4036
λz (h^−1)	0.088 ± 0.004	0.084 ± 0.002	0.4742
T1/2λ (h)	8.13 ± 0.60	8.25 ± 0.08	0.3000
MRT (h)	10.94 ± 0.24	12.93 ± 0.32	*0.0044

*p < 0.05.

Discussion

Traditional herbal medicines can act through a variety of mechanisms to alter the pharmacokinetic profile of the co-administered drug (Fugh-Berman, 2000). It is important to be aware of the interaction potency of commonly used herbal medicines in order to avoid a reduction in efficacy or an increase in the side-effects of concomitantly administered drugs (Tang et al., 2009). Further, the clinical consequence of interactions may be unforeseen side effects and non-compliance, and it is, therefore, of major importance for patient outcome (Landmark & Patsalos, 2008).

To best of our knowledge, this is the first report which explored the effect of some Asian traditional herbs on the disposition of theophylline after oral administration using an animal model. Our study has shown that all tested herbs interfere with the theophylline pharmacokinetic to varying degrees. Fenugreek treatment for 7 d caused a decrease in C_max and AUC_0-t. The T_1/2λ whoever did not change significantly following fenugreek treatment and this can be explained by a similar simultaneous reduction in CL and V_ss following the herb administration in beagle dogs. It is possible that one or more of fenugreek constituents interfere with theophylline tissue distribution, resulting in smaller V_ss. In a recent study (Alkharfy et al., 2013a) fenugreek caused a significant reduction in the calculated C_max and AUC_0–t (∼ 50%) as compared with controls.

In our study, garden cress produced no significant effect on C_max but caused an increase in T_max. Theophylline AUC_0–∞ also increased significantly following garden cress administration in beagle dogs, and the relative bioavailability of theophylline was 37.44% more when it was concurrently administered with the herbal product. Our results are in accordance with recently published data, where garden cress increased AUC_0–t and AUC_0–∞ of cyclosporine by about 40% (Al-Jenoobi et al., 2013). In contrast, black seed did not produce any remarkable effect on the disposition of theophylline in beagle dogs at the doses used in this study.

CYP1A2 is an important CYP enzyme subfamily, accounting for 13% of the total CYP enzyme in humans (Chien et al., 2010). In the current study, theophylline was a probe drug for CYP1A2, and showed some evidence of potential herbal interactions. Hence, many CYP1A2-metabolized drugs such as caffeine, theophylline, clozapine, and tacrine (Faber et al., 2005) could interact with fenugreek and garden cress. It would be interesting to identify herbs' specific constituent(s) responsible for these effects.

Conclusion

These findings have shown that fenugreek and garden cress have the potential to interfere with theophylline disposition. Therefore, it would be important to consider such types of interactions when dealing with individuals on theophylline therapy. In addition, these interactions should be further evaluated to assess their significance and clinical consequence in humans.

Declaration of interest

The authors report no declarations of interest. This study was supported by the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia (Grant number: DSR-AR-2-20).