INTRODUCTION:

Interactions among the concomitantly administered drugs which are undesired or unknown are truly challenging for both physicians and patients as they may either result in untoward effects or unsafe reactions. Hence the screening of such potential interactions became significantly important for the clinical pharmacists as well as physicians which is a very cumbersome process that need lot of attention with all respects¹. It is well reported that most of the drugs have multiple pharmacological effects and when administered along with other drugs particularly which have related effects are prone to show variation in the expected clinical effect².

With the modern living style, administration of multiple drugs is evident which further enhanced the risk of interactions and adverse reactions resulting in more hospitalization, incidence of untoward effects, increase in the health care expenses and lack of patient safety³. This made the researchers focus more on such interactions like drug-drug, drug-excipient, and drug-food interactions^4-7. Several screening techniques are introduced for such studies in order to identify and report the serious drug interactions. The information that is generated with respect to such interactions has more importance and valuable for better health care management⁸. Drugs interactions are reported to be possible in 3-7% of patients taking around 10 medications and in 20% patients who are taking around 10-20 medications. On an around there are 2.8% of hospitalization cases due to these drug interactions⁹. Now-a-days, awareness on herbal formulations or nutraceuticals has been tremendously increased and forcing the patients to take them in addition to the medication prescribed by a physician¹⁰. This may in turn result in any undesired effects due to drug-nutraceutical interactions. Since, diabetes is one of the most prevailing disorder across the globe, multi drug therapy and drug-nutraceutical interactions are expected to happen^11,12. Hence, in the present investigation, it is proposed to study the pharmacokinetic interactions of concomitant administration of eugenol with gliclazide in diabetic induced rabbits.

MATERIALS AND METHODS:

Gliclazide was obtained as gift sample from Dr. Reddy’s Labs, Hyderabad, India. Eugenol and Streptozotocin were purchased from Sigma Aldrich Pvt. Ltd., Mumbai, India. Tris buffer, ethylenediamine tetra acetate (EDTA), HPLC grade methanol, ethyl acetate, glacial acetic acid, formic acid, acetonitrile and all other chemicals were obtained from E. Merck, Mumbai, India. All the chemicals used were of analytical grade.

Experimental animals:

Albino rabbits of either sex weighing between 1.5 - 2kg procured from Chalapathi Institute of Pharmaceutical Sciences, Guntur, Andhra Pradesh, India were used in the study¹³. The rabbits were housed in rabbit cages at room temperature (25±3°C) and were maintained with a normal photoperiod of 12 h light and 12 h darkness^14,15. They were fed with a balanced diet and water ad libitum. The study protocol was approved by the Institutional Animal Ethical Committee of Chalapathi Institute of Pharmaceutical Sciences, Guntur, Andhra Pradesh, India (Ref. No. 03/IAEC/CIPS/2016-17; Dt. 05/04/2016; Regd. No. 1048/PO/Re/S/07/CPCSEA).

Study design^16-19:

Rabbits were divided into four groups and each group is comprised of 6 rabbits.

Group-A (Diabetic control: Diabetic rabbits administered with only vehicle)

Group-B (Treatment-1: Diabetic rabbits administered with gliclazide

Group-C (Treatment-2: Diabetic rabbits administered with eugenol)

Group-D (Treatment-3: Diabetic rabbits administered with gliclazide and eugenol)

The rabbits were fasted for a period of 18 h prior to experimentation and water supplied ad libitum. Both water and food were removed during the experiment.

Based on previous reported investigations, the doses used in the present investigation are streptozotocin 50 mg/kg body weight, gliclazide 5.6mg/1.5kg body weight and eugenol 10mg/kg body weight. Streptozotocin, 50mg/kg body weight, was used for induction of diabetes using 0.1 M citrate buffer pH 4.5 by injecting intraperitoneally^16-19. After 48 h, diabetes was confirmed by estimation of serum glucose levels using glucose kit. Rabbits showing glucose levels of 200-250mg/dl were used in the experiment as diabetic.

Collection of blood samples:

Blood samples were withdrawn from the marginal ear vein of each rabbit. Blood samples were collected at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 h from the marginal ear vein of the rabbits and processed for different estimations like glucose levels were estimated by GOD/POD method using commercial glucose kits and gliclazide concentration in plasma using developed RP-HPLC method.

Estimation of plasma gliclazide concentration by RP-HPLC method:

A developed and validated bioanalytical method was used for the determination of plasma gliclazide by using RP-HPLC²⁰. The Agilent HPLC system (Agilent 1200 liquid chromatography system, Agilent Technologies Inc., USA.) equipped with an isocratic pump (1200 series) and analytical column of Hyper chrome ODS-BP (5μm, 4.6mm x 250mm) was used for the study. The mobile phase consisting of 0.1% formic acid: acetonitrile (70:30, v/v) and the flow rate of 1.0ml/min was used. The mobile phase was filtered through 0.45 µm membrane filter and degassed by ultrasonication. The drug was monitored at 228nm using variable wavelength detector (VWD). The data analysis and quantitation were performed with Agilent Chemstation software (Agilent Technologies). The retention time was found to be 4.2±0.3min.

Treatment of plasma samples:

Liquid-liquid extraction method was used to isolate drug from rabbit plasma. For this purpose, 100μl of plasma sample was added into labelled polypropylene tubes containing anticoagulant and vortexed briefly. Further 100μl of 0.5 N sodium carbonate, 2.5ml of extraction solvent (methyl tertiary butyl ether) was added and vortexed for 10 min. Then the samples were centrifuged at 4,000rpm for 10 min at 20°C. Subsequently, the supernatant from each sample was transferred into respective polypropylene tubes. All the samples were kept for evaporation under nitrogen at 40°C. The dried residue was reconstituted with 500μl of reconstitution solution (mobile phase) and vortexed briefly. Finally, the extracted sample was injected into HPLC for analysis.

Pharmacokinetic analysis:

The peak concentration of drug in plasma, C_maxand concentration peak time, T_max were directly read from the concentration-time data. Other pharmacokinetic parameters were determined on subjecting the concentration-time data to non-compartmental analysis using PK SOLVER software. The elimination rate constant, K_E and half-life, t_1/2 were determined by linear regression analysis of the log-linear part of the plasma drug concentration-time curve. Absorption rate constant, K_a was calculated using Wagner Nelson method. V_d and Cl_T were calculated. Area under the concentration-time curve [AUC]_0-24 was calculated by use of the linear trapezoidal rule and [AUC]_0-_a with extrapolation to infinity by dividing the last measured concentration by K_E. [AUMC]_0-_a was calculated using ct vs time curve. The mean residence time, MRT was calculated using the formula, MRT = [AUMC]_0-_a/ [AUC]_0-_a.

Statistical analysis:

The data was presented as mean±standard error mean (SEM). The statistical analysis was performed by means of student’s paired t-test. A value of P<0.05 was considered for statistical analysis. Data was processed with Microsoft Excel.

RESULTS AND DISCUSSION:

Pharmacokinetics:

From the calibration curve of peak area vs concentration of gliclazide in rabbit plasma, based on regression line equation, the collected blood samples from the experimental studies were used for the estimation of plasma drug concentration of gliclazide. The obtained results are depicted in Table 1 and comparative graphical representation is given in Fig. 1.

Table 1: Average plasma drug concentrations of gliclazide with treatment of gliclazide and combination treatment of gliclazide and eugenol in diabetic rabbits (n=6)

Time (h)	Group B	Group D
0	0.00	0.00
0.5	13.92±2.23	34.77±3.64
1	26.02±2.13	69.76±9.72
2	79.60±5.50	117.01±10.05
3	65.65±5.20	88.69±9.88
4	32.69±2.35	66.19±9.51
6	23.62±1.64	45.28±5.07
8	16.96±0.74	33.77±4.39
10	14.20±0.80	29.18±4.50
12	7.93±0.71	18.60±1.89
16	3.42±0.51	12.90±1.19
20	1.48±0.19	9.08±1.13
24	0.82±0.21	7.04±1.32

Note: Data presented as mean±SEM

Pharmacokinetic parameters:

Different pharmacokinetic parameters calculated using PK SOLVER software for Group B (diabetic rabbits treated with gliclazide alone) and Group D (diabetic rabbits treated with combination of gliclazide and eugenol) are presented in Table 2. As per students paired t-test, there is a significant difference in K_a, K_E, t_1/2, C_max, AUC_0-t, AUC_0-_a, AUMC_0-_a, and MRT between Group B and Group D treatments indicating that there is an improved bioavailability of gliclazide when administered along with eugenol compare with gliclazide alone. Whereas there is no significant difference in the pharmacokinetic parameters T_max, V_d/F, and Cl_T/F.

Fig. 1: Mean plasma drug concentrations of gliclazide with treatment of gliclazide alone and combination of gliclazide with eugenol in rabbit rabbits (n=6), mean±SEM

Table 2: Mean pharmacokinetic parameters of treatment with gliclazide alone (Group B) and combination of gliclazide with eugenol (Group D) in diabetic rabbits (n=6)

Pharmacokinetic Parameter	Unit	Group B	Group D
K_a	1/h	0.45±0.01	0.80±0.10*
K_E	1/h	0.22±0.03	0.09±0.01*
t_1/2	h	3.27±0.29	8.47±1.30*
T_max	h	2.00±0.00	2.17±0.17
C_max	μg/ml	79.60±4.49	123.28±5.45*
AUC_0-t	μg/ml.h	375.36±7.94	748.93±40.66*
AUC_0-_a	μg/ml.h	379.54±8.02	839.44±56.69*
AUMC_0-_a	μg/ml.h²	2198.36± 44.66	8637.66± 1237.26*
MRT	h	5.80±0.11	10.04±0.92*
V_d/F	(mg)/ (μg/ml)	0.07±0.01	0.08±0.01
Cl_T/F	(mg)/ (μg/ml)/h	0.01±0.00	0.01±0.00

Note: *Statistically significant at P<0.05, Data presented as mean±SEM

Pharmacodynamics:

Glucose levels of the collected blood samples from the rabbits were determined by following GOD/POD method using commercial glucose kits. The obtained results are depicted in Tables 3 and 4 and comparative graphical representation is given in Fig. 2 and 3.

Table 3: Average blood glucose levels (Mean and SEM) upon treatment with vehicle (Group A), gliclazide (Group B), eugenol (Group C), and their combination (Group D) in diabetic rabbits (n=6)

Time (h)	Group A	Group B	Group C	Group D
0	322.00±8.13	338.50±1.04	334.00±3.58	339.75±1.31
0.5	321.00±1.00	290.25±5.39*	322.25±4.73	281.50±5.52*^$
1	325.50±7.27	254.75±4.15*	312.50±5.81*	228.50±4.43*^$
2	328.50±8.45	205.25±4.61*	296.50±3.97*	178.75±4.59*^$
3	328.00±8.22	218.25±1.44*	284.25±3.50*	200.00±1.47*^$
4	327.50±8.51	240.50±2.96*	288.75±0.85*	222.75±2.39*^$
6	322.75±10.96	262.25±1.70*	301.75±3.90	245.00±3.65*^$
8	325.25±7.26	275.50±2.78*	316.25±3.20	266.50±3.80*
10	326.50±6.38	290.00±3.34*	317.25±1.89	285.00±5.61*
12	323.25±7.09	309.25±3.82	318.25±2.02	297.50±4.37
16	328.00±6.78	318.75±2.29	320.25±0.63	307.25±3.33
20	326.00±7.43	332.50±3.18	322.25±2.25	325.25±2.02
24	329.25±7.03	335.75±2.06	328.25±3.92	327.50±2.33

*Significant at P<0.05 compared with diabetic control; ^$Significant at P<0.05 compared with diabetic treatment with gliclazide;

Data presented as mean±SEM

Table 4: Average percent blood glucose reduction (Mean and SEM) upon treatment with gliclazide (Group B), eugenol (Group C), and their combination (Group D) in diabetic rabbits (n=6)

Time (h)	Group B	Group C	Group D
0	0.00±1.04	0.00±3.58	0.00±1.31
0.5	14.25±5.39	3.52±4.73	17.14±5.52*
1	24.74±4.15	6.44±5.81	32.74±4.43*
2	39.36±4.61	11.23±3.97	47.39±4.59*
3	35.52±1.44	14.90±3.50	41.13±1.47*
4	28.95±2.96	13.55±0.85	34.44±2.39*
6	22.53±1.70	9.66±3.90	27.89±3.65*
8	18.61±2.78	5.31±3.20	21.56±3.80*
10	14.33±3.34	5.01±1.89	16.11±5.61*
12	8.64±3.82	4.72±2.02	12.44±4.37
16	5.83±2.29	4.12±0.63	9.57±3.33
20	1.77±3.18	3.52±2.25	4.27±2.02
24	0.81±2.06	1.72±3.92	3.61±2.33

*Significant at P<0.05 compared with gliclazide treatment Data presented as mean±SEM

Fig. 2: Mean blood glucose concentrations with vehicle, gliclazide, eugenol and their combination in diabetic rabbits (n=6), mean±SEM

Fig. 3: Mean % blood glucose reduction with gliclazide, eugenol and their combination in diabetic rabbits (n=6), mean±SEM

CONCLUSION:

In the present investigation, the results clearly indicated that there is a statistically significant improved bioavailability of gliclazide on administration along with eugenol in diabetic rabbits. Pharmacodynamic studies also clearly revealed that there is an improved reduction in the blood glucose levels with combined treatment of gliclazide and eugenol compared with treatment of gliclazide alone. Hence, it can be concluded that there is a pharmacokinetic and pharmacodynamic advantage of administering gliclazide along with eugenol. However, further studies in multiple dosing as well as single and multiple dose studies in another animal models are required to obtain more information on these interactions between gliclazide and eugenol^21-27.

ACKNOWLEDGEMENT:

The authors are grateful to the Chalapathi Institute of Pharmaceutical Sciences for providing necessary facilities to conduct the research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 03.12.2020 Modified on 09.03.2021

Research J. Pharm.and Tech 2022; 15(2):787-791.

DOI: 10.52711/0974-360X.2022.00131