PHARMACOKINETIC AND PHARMACODYNAMIC INTERACTIONS OF ATORVASTATIN WITH GUGGULSTERONE IN HYPERLIPIDEMIC RATS

PHARMACOKINETIC AND PHARMACODYNAMIC INTERACTIONS OF ATORVASTATIN WITH GUGGULSTERONE IN HYPERLIPIDEMIC RATS

Kalam Sirisha ^{* 1}, Puchchakayala Goverdhan ², Akunuri Swarnalatha ² and Akina Sravanthi ²

Department of Pharmaceutical Analysis ¹, Department of Pharmacology ², Vaagdevi College of Pharmacy, Ram Nagar, Hanamkonda, Warangal - 506001, Andhra Pradesh, India.

ABSTRACT: Atorvastatin is an HMG-CoA reductase inhibitor used in the treatment of hyperlipidemia. Guggulsterone is a herbal product used in hyperlipidemia. The present work was aimed to study the pharmacokinetic and pharmacodynamic interactions of atorvastatin with guggulsterone in hyperlipidemic rats. Wistar albino rats with induced hyperlipidemia were divided into six groups (n=6). Blood samples were collected at predetermined time intervals from the groups treated with atorvastatin alone and in combination with guggulsterone for kinetic analysis and lipid profiles. Plasma samples were quantified for atorvastatin concentration by HPLC, and then the concentration-time data were analyzed. C_max, t_1/2, AUC, and MRT were significantly (p<0.05) decreased during kinetic study, and clearance was significantly (p<0.05) increased in comparison with atorvastatin alone. Further, there was no significant difference in T_max in all groups. The study revealed lower values [C_max AUC, T_1/2, and MRT] of atorvastatin in guggulsterone treated groups. The combination of atorvastatin and guggulsterone in hyperlipidemic rats produced a significant change in lipid profiles when compared with hyperlipidemic control, but less than atorvastatin alone.

Atorvastatin, Guggulsterone, Hyperlipidemia, HPLC, Pharmacokinetics, Pharmacodynamics

INTRODUCTION: Atorvastatin (AT) is a synthetic lipid-lowering agent. It is a selective competitive inhibitor of 3-hydroxy- 3-methyl glutaryl coenzyme A reductase (HMG-CoA), which catalyzes the conversion of HMG-CoA to mevalonate, an important rate-limiting step in cholesterol biosynthesis ¹. It is used in the treatment of hyperlipidemia ². It is a substrate for the CYP3A4 isoenzyme. It has shown susceptibility to inhibitors and inducers of CYP3A4 to produce increased or decreased plasma concentrations, respectively ³.

The usage of herbal therapies, along with prescription and Over The Counter (OTC) medications is increasing day by day. Guggulsterone from guggul resin (Commiphora mukul) is a well-known herbal product that has been targeted for its hypolipidemic activity ⁴. The farnesoid X receptor (FXR) antagonism by guggulsterone has been proposed as a mechanism for its hypolipidemic effect. It inhibits lipogenic enzymes and HMG-CoA reductase in the liver ⁵. It also increases biliary and fecal excretion of cholesterol ⁶. Guggulsterone is reported to be more beneficial in reducing blood cholesterol, triglycerides levels, and to reduce the risk of cardiovascular disease without any apparent risk of liver damage ^{7, 8}.

The present work was aimed at studying the pharmacokinetic and pharmacodynamic herb-drug interactions of guggulsterone with atorvastatin in hyperlipidemic rats. The standard cholesterol diet ⁹ has been successfully used to induce hyper-lipidemia in rats, due to its convenience, reproducibility, and availability.

MATERIALS AND METHODS:

Drugs and Chemicals: Atorvastatin pure drug was a kind gift from Stallion Laboratories Pvt. Ltd., Bavla, Ahmedabad, Gujarath. Commiphora mukul standardized extract (2.6% w/w total guggulsterones) was procured from Natural Remedies Pvt. Ltd, Bangalore, India. HPLC grade acetonitrile, methanol and water were purchased from Finar Chem. Ltd., Hyderabad, India. Diclofenac sodium was a kind gift from Farmson Pharmaceutical Pvt. Ltd. Gujarat. Cholesterol kit (Enzymatic Method), Triglycerides kit and HDL-C kit were procured from Excel Diagnostics Pvt. Ltd., Hyderabad, India.

Experimental Animals: Wistar albino adult male rats weighing 170-200g were purchased from Mahaveer Enterprises, Hyderabad and housed in polypropylene cages in a room where the congenial temperature was 27 ± 1 °C and 12 h light and dark cycles were maintained. The animals were allowed to acclimatize to the environment for 7 days and supplied with a standard pellet diet and water ad libitum. The Institution Animals Ethics Committee (IAEC), Vaagdevi College of Pharmacy, India had approved the experimental protocol (VCOP/2012/ 11/1/S-II/6), and care of animals was taken as per guidelines of CPCSEA, Department of Animal Welfare, and Government of India (1047/ac/07/ CPCSEA). Before induction of hyperlipidemia, the weight of the individual animal and plasma cholesterol levels were estimated. The standard cholesterol diet, along with butter (0.5 ml twice a day) was administered for 30 days to induce hyperlipidemia.

At the end of one month, the blood was withdrawn from retro-orbital sinuses to analyze for lipid profiles (TC, TG, LDL-C and HDLC levels) to confirm the induction of hyperlipidemia ¹⁰.

Anti-Hyperlipidemic Studies: The hyperlipidemic rats were divided into six groups of six rats each and dosed as below.

Group I: (HL) 1% Tween 80 (hyperlipidemic control).

Group II: (AT) atorvastatin (10 mg/kg p.o).

Group III: (GE) guggulsterone standardized extract (25 mg/kg p.o.).

Group IV: (GC) guggulsterone marketed preparation (capsule) (25 mg/kg p.o.).

Group V: (AT-GE) pretreated with guggulsterone standardized extract (25 mg/kg p.o.) for seven days and administered atorvastatin on the 8^th day (10 mg/kg p.o.).

Group VI: (AT-GC) pretreated with guggulsterone marketed preparation (25 mg/kg p.o.) for seven days and administered atorvastatin (10 mg/kg) on 8^th day ¹¹.

Collection of Blood Samples: On 8^th day, blood samples of 0.5 mL were withdrawn at different time intervals through retro-orbital sinus into heparinized Eppendorf tubes at 0, 1, 2, 4, 8, 12 and 24 h and equal amount of saline was administered intraperitoneally to replace blood volume at every blood withdrawal time ¹². Plasma was obtained by immediate centrifugation of blood samples using REMIULTRA cooling centrifuge at 3000 rpm for 10 min at room temperature. All samples were stored at -20 °C until analysis.

Preparation of Standard Graph: Stock solution of atorvastatin was prepared by dissolving it in methanol at a concentration of 1 mg/ml. This solution was diluted with methanol to obtain the working standard solutions in the range of 0.05-5 µg/ml. Chromatogram of Blank rat plasma is shown in Fig. 1. Plasma standards were prepared in the range of 0.05-5 µg/ml by taking 100 µl of working standard solutions in a disposable tube, evaporating to residue by heating at 45 ºC. To the residue, 1 ml of pooled untreated plasma was added and mixed in a vortex mixer for 30 sec and used for standard curve preparation. A standard curve was prepared from working plasma standard in the range of 0.005-5 µg/ml.

After extraction of the drug, standard samples were injected into the HPLC system, and peak areas were recorded and plotted against respective known concentrations of plasma atorvastatin to obtain a linear regression line (r²=0.991).

FIG. 1: BLANK RAT PLASMA CHROMATOGRAM

Measurement of Plasma Atorvastatin Concentration: To the plasma sample (100 µl), internal standard (Diclofenac sodium) (20 µl) and methanol (400 µl) was added and vortexed for 1 min. After centrifugation (4000 rpm for 10 min), the supernatant was collected into a fresh eppendroff’s tube and evaporated to dryness and then reconstituted in 0.2 ml of methanol, 20 μl of this was injected into the High-Performance Liquid Chromatography (HPLC) system (Shimadzu LC-10AD) containing SPD-10A VP solvent delivery UV-Visible spectrophotometer detector.

The same extraction procedure was applied for the standard samples as well as those to be assayed for pharmacokinetic analysis ¹³. The chromatography column was C-18 (Phenomenox, USA; 100A size 250 × 4.60 mm) coated with 0.5 µ silica gel. The mobile phase used was an isocratic solution of acetonitrile: water of 60:40 v/v ¹⁴, which was filtered through 0.2-µm nylon filter paper. The flow rate was adjusted to 1 ml/min. The peak was detected at 247 nm. The retentions times of atorvastatin and diclofenac sodium (internal standard) were 6.09, 10.19 min, respectively Fig. 2.

FIG. 2: CHROMATOGRAM OF STANDARD ATORVASTATIN WITH 5μg/ml IN RAT PLASMA

Biochemical Analysis: Plasma lipid levels (TC, TG, and HDL-C) were determined using respective diagnostic commercial kits ^{15, 16} from Excel Diagnostics Pvt. Ltd., Hyderabad, India and LDL-C in plasma was calculated as per Friedewald estimation^{17, 18}, LDL-C (mg/dl)= TC (TG/5+HDL)-C.

Statistical Analysis: The pharmacokinetic data were statistically analyzed by Student t-test; KINETICA (version 5.0). Other data were analyzed statistically by one-way Analysis of Variance (ANOVA) with Dunnett’s test with significance level at 0.05 using Graph pad prism version 6.0.

RESULTS AND DISCUSSION:

Pharmacokinetic Analysis: Pharmacokinetic parameters like area under the curve [AUC], elimination half-life [t_1/2], volume of distribution [V_d], total clearance [CL], peak plasma concentrations [C_max], mean residence time [MRT] and time to reach peak plasma concentrations [T_max] were calculated for each subject using non compartmental pharmacokinetic model “KINETICA” (5.0 version). The plasma levels (mg/ml) of atorvastatin in groups treated with atorvastatin, atorvastatin-guggulsterone extract, and atorvastatin-guggulsterone capsules at different time points, on day 8^th were shown in Table 1, Fig. 3 and respective pharmacokinetic parameters were shown in Table 2, 3.

TABLE 1: COMPARISON OF MEAN ± SD PLASMA CONCENTRATION-PROFILE OF AT ALONE, AT+GE, AT+GC IN HYPERLIPIDEMIC RATS

All values expressed as Mean ± SD, n=6 ^a p<0.05 significant, ^b p<0.01 highly significant, ^c p<0.001 very highly significant

AT= Atorvastatin, GE= Guggulsterone standardized extract, GC= Guggulsterone capsule 

TABLE 2: PHARMACOKINETIC PARAMETERS OF PLASMA ATORVASTATIN ALONE AND IN COMBINATION WITH GUGGULSTERONE STANDARDIZED EXTRACT, GUGGULSTERONE CAPSULE ON DAY 8 (n=6)

 All values expressed as Mean ± SD, n=6 ^a p<0.05 significant, ^b p<0.01 highly significant, ^c p<0.001 very highly significant (Compared by one way ANOVA followed by Dunnett’s test).

FIG. 3: MEAN PLASMA CONCENTRATION (µg) OF ATORVASTATIN ALONE AND COMBINATION WITH GUGGULSTERONE STANDARDIZED EXTRACT AND GUGGULSTERONE MARKETED PREPARATION ON DAY 8^TH IN HYPERLIPIDEMIC RATS (n=6)

TABLE 3: MEAN PLASMA LIPID PROFILE CONCENTRATION (mg/dl) IN HYPERLIPIDEMIC RATS

All values expressed as Mean ± SD, n=6 ^a p<0.05 significant, ^b p<0.01 highly significant, ^c p<0.001 very highly significant

AT= Atorvastatin, GE= Guggulstearone standardized extract, GC= Guggulstearone capsule.

(TC=Total Cholesterol, TG=Triglycerides, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, VLDL-C=very low-density lipoprotein cholesterol) (Compared by one way ANOVA followed by Dunnett’s test)

FIG. 4: MEAN PLASMA LIPID PROFILE CONCENTRATION (mg/dl) IN HYPERLIPIDEMIC RATS. ^ap<0.05 significant, ^bp<0.01 highly significant, ^cp<0.001 very highly significant

Herb-Drug interactions have both a pharmacokinetic and pharmacodynamic basis, most of which is attributed to the induction or inhibition of hepatic and intestinal microsomal enzymes (primarily cytochrome P450), and drug transporters ¹⁹. Although, popular belief about the herbal products is that many of these preparations are considered natural and safe, they require attention for potential risk as they are pharmacologically active. Most of these herbal remedies can interact with allopathic drugs, resulting in altered activity and toxicity.

Some of the herbs that interact with Atorvastatin are red yeast Rice (Monascus purpureus), grapefruit, grape fruit juice, Garlic (Allium sativum) ¹¹.

Atorvastatin lowers plasma cholesterol and lipoprotein levels by inhibiting HMG CoA reductase and cholesterol synthesis in the liver and by increasing the number of hepatic low-density lipoprotein (LDL) receptors on the cell surface for enhanced uptake and catabolism of low-density lipoprotein (LDL), it also decreases triglycerides (TG) levels but, increases high-density lipoprotein (HDL) levels ²⁰. It was reported that the atorvastatin therapy produced statistically significant changes in total cholesterol, LDL-C, TG, and HDL-C within 24 hours ²¹.

The present study investigated the pharmacokinetic and pharmacodynamic interactions of atorvastatin with guggulsterone standardized extract and guggulsterone marketed preparation in hyper-lipidemic rats.

The observed peak plasma concen-trations (C_max) of atorvastatin was 2.43 ± 0.97 µg/ml at 2 h after oral administration on day 8, but in combination with guggulsterone, standardized extract or guggulsterone marketed preparation it was significantly changed to 0.88 ± 0.17, 0.68 ± 0.13 µg/ml (p<0.001) respectively.

The AUC of atorvastatin was significantly decreased to 63.45%, 82.27% when it was combined with guggulsterone standardized extract or guggulsterone marketed preparation.

While there was an increase in the clearance (CL) of atorvastatin in combination with guggulsterone standardized extract or guggulsterone marketed preparation. These results suggest that there is a significant pharmacokinetic interaction between atorvastatin and guggulsterone. In particular, guggulsterone (a CYP inducer) probably enhanced the metabolism of atorvastatin. Further, there was also a significant decrease in lipid levels of AT+GE, AT+GC with hyperlipidemic control, but the results were found to be less significant than atorvastatin alone.

CONCLUSION: In conclusion, the therapy of atorvastatin with guggulsterone alters the pharmacokinetics of atorvastatin in hyperlipidemic rats producing a significant change in lipid profiles when compared with hyperlipidemic control.

However, the hypolipidemic effect resulted from the combination therapy was found to be less significant than atorvastatin alone. Thus, it can be suggested that the combination of atorvastatin with guggulsterone has no potential benefits in safety, efficacy, and tolerability than individual drugs.

ACKNOWLEDGEMENT: The authors are thankful to the Principal and Management of Vaagdevi College of pharmacy for providing the necessary facilities.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Katrapally PK, Janki A, Reddy ARN and Reddy YN: Pharmacokinetic and pharmacodynamics of Atorvastatin alone and in combination with lercandepine in hyperlipidemic rats. Journal of Pharmacy Research 2009; 2: 66-70.
2. Jose MA, Anandkumar S, Narmadha MP and Sandeep M: A comparative effect of atorvastatin with other statins in patients of hyperlipidemia. Indian Journal of Pharmacology 2012; 44: 261-63.
3. Willrich MAV, Rodrigues AC, Cerda A, Genvigir FDV, Arazi SS, Dorea EL, Bernik MMS, Bertolami MC, Faludi A, Largura A, Baudhuin LM, Bryant SC, Hirata MH and Hirata RDC: Effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and CYP3A activity in hypercholesterolemic patients. Clinica Chimica Acta 2013; 421: 157-63.
4. Ramachandran C, Nair SM, Quirrin KW and Melnick SJ: Hypolipidemic effects of a proprietary Commiphora mukul gum resin extract and medium-chain triglyceride preparation (GU-MCT810). Journal of Evidence-Based Complementary & Alternative Medicine 2013; 18: 248-56.
5. Ji G, Zhao X, Leng L, Liu P, and Jiang Z: Comparison of dietary control and atorvastatin on high-fat diet-induced hepatic steatosis and hyperlipidemia in rats. Lipids in Health and Disease 2011; 10: 10-23.
6. Jurre Hageman J, Herrema H, Groen AK and Kuipers F: ATVB in focus: nuclear receptors in metabolism and cardiovascular disease-a role of the bile salt receptor fxr in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology 2010; 30: 1519-28.
7. Siddiqui MZ and Mazumder PM: Comparative study of the hypolipidemic profile of resinoids of Commiphora mukul/Commiphora wightii from different geographical locations. Indian Journal of Pharmaceutical Sciences 2012; 74: 422-27.
8. Siddiqui Z: Guggul: an excellent herbal panacea. Asian Journal of Pharmaceutical and Health Sciences 2011; 35-39.
9. Kumar KP, Reddy ARN, Anbu J and Reddy YN: Simultaneous determination of atorvastatin and lercanidipine in rat plasma by HPLC and pharmacokinetic studies. Asian Journal of Pharmacokinetics and Pharmacodynamics 2008; 8: 299-04.
10. Gulati R, Agarwal DK, Hossain MM, Ali R and Srivastava U: Study of serum lipid profile changes in Met-Enkephalin treated rats. Indian Journal of Physiology and Pharmacology 2003; 47: 357-60.
11. Reddy GD, Reddy AG, Rao GS and Kumar MV: Pharmacokinetic interaction of garlic and atorvastatin in dyslipidemic rats. Indian Journal of Pharmacology 2012; 44: 246-52.
12. Eliot LA and Jamali F: Pharmacokinetics and pharmacodynamics of nifedipine in untreated and atorvastatin-treated Hyperlipidemic rats. Journal of Pharmacology and Experimental Therapeutics 1999; 291: 188-93.
13. Bahrami G, Mohammadi B, Mirzaeei S and Kiani A: Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2005; 826: 41-45.
14. Sulthana N, Arayne MS and Naveed S: Simultaneous determination of enalapril and statin’s in pharmaceutical formulations by RP-HPLC. Journal of the Chilean Chemical Society 2011; 56: 734-36.
15. Mohamadin A, Elberry AA, Gawad HSA, Morsy GM and Al-Abbasi FA: Protective effects of simvastatin, a lipid lowering agent, against oxidative damage in experimental diabetic rats. Journal of Lipids 2011; 1-13.
16. Fukuyama N, Homma K, Wakana N, Kudo K, Suyama A, Ohazama H, Tsuji C, Ishiwata K, Eguchi Y, Nakazawa H and Tanaka E: Validation of the Friedewald Equation for Evaluation of Plasma LDL-Cholesterol. Journal of Clinical Biochemistry and Nutrition 2008; 43: 1-5.
17. Friedewald WT, Levy RI and Fredrickson DS: Estimation of low-density lipoprotein cholesterol in plasma, without the use of the preparative centrifuge. Clinical Chemistry 1972; 18: 499-02.
18. Stern RH, Yang BB, Hounslow NJ, MacMohan M, Abel RB and Olson SC: Pharmacodynamics and pharmaco-kinetic-pharmacodynamic relationships of atorvastatin, an HMGCOA reductase inhibitor. Journal of Clinical Pharmacology 2000; 40: 616-23.
19. Thomas AS, Varughese P, Shirwaikar A and Shirwaikar A: Herb-drug interactions: a review. J. D. Med. 2012; 4: 33-40.
20. Bae SK, Kim EJ, Chung SJ, Kim SG and Lee MG: Pharmacokinetics interaction between oltipraz and dimethyl-4-4-dimethoxy-5-6-5-6-dimethylene dioxybiphenyl-2-2-dicarboxylate (DDB) after single intravenous and oral administration to rats. Journal of Pharmacy and Pharmacology 2003; 55: 1241-49.
21. Pitt B, Loscalzo J, Monyak J, Miller E and Raichlen J: Comparison of lipid-modifying efficacy of rosuvastatin versus atorvastatin in patients with the acute coronary syndrome (from the LUNAR study). American Journal of Cardiology 2012; 109: 1239-46.
    

    ---

    How to cite this article:

    Sirisha K, Goverdhan P, Swarnalatha A and Sravanthi A: Pharmacokinetic and pharmacodynamic interactions of atorvastatin with guggulsterone in hyperlipidemic rats. Int J Pharm Sci & Res 2014; 5(10): 4262-68. doi: 10.13040/IJPSR.0975-8232.5(10).4262-68.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---