Effect of Verapamil on Serum Level of Salinomycin in Diabetic Rats

Problem statement: The aim of present study was evaluation function o f P-glycoprotein with or without verapamil in normal and diabetic ra ts; which the function of P-glycoprotein was indirectly evaluated by detection of serum slainomy cin concentration with HPLC method. Approach: This study was carried in 4 groups of rats includi ng normal rats which received salinomycin and verapamil together salinomycin; and diabetic rats which received salinomycin and verapamil together salinomycin. Serum concentration of salinomycin was measured by HPLC after 3 h from its administration. Results: Results show the serum concentration of salinomyci n significantly elevated in diabetic rats which received verapamil together salinomycin; while this concentration did not significantly change in other groups. Conclusion: Since the p-glycoprotein activity decreases in diabetic conditions and verapamil inhibits it; prob ably transport of salinomycin from blood to tissues or its elimination was decreased that caused its el evated serum concentration.


INTRODUCTION
P-glycoproteins (P-gp) are transmembrane carrier that prevents the cellular accumulation of some xenobiotics and endogenous compounds and is responsible for certain multidrug resistance mechanism in tumor cells (Aller et al., 2009). P-gp is belonged to ATP-binding cassette transport protein superfamily (ABCB1/MDR1) (De Lange, 2004). This pump is mainly expressed in organs that have an excretion (liver, kidney), absorption (intestine) or barrier function, such as the blood-brain barrier (Mercier et al., 2004;Hsiao et al., 2008). For example, P-gp inhibition at the rat BBB has been determined, when the rat is pretreated with cyclosporine A (as p-gp inhibitor) the brain/plasma ratio of verapamil is increased (Hsiao et al., 2006;Hendrikse and Vaalburg, 2002). Based on these data and others, it has been widely postulated that P-gp plays a vital role in limiting drug distribution at bloodbrain barrier and drug interactions will result in an important increase in brain concentrations of the affected drugs and, therefore, their CNS efficacy or toxicity (Neuhaus et al., 2010).
Salinomycin is a polyether antibiotic belonging to the group of ionophores. Salinomycin is extensively used as a coccidiostat in poultry and other livestock (Rajaian et al., 2009). Severe human poisoning with salinomycin has also been reported (Story and Doube, 2004). P-gp activity may affect the toxic exposure to salinomycin. Individuals with reduced or absent P-gp activity could therefore be more susceptible to salinomycin toxicity (Lagas et al., 2008). Verapamil, a calcium channel blocker, is substrate and inhibitor of pgp (Sulova et al., 2008).
This study was carried to evaluation function of Pglycoprotein with or without verapamil in normal and diabetic rats; which the function of P-gp was indirectly evaluated by detection of serum slainomycin concentration with HPLC method.

MATERIALS AND METHODS
This study was carried in 4 groups of rats (10 rats in each group). The male Wistar rats were purchased from laboratory animal center Jundishapour of Ahvaz. The age of rats was 12 weeks. The animals access to food and water ad libitum under 12h light and 12 dark conditions. Two groups of normal rats received salinomycin (1mg kg −1 orally) and verapamil (25mg kg −1 orally) (Bansal et al., 2009) and 1 h later salinomycin (1 mg kg −1 orally). Two groups of rats received sterepozocin (45mg kg −1 intravenously) for diabetes induction. One group of diabetic rats (those had blood glucose more than 600mg dl −1 ) received salinomycin (1mg kg −1 orally) and another group received verapamil (25mg kg −1 orally) and 1 h later salinomycin (1mg kg −1 orally). Serum concentration of salinomycin was measured by HPLC after 3 hrs from its administration in all rats. HPLC system was used from Shimadzu model (Japan) with column C 18 . Column temperature was set at 40c 0 . HPLC mobile phase A was water/acetonitrile (95:5, v/v), containing 0.1% formic acid; mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was 0.6 ml min −1 and the injection volume was 20 ml.

RESULTS
Results show minimum level of salinomycin was detected in serum of diabetic rats. Although this level did not significantly differ with normal group. The serum concentration of salinomycin significantly was enhanced in diabetic rats which received verapamil together salinomycin; while this concentration did not significantly change in other groups. The mean of serum concentration of salinomycin (± standard error) was illustrated at Fig. 1.

DISCUSSION
Our study shows the interaction salinomycin and verapamil changes in diabetic condition and increases serum level of salinomycin. It seems the some parts of this interaction are related to p-gp transporting. P-gp function alters in diabetic status (Wu et al., 2009). Nawa et al. (2011) evaluated p-gp expression and function in diabetic rats. They demonstrated the expression and function of p-gp significantly decreased after 9 days induction of diabetes (Nawa et al., 2011). Also Liu et al. (2006) and Maeng et al. (2007) demonstrated the function of p-gp decreased in the blood-brain barrier of streptozotocin-induced diabetic rats (Liu et al., 2006;Maeng et al., 2007). Insulin therapy restores impaired function and expression of Pgp in blood-brain barrier of experimental diabetes (Liu et al., 2008). But we did not demonstrate significant change at serum concentration of salinomycin in diabetic and normal rats. This may be related dieresis in diabetic rats and more excretion of salinomycin.
Salinomycin is a p-gp inhibitor (Riccioni et al., 2010). This drug inhibited p-gp in leukemic cells and increased cell death (Fuchs et al., 2010). In other hands, Verapamil is P-gp inhibitor (Bansal et al., 2009;De Klerk et al., 2010). Administration of verapamil enhanced uptake of dextromethorphan in the CNS of rats (Marier et al., 2005). We demonstrated coadministration of salinomycin and verapamil significantly increased serum level of salinomycin in diabetic rats. We think this enhancing may be related inhibition of salinomycin transport into kidney and CNS by verapamil and diabetic condition. But exact conclusion deeds more detailed studies.

CONCLUSION
Since the p-glycoprotein activity decreases in diabetic conditions and verapamil inhibits it; probably transport of salinomycin from blood to tissues or its elimination was decreased that caused its elevated serum concentration.

ACKNOWLEDGMENT
The researchers wish to express their gratitude to the research council of Shahid Chamran University for their financial supports.