Synthesis, characterization, and pharmacological evaluation of some metal complexes of quercetin as P-gp inhibitors

Six different metal complexes of quercetin (Cu, Zn, Co, Vd, Mo, Ni) were synthesized, purified, and characterized by their physical and spectral (UV, IR) data. They were evaluated for their P-gp (permeability glycoprotein) inhibitory activity by in vitro everted sac method in rats. The apparent permeability of atorvastatin (P-gp substrate) from everted sac of the rat intestine was determined in control, standard (verapamil), and groups treated with quercetin-metal complexes. The drug contents were analyzed by validated RP-HPLC method using a mixture of acetonitrile and water (60:40 v/v) adjusted to pH 2.8 with phosphate buffer as mobile phase. In vitro studies revealed that the apparent permeability of atorvastatin (P-gp substrate) across the small intestine is much affected by the treatment with Cu/Co/Ni complexes of quercetin. The mean ± SD and apparent permeability of atorvastatin decreased after pre-treatment with these metal complexes. The quercetin Cu/Co/Ni complexes could inhibit P-gp and increase the atorvastatin absorption. Hence, they could be considered P-gp inhibitors.


Background
Cancer is a dreadful disease, killing a large number of the population worldwide. More than 100 different types of cancer are reported to affect humans [1,2]. Chemotherapy is widely used for cancer treatment but it is hindered mostly due to the resistance of tumor cells to anticancer drugs [3,4]. Several mechanisms underlying drug resistance were identified. Increased efflux of drugs by cancerous cells, due to over expression of membrane transporter proteins (efflux pumps) is one of the major mechanisms documented. P-glycoprotein (P-gp) is the first discovered multidrug transporter that pumps drugs out of tumor cells, resulting in decreased intracellular drug concentrations and thus reducing the efficacy of drugs [5]. It is present in several normal tissues like intestinal lining epithelium, endothelial cells, and bone marrow.
Quercetin (Q) is a major naturally occurring flavonoid, belonging to the class of flavonols. It is ubiquitously found in a wide variety of plant products like coffee, tea, dyes, vegetables, and fruits [6]. The beneficial effects of quercetin are mostly due to its free radical scavenging or antioxidant property and its ability to chelate metal ions (Fe 2+ and Fe 3+ , Cu 2+ , Ni 2+ ) [7][8][9][10][11][12]. Quercetin and some of its metal complexes displayed various biological actions such as antimicrobial, antiulcer, antiallergic, anti-Alzheimer's, and anticancer [13][14][15][16][17][18]. It was reported that quercetin could competitively inhibit the members of MDR family, P-gp, MRP1, and BCRP [19][20][21][22][23]. But, hitherto, there are no reports on the P-gp inhibitory activity of quercetin-metal complexes. In this regard, the present work was aimed to synthesize, characterize, and pharmacologically evaluate the metal (M) complexes of quercetin (M=Cu, Zn, Mo, VO, Ni, Co) for their P-gp (Pglycoprotein) inhibition activity by in vitro rat everted sac method in Wistar albino rats [24,25], as this model despite a few limitations is an economic, simple, and highly efficient tool in investigating the effect of P-gp inhibitors across the small intestine of rats and serves to predict the drug absorption in humans.

Drugs and chemicals
The drug atorvastatin was purchased from Stallion Laboratories Pvt. Ltd., Ahmedabad; verapamil was purchased from Ranbaxy Laboratories Ltd., Hyderabad; and quercetin was obtained from Sigma Aldrich Chemicals, Bangalore. HPLC-grade methanol and acetonitrile and BDH-grade of the chemicals copper chloride dihydrate, zinc chloride hexahydrate, ammonium molybdate, vanadyl sulphate monohydrate, nickel chloride hexahydrate, cobaltous chloride hexahydrate, and hydrochloric acid were purchased from Qualigens Fine Chemicals, Mumbai. Dimethyl sulfoxide, sodium hydroxide, and hexane were obtained from Finar Chemicals, Mumbai. Dulbecco's phosphate buffer pH of 7.4 was purchased from Hi Media Ltd., Mumbai, and HPLC-grade water was procured from S.R. Enterprises, Hanamkonda.

Spectral analyses of quercetin complexes
UV-visible study of quercetin and its metal complexes (Cu, Zn, Mo, VO, Ni, Co) were carried out in the wavelength range of 200 to 500 nm by using UV-visible spectrophotometer. Infrared spectral study of these complexes was performed using IR spectrophotometer and the obtained spectral data were analyzed [9,26,27].

Preparation of quercetin-metal complexes
The quercetin-metal complexes have been synthesized and prepared according to previously reported method [28].The Cu(II), Zn(II), Ni(II), and Co(II) complexes were prepared using respective metal chlorides; VO(IV) complex was prepared using vanadyl sulphate; and Mo(IV) complex was prepared using its ammonium salt.
In the preparation of all the metal complexes, the metal and the quercetin compounds were combined in 1:2 mole ratio (the metal being in slight excess of the desired ratio) using appropriate quantities of aqueous methanolic quercetin and methanolic or aqueous metal salt solutions, respectively. The pH of the solutions was adjusted to 10 by adding sodium hydroxide solution.
General procedure for the preparation of quercetin-metal complexes

Pharmacological evaluation Everted rat sac method
The in vitro everted sac technique was introduced by Wilson and Wiseman [29] and later modified into modern everted sac technique. It is a simple, renowned tool to study various pharmacokinetic phenomena like absorption, metabolism, and interaction of drugs and to carry out studies related to multidrug resistance (MDR), role of efflux transporters, and the effect of MDR modulators on drug absorption in the gut. This model involves the gentle eversion of a freshly excised portion of rat small intestine using a glass rod, clamping, and tying one end of the segment and filling it with appropriate solution, sealing the other end tightly, immersing the obtained sac in physiological solution maintained at 37°C, collecting the samples at different time intervals, and analyzing the results obtained. The everted sac technique measures the serosalto-mucosal transport of drug substrates [30,31] unlike the non-everted sac technique which measures the mucosal to serosal transport of drug substrates [32].
The animals were overnight fasted and anaesthetized using isoflurane (2-3% by drop jar method). After anaesthetizing the animals, they were euthanized with cervical decapitation and the whole small intestine of each animal was isolated and flushed with 50 ml of ice-cold saline. The small intestine was divided into duodenum, jejunum, and ileum (n = 3). Segments were everted, and 10-cm-long sacs were prepared. About 1 ml of atorvastatin    [33,34]. The Everted sac model, i.e., intestinal serosal-to-mucosal transport of atorvastatin was measured by the periodic collection of samples from the mucosal medium at different time intervals (0, 10, 30, 45, 60 min). All the samples were mixed with acetonitrile in 2:1 ratio, vortexed (2 min), and centrifuged at 3000 rpm (15 min). Aliquots of supernatant were separated, filtered through membrane filter (0.22-μm pore size) and preserved at -20°C until analysis. Twenty microliters of it was injected into the HPLC column using a Hamilton syringe. The transport of atorvastatin in the absence of P-gp inhibitor (control), in the presence of Q-M complexes (test) or verapamil (standard) was measured by validated reverse-phase high-performance liquid chromatography (RP-HPLC) method [35]. Apparent permeability coefficients (P app ) of atorvastatin were calculated from the equation [36] given: where dQ/dt is the steady-state appearance rate on the acceptor solution A is the surface area of the intestinal sacs (7.85 cm 2 / sac) C 0 is the initial concentration of drug (inside the sac)

Sensitive RP-HPLC method Chromatographic conditions
Acetonitrile and water in the proportion of 60:40 (v/v) adjusted to pH 2.8 with phosphate buffer was used as mobile phase, stationary phase was a C-18 reverse-phase column of 250 × 4.6 mm dimension, and 5 μ particle size, flow rate was 1 ml/min at λ max of 247 nm, run time was < 15 min, injection volume was 20 μl, and AUFS (absorbance units full scale) was 0.005.

Preparation of calibration curve of atorvastatin Preparation of stock solutions
A stock solution representing 1 mg/ml of atorvastatin was prepared in methanol. The working standard solutions were prepared prior to use from stock solution by sequential dilution with methanol to yield final concentrations of 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 5μg/ml of atorvastatin.

Construction of calibration curve
The calibration curve was constructed using peak height ratios of the drug versus concentration [37]. The slope of the plot determined by the method of linear regression analysis was used to calculate the atorvastatin

Statistical analysis
The in vitro results were compared by Student's paired t-test using Graph Pad Instant Software version-5.0. A value of p < 0.05 was considered statistically significant.

Chemistry
Addition of aqueous sodium hydroxide during the synthesis of metal complexes resulted in the deprotonation of the OH groups and thus favored the stability of quercetin at pH 10. All the metallic salts were soluble in the reaction mixture except cobaltous chloride hexahydrate which was dissolved using dimethyl formamide. All the quercetin-metal complexes were obtained in good yields ranging from 80.39 to 84.64% as indicated in Table 1.

UV-visible study of the quercetin-metal complexes
The UV-visible spectrum of quercetin in DMSO and phosphate buffer shows two major absorption bands due to π-π* transitions at 369 (band-I) and 252 nm (band-II) ( Table 2). When the solution of a metallic salt was added, bands I and II gradually shifted to longer wavelengths, accompanied with slight decrease in absorption. The results indicated formation of a complex between quercetin and respective metal ions. Band-I bathochromic shift (401 nm) is a common feature observed with all the quercetin-metal complexes. It could be due to the interaction of metal ions (Cu, Zn, Co, VO, Mo, and Ni) with the 3-hydroxy and 4-

RP-HPLC method of analysis
In the present study, the atorvastatin concentrations were estimated by a sensitive RP-HPLC method ( Table  3). The calibration curve of atorvastatin was plotted with concentration versus peak height as shown in Fig. 1. The typical chromatograms corresponding to solvent buffer and atorvastatin in the absence and presence of verapamil and in the presence of quercetin-metal complexes at different time points of the experimental study are depicted in Figs. 2, 3, 4, 5, and 6. The separation of both peaks (solvent and atorvastatin) was satisfactory, with retention times of 2.46 min and 3.30 min, respectively. Tables 4, 5, and 6 reveal the apparent permeability coefficients (P app ) of atorvastatin alone (control) and in presence of verapamil (standard) and the quercetinmetal complexes (test) in the everted duodenum, jejunum, and ileum, respectively.

Discussion
Clinically, multidrug resistance (MDR) is one of the major causes for chemotherapeutic treatment failure in cancer patients. Several strategies for overcoming drug resistance have been studied, including (1) developing reversal agents which target drug resistance markers such as P-gp and MRP1, (2) developing new agents to which drug-resistant cancer cells are sensitive, (3) employing agents to which drug-resistant cancer cells show collateral sensitivity, and (4) developing antiangiogenesis therapy and gene therapy for drug-resistant cancer cells [39]. Among them, developing reversal agents which target P-gp has been a principal strategy.
Multidrug resistance of cancer cells is often associated with over expression of P-gp, a plasma membrane transporter that extrudes chemotherapeutic drugs by using ATP hydrolysis as the energy source. P-gp is encoded by the MDR1 gene and functions as an energy-dependent multidrug membrane transporter that rapidly extrudes a variety of hydrophobic anticancer drugs from target cancer cells and thereby prevents the drugs from exerting cytotoxic effects. Since the first P-gp inhibitor, verapamil, was found in 1981, a variety of agents including cyclosporin A (CysA) and others have been reported as agents for overcoming MDR [40].
However, the clinical trial results of these drugs are very disappointing so far, due to dose-limiting toxicity. Therefore, it is necessary to find new reversing agents with lesser side effects. Natural sources come under this category of having no dose-limiting toxicity and lesser side effects [41,42]. Several members of flavonoids were found to be able to modulate drug efflux in MDR cancer cells, such as genistein, quercetin, and naringenin [43,44]. The flavonoid quercetin was shown to inhibit P-gp-mediated drug transport by inhibiting the ATP binding site of P-gp. It was reported to decrease the expression of P-gp significantly in multidrug-resistant human cervical carcinoma (KB-V1) cells at the concentration of 30 μM [45].
Experimental studies proved that quercetin exhibits both P-gp inhibitory and antitumor activities and it also  has the ability to chelate metal ions [16]. Further, some metal complexes show P-gp inhibitory activity [46][47][48]. But till now, there are no reports on P-gp inhibitory activity of quercetin-metal complexes.
In the present study, we have synthesized metal (Cu, Co, Ni, Zn, Mo, VO) complexes of quercetin in high yields and purity and characterized them by using UV-visible and IR spectral data. From spectroscopic studies, quercetin was found to chelate metal ions (Cu, Co, Ni, Zn, Mo, VO) at the 3-hydroxy and 4-carbonyl groups. They were pharmacologically evaluated for their P-gp inhibitory activity by in vitro everted sac method in rats by comparing the apparent permeability of P-gp substrate (atorvastatin) with that of standard P-gp inhibitor, verapamil.
Results of this in vitro study revealed that the transport of atorvastatin (P-gp substrate) across the small intestine is much affected by the Q-Cu, Q-Co, and Q-Ni complex treatment. The mean ± SD and apparent permeability of atorvastatin decreased in the presence of the Q-Cu, Q-Co, and Q-Ni complexes. A time-dependent effect was observed. Also, a significant reduction in the Papp values was observed in the everted sacs treated with Q-Cu, Q-Co, and Q-Ni complexes as compared with controls. All the three metal complexes were found to be more potent than the standard verapamil at all three sites of small intestine (i.e., duodenum, jejunum, and ileum) at only one-tenth dose of verapamil. Among the three complexes, Q-Ni complex exhibited superior inhibitory activity on the permeability glycoprotein (P-gp). Next to it, Q-Co complex significantly reduced the transport of atorvastatin across the intestinal membranes. The rest of the complexes (Q-Mo, Q-Zn, Q-VO) did not show any effect on the transport of atorvastatin and hence considered inactive. The study revealed that Q-Cu, Q-Co, and Q-Ni metal complexes could inhibit P-gp (at much lesser concentrations than verapamil) and increase the absorption of atorvastatin.

Conclusion
The transport of atorvastatin (P-gp substrate) across the small intestine is much affected by the Q-Cu, Q-Co, and Q-Ni complexes treatment. The treatment of sacs with these metal complexes decreased the intestinal permeation of atorvastatin (P-gp substrate) when compared with that of standard P-gp inhibitor and control, which may be due to inhibition of P-glycoprotein. Hence, they could be considered P-gp inhibitors and studied further.