Formulation
Quercetin (batch no.: 45092, formula: C15H10O7, CAS no. 117-39-5) and its lecithin formulation Quercetin Phytosome® (QUERCEFIT™, batch no.: 06/16/PA) used in the study were prepared and provided by Indena SpA (Milan, Italy). Quercetin Phytosome consists of quercetin and sunflower lecithin in a 1:1 weight ratio along with about a fifth part of food-grade excipients that are added to improve the physical state of the product and to standardize it to a HPLC-measured total quercetin content of about 40% (patent application no. 171816341).
For the clinical study, 500 mg of quercetin were formulated by Indena SpA into film-coated tablets containing anhydrous calcium phosphate (Di-Cafos® A150, Budenheim, Germany), hydroxypropylmethylcellulose (Methocel™ E15, Dow, Germany), silicon dioxide (Syloid® 244FP, Grace GmbH, Germany), polyvinylpolypyrrolidone (Kollidon® CL, BASF, Germany), talc (Microtalc Pharma 50, Mondo Minerals BV, Netherlands), and magnesium stearate (Ligafood®, Peter Greven, Netherlands). For the clinical study, 250 mg of Quercetin Phytosome were formulated by Indena SpA into film-coated tablets containing anhydrous calcium phosphate, Isomalt (GalenIQ™ 960, Beneo GmbH, Germany) polyvinylpolypyrrolidone, silicon dioxide, talc, and magnesium stearate.
All tablets were coated with a hydroxypropylmethylcellulose-based film coating system (Opadry White, Colorcon Inc., USA). Before releasing the film-coated tablets containing quercetin (batch no. 89107) or Quercetin Phytosome (batch no. 89108), their appearance, average mass, uniformity of mass, HPLC-measured content of quercetin, disintegration time, and microbiological quality were tested.
Solubility Study
The solubility of Quercetin Phytosome was determined under saturation conditions and compared with that of unformulated quercetin under the same saturation conditions and in the following simulated gastrointestinal media: FaSSGF pH 1.6 (fasted-state simulated gastric fluid), FaSSIF pH 6.5 (fasted-state simulated intestinal fluid), and FeSSIF pH 5.0 (fed-state simulated intestinal fluid). In order to clarify the influence of the manufacturing process of Quercetin Phytosome on the solubility of quercetin, a physical mixture with the same quali/quantitative composition as Quercetin Phytosome was also prepared and submitted to the solubility study. Biological media (Biorelevant.com, London, UK) were prepared according to the manufacturer’s instructions. In order to ensure that the quercetin concentrations in all of the samples were very similar, about 20 mg of quercetin, about 50 mg of Quercetin Phytosome and about 50 mg of the physical mixture, with the same composition as Quercetin Phytosome, were added to 10 ml of each simulated biological fluid. The resulting suspensions were left for 2 h at room temperature under constant magnetic stirring. After that period, an aliquot of each suspension was filtered through a 0.2-µm polytetrafluoroethylene (PTFE) syringe disposable filter, and 1 µl of the clarified solution was injected and analyzed for its content of quercetin by ultra performance liquid chromatography (UPLC). Chromatographic separation of quercetin was achieved by a reversed-phase UPLC method, with UV detection at 371 nm, using a Waters Acquity UPLC system. Briefly, a Waters Acquity BEH C18 column (100 mm × 2.1 mm, particle size 2.7 µm) kept at 27 °C was eluted at constant flow of 0.368 ml/min with a gradient of H3PO4 0.3% in water as solvent A and acetonitrile as solvent B according to the following timetable (linear gradient): initial conditions B 15%; 1.55 min B 15%; 5.33 min B 44%; 7.59 min B 50%; 8.73 min B 55%; 9.10 min B 95%; 10.60 min B 95%; total run time (including the re-equilibration step): 12.00 min. The samples were kept at 5 °C in silanized glass vials prior to analysis.
Clinical Study
A single-dose, randomized, six-sequence/three-period crossover clinical trial (3 × 3 × 3 crossover design) with a balanced carryover effect was performed in healthy volunteers under fasting conditions to evaluate the oral absorption of the Quercetin Phytosome in comparison to that of quercetin.
Subjects
Healthy volunteers of both sexes who were within the age range of 18–50 years inclusive and had body mass index values within the range 18.5–27 participated in the study. No evidence of significant organic or psychiatric diseases (based on history, physical examination, and additional tests) was observed, and negative serology for hepatitis B (HBV) and C (HCV) viruses as well as for human immunodeficiency virus (HIV) was verified. Laboratory tests (blood count, biochemistry, and urine sediment) were performed according to the normal reference values of the laboratory of biochemistry, University Araba Hospital, Txagorritxu headquarters, Vitoria-Gasteiz. Whether variations were acceptable depended on the clinical judgment of the investigator. Vital signs (blood pressure, heart and respiratory rate, temperature) and ECG results were monitored to ensure that they were within normal limits, and pregnancy tests were performed in the women by determining plasma human β-chorionic gonadotropin (β-HCG) levels during the selection phase and in urine before each experimental period.
The study was carried out in accordance with the relevant guidelines of the Declaration of Helsinki (1964) and its amendments and the general principles of the ICH Harmonised Tripartite Guidelines for Good Clinical Practice (ICH Topic E6, CPMP/ICH/135/95). At the beginning of the study, written informed consent (reviewed by the ethics committee) was obtained from all individual participants included in the study, which was performed in accordance with the ICH-GCP, the Declaration of Helsinki, and the regulatory and legal requirements of Spain. The Spanish Research Ethics Committee of Araba University Hospital (Vitoria-Gasteiz, Alava, Spain) approved the study on 20th January 2017.
Study Design
A randomized crossover pharmacokinetic clinical study of the three different treatments of quercetin administered in a single dose to healthy volunteers under fasting conditions was performed. Patients were told not to consume quercetin-containing foods from at least 72 h prior to day 1 until the end of the study. There was no control group who used a placebo or another treatment; each individual acted as his/her own control. The volunteers were selected, and in each case an anamnesis was compiled and a physical examination, ECG, and analytical tests were performed. Participants were asked to refrain from excessive consumption of quercetin-containing foods (a list of quercetin-containing foods was provided to them) at least from 72 h prior to day 1 until the end of the study. Each experimental session required hospitalization, and participants remained in hospital under the supervision of qualified personnel for up to 12 h after the administration of the product, with subsequent monitoring at 24 h post-administration. Additional tests for substance abuse and pregnancy were performed before the administration of the product.
On each experimental day (a day when the product was given), an indwelling cannula for blood sample collection in a forearm vein was placed, the product was administered orally, and blood sampling was performed at the predefined times.
At least 1 week after completing the third experimental period, the final examination was performed. This involved a new clinical and analytical evaluation of all participants, including a new physical examination, ECG, and analytical tests that were similar to the original set of tests but with the serology (hepatitis, HIV), substance abuse test, and β-HCG determination omitted. Therefore, clinical safety (evaluation of vital signs and adverse systemic effects) and biological safety (evaluation of each subject’s blood count and blood chemistry results) were monitored to determine the tolerability of the treatments.
Each volunteer received one film-coated tablet of quercetin 500 mg (treatment A, batch no. 89107), one film-coated tablet of Quercetin Phytosome 250 mg (treatment C, batch no. 89108), and two film-coated tablets of Quercetin Phytosome 250 mg (500 mg total; treatment B, batch no. 89108) on the three experimental days, administered according to a previously randomized sequence. Treatments were known to the investigator (the clinical research products were identified as A, B, or C), but the aliquoted samples for pharmacokinetic analysis were not labeled according to the treatment applied, i.e., the analyst was blinded to the treatment associated with each sample.
A balanced carryover effect in healthy volunteers was realized, with washout periods of at least one week between the three treatment periods.
In order to determine quercetin levels, blood samples were collected at the following 12 time points: before dosing (time 0), at 15, 30, 45, and 60 min, and at 2, 3, 4, 6, 8, 12, and 24 h after the administration of the compounds.
Sample Preparation and Analysis
Blood and blank samples (8 ml each) were transferred to pre-labeled K2-EDTA Vacutainer tubes on ice. After centrifugation, 1 ml of each plasma sample was mixed with 100 µl of ascorbic acid solution 10% v/v as a preservative and stored at − 80 °C until it was analyzed. Pretreatment of the blank human plasma with ascorbic acid (10% v/v) was necessary to stabilize the quercetin before the extraction process. Samples (220 µl) were analyzed using a method previously validated by Kymos Pharma Services S.L. Briefly, the free quercetin component was determined by HPLC MS/MS (HPLC: Agilent series 1100 with a Luna C18(2) column, 5 μm 4.6 × 50 mm, Phenomenex 008-4252-EO; MS: MDS Sciex API-3200 equipped with a TurboIonSpray ion source, used in conjunction with the Analyst software package, version 1.4.2) after a liquid–liquid extraction with ethyl acetate. The same procedure was applied for total quercetin (free and conjugated), but the samples were subjected to enzymatic hydrolysis by β-glucuronidase from Helix pomatia (BBI Enzymes GH2G) before liquid extraction. Analysis was performed in the presence of the internal standard quercetin-d3. The concentration range of the method, in which a linear fitting model (1/χ2) was applied, was set from the lower limit of quantification (1 ng/mL) to 1000 ng/mL. The method proved to be selective, linear, precise, and accurate when applied to quercetin determination. The plasma samples were analyzed in four chromatographic batches for free quercetin and six chromatographic batches for total quercetin. Each batch included a set of calibration standards (concentration range: 1–1000 ng/mL), blanks (blank human plasma), a zero sample (blank spiked with internal standard), and quality control samples at three different concentrations (nominal concentrations of 3, 30, and 800 ng/mL). The chromatographic batches were accepted if they complied with the acceptance criteria defined for the calibration curve and quality control samples. Quality control samples corresponding to three concentration levels (3, 30, and 800 ng/mL) were prepared in each validation batch. In each chromatographic batch, the minimum number of quality control samples (in multiples of three) was at least 5% of the number of unknown samples or six quality control samples, whichever was greater.
Plasma concentrations were analyzed by a noncompartmental model, and the following major pharmacokinetic parameters were calculated by Phoenix WinNonlin (v.6.4; Pharsight): Cmax (maximum plasma concentration), Tmax (time to achieve Cmax), AUClast (area under the plasma concentration vs time curve), t1/2 (elimination half-life), and MRT (mean residence time).
Statistical data analysis was performed by two-way ANOVA with repeated measures followed by post hoc analysis (Tukey’s test).