Pharmacokinetics and Tissue Distribution of a Water-Soluble Flavonol Triglycoside, CTN986, in Mice

• Abstract
• Introduction
• Materials and Methods
• Chemicals and reagents
• Animals
• Pharmacokinetic profile and absolute bioavailability
• Tissue distribution
• Analytical technology
• Data analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

The pharmacokinetics and bioavailability of CTN986, a highly water-soluble flavonol triglycoside that has shown interesting antidepressant effects, were determined in mice after intravenous (i. v.), intraperitoneal (i. p.) and oral administrations. Concentrations of CTN986 in the biological samples were determined by a validated LC/MS/MS method. Non-compartmental methods were used to perform pharmacokinetic data analysis. The dose-dependent pharmacokinetics of CTN986 was characterized after i. v. administrations (0.16, 0.4 and 1.0 mg/kg) to mice. There was no significant difference in clearance (CL) with increasing dose [252.66 ± 42.82 mL/h (0.16 mg/kg) versus 241.73 ± 14.93 mL/h (1.0 mg/kg)] after i. v. administrations. The absolute bioavailability of CTN986 after i. p. and oral administrations was 96.57 % and 1.31 %, respectively. CTN986 was found to distribute widely in the internal organs of mice 10 min after oral dosage, with tissue concentrations in the order of duodenum, stomach, small intestine, kidney, lung, liver, brain, heart and spleen (from the highest to the lowest). In conclusion, CTN986 could be absorbed and extensively distributed into tissues as its intact form in mice.

Introduction

Flavonoids are a large group of natural polyphenols that exist ubiquitously in the plant kingdom [1]. A variety of beneficial pharmacological properties have been attributed to flavonoids, including antioxidant, anti-inflammatory, anticarcinogenic, antidepressant, chemopreventive, and cytochrome P450-inhibitory activities [2], [3]. Minor differences in the structures of flavonoids often have profound effects on their biological activities. Much attention is now being paid to flavonoids because of their pleiotropic effects. More recently, research has been focused on the absorption and bioavailability of flavonoids. Serial reviews on the absorption, metabolism, and bioactivity of flavonoids have been published [4], [5], [6]. The extent and the form of absorption of flavonoids, after oral administration, are important unsolved problems in studying their potential health effects. However, data on flavonoid bioavailability are still contradictory and the mechanism and efficacy of their absorption remains uncertain [7], [8]. Furthermore, it has been reported that the sugar moiety and plant matrix affect significantly the amount of intestinal absorption of quercetin [9], [10], [11].

Quercetin 3-O-β-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (code name CTN986, [Fig. 1]), a highly water-soluble flavonol triglycoside isolated from glandless cotton seeds in our laboratory [12], showed notable antidepressant effects in forced swimming experiments on mice [13]. It may be worth examination as a new potential antidepressant. To further understand the biological effects of CTN986, information about its pharmacokinetics and metabolism are required. Because CTN986 is present as a glycoside conjugate, a major concern is whether its absorption can occur in the small intestine without splitting of the β-glycoside bond. Despite extensive research on the pharmacological activities of flavonoids, little is known about the pharmacokinetics of flavonol triglycosides. The main cause hindering the pharmacokinetic study was possibly the lack of highly sensitive quantification methods. In particular, CTN986 is more hydrophilic and structurally more complex than the flavonoids that have been studied, so the research about its metabolism and pharmacokinetics in vivo is a challenging task. We have developed an LC/MS/MS method for quantitating CTN986 and its two deglycosylation products, namely rutin and hirsutin, in serum [14]. In this paper, we report the results of pharmacokinetics and tissue distribution studies of CTN986 in mice using the established LC/MS/MS method. The results obtained in the present study should be helpful for the better understanding of its pharmacological activities and underlying mechanisms, and should provide more information about the pharmacokinetics of flavonol triglycosides.

Materials and Methods

Chemicals and reagents

CTN986, CTN987 (internal standard, IS) and hirsutin were isolated from glandless cotton seeds in our laboratory, and their structures were confirmed by ¹H- and ¹³C-NMR spectroscopy. Their purities were all above 98 % as checked by RP-HPLC. Rutin was purchased from Acros Organics. Bakerbond SPE cartridges (C₁₈, 100 mg, 1 mL) were purchased from J.T. Baker Corporation. Methanol and 2-propanol (HPLC grade) were purchased from Fisher Scientific. All other chemicals and solvents used were of analytical grade and were purchased from Beijing Chemical Reagent Company. Milli-Q deionized water was used throughout the study.

Animals

ICR mice (male and female, 18 - 22 g) were purchased from the Animal Center of the Academy of Military Medical Sciences, Beijing. The experimental protocol was approved by the Institutional Ethic Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals. Mice were housed under standard environmental conditions (22 ± 2 °C, relative humidity 55 ± 5 %, 12-h light-dark cycle) with free access to standard diet and water ad libitum.

Pharmacokinetic profile and absolute bioavailability

Mice were randomized into five treatment groups: Group 1 (n = 33) received 0.16 mg/kg CTN986 i. v. via the tail vein as a bolus injection. Group 2 (n = 33) received 0.4 mg/kg CTN986 i. v. via the tail vein as a bolus injection. Group 3 (n = 33) received 1.0 mg/kg CTN986 i. v. via the tail vein as a bolus injection. Group 4 (n = 33) received 1.25 mg/kg CTN986 i. p. as a bolus injection. Group 5 (n = 38) received 80.0 mg/kg CTN986 orally by means of a stainless steel gastric intubation tube. Solutions were prepared freshly before treatment by dissolving CTN986 in physiological saline.

Three groups of mice were injected intravenously without fasting, and blood samples were collected before and post-dosing at 0.03, 0.08, 0.17, 0.33, 0.67 and 1.0 h. The other two groups were administered after an overnight fast. Blood samples were collected before dosing and at 0.08, 0.17, 0.33, 0.67, 1.0 and 2.0 h after i. p. administration, and at 0.08, 0.17, 0.33, 0.67, 1.0, 2.0 and 4.0 h after oral administration, respectively. Serum was separated from each sample by centrifugation at 5000 × g for 10 min, and kept frozen at -20 °C until analysis.

Tissue distribution

Tissue samples were taken from the internal organs of the orally administered mice at 0.17, 0.5, 1.0 and 3.5 h after a single oral dose of 80 mg/kg CTN986. The important tissues including heart, brain, liver, kidney, lung, spleen, stomach and intestine were collected immediately after cervical dislocation. Samples of tissue were rinsed, dried, minced and homogenized in methanol (g : mL = 1 : 3). After centrifugation, aliquots of supernatants were collected and stored at -20 °C until analysis.

Analytical technology

The concentrations of CTN986 and its two deglycosylation products rutin and hirsutin in serum were determined using our previously developed LC/MS/MS method [14]. In brief, the serum was acidified with 0.25 M phosphoric acid and then extracted using C₁₈ extraction cartridges. Tissue homogenates were treated in the same way. The analytes were injected onto a reversed phase C₈ column using a mobile phase consisting of methanol/2-propanol/water/formic acid (20 : 10 : 70 : 0.1, v/v/v/v). Drug and IS were detected by an API 3000 mass spectrometer (Applied Biosystems/ MDS Sciex). The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring in the eletrospray ionization source. The assay was linear up to a concentration of 1000 ng/mL with a lower limit of quantitation of 2 ng/mL.

Data analysis

The pharmacokinetic parameters were calculated by non-compartmental methods using WinNonlin software version 4.1 (Pharsight Corporation). The absolute oral and intraperitoneal bioavailability (F) was determined as F = (AUC_po/AUC_iv) × 100 % and F = (AUC_ip/AUC_iv) × 100 %, respectively. Differences in pharmacokinetic parameters and the regression analysis of the AUC _0-t -dose plot were assessed using SPSS software (version 10.0).

Results

The mean serum concentration-time profiles of CTN986 after i. v. administration of different doses to mice are shown in [Fig. 2]. The pharmacokinetic parameters obtained by a non-compartmental model analysis using the WinNonlin software are summarized in [Table 1]. After administration of 0.16, 0.4 and 1.0 mg/kg of CTN986, a good linearity (r = 0.993, P < 0.05) was found in the regression analysis of the AUC _0-t -dose plot. The dose-dependent pharmacokinetics of CTN986 was characterized after i. v. administration to mice. No significant difference was observed in CL with increasing dose [252.66 ± 42.82 mL/h (0.16 mg/kg) versus 241.73 ± 14.93 mL/h (1.0 mg/kg)]. The CL of CTN986 in mice was high, indicating a high clearance rate of CTN986 in vivo.

The curve of mean serum concentration versus time of CTN986 after i. p. administration to mice is shown in [Fig. 2] and the pharmacokinetic parameters are listed in [Table 1]. The absolute i. p. bioavailability of CTN986 was high (96.57 %). After a single i. p. administration of CTN986, rapid and nearly complete i. p. absorption in mice can be defined with the mean T_max of 0.08 h and the mean i. p. bioavailability of 96.57 %. The rapid absorption of CTN986 in mice was also indicated by the short difference in MRT values obtained after i. v. and i. p. dosing (approximately a difference of 0.1 h for mean MRT_0-∞).

The flavonol triglycoside CTN986 was detected in its original form after the oral administration and no deglycosylation products, namely rutin and hirsutin, were detected in the serum samples. The mean serum concentration-time profile ([Fig. 2]) and the pharmacokinetic parameters ([Table 1]) after oral administration of 80 mg/kg CTN986 to mice showed that a peak value (122.90 ng/mL) appeared shortly after the administration (0.27 h). The concentration of CTN986 in serum dropped rapidly with a half-life of 0.82 h. The absolute oral bioavailability of CTN986 in mice was low (1.31 %). This was consistent with the previous pharmacological results of CTN986 in mice that the oral dose markedly exceeded the i. v. dose (the i. p. dose was similar to the i. v. dose) to achieve equal biological activity. Thus, a pharmaceutical strategy for promoting its oral bioavailability should be designed to develop CTN986 as a new drug candidate.

After oral administration of CTN986 to mice with a single dose of 80 mg/kg body weight, the concentration of CTN986 in the internal organs was measured at 0.17, 0.5, 1.0 and 3.5 h, and the results are shown in [Table 2]. No deglycosylation products, rutin and hirsutin, were detected in the biological samples. The peak levels in the tested organs were observed at 0.17 h, which indicated a very rapid uptake of CTN986 into internal tissue. The drug concentrations in the stomach, duodenum and small intestine were relatively high. The concentrations in the liver, kidney and lung were some lower, and the concentrations in the heart, brain and spleen were much lower. The drug levels in the liver, kidney and lung decreased by 83, 65 and 77 %, respectively, at 0.5 h after the dosing. These data indicated that the distribution of CTN986 in the bodies of mice was rapid and extensive, followed by a rapid elimination from the tissues.

Discussion

CTN986 could be detected as its intact form in mice after oral administration followed with low oral bioavailability, indicating there was minor absorption and systemic distribution from the small intestine. This is consistent with the experimental result of Matsumoto et al, in which it was demonstrated that αG-rutin, a flavonol triglycoside of high water-solubility, was absorbed as the intact form in the isolated gastric and intestinal mucosa [15]. The mechanism of how CTN986 can be transported through the surface membrane of the intestine is yet unknown. It has been shown that quercetin 3-O-glucoside is absorbed as the intact form into the mucosal cells by the way of sodium-dependent glucose transport [16], [17]. However, it is uncertain whether CTN986, consisting of three sugar moieties in the structure, passes through the brush border membrane as the intact form by way of sodium-dependent glucose transport. CTN986 may be transported via the tight junction between the intestinal epithelial cells by diffusion. It has been reported that fluorescein isothiocyanate-dextran-4 (MW 4400) can be transported via the tight junction. This compound is used as a paracellular passage marker and has much higher molecular weight than CTN986 [18].

The pharmacokinetics of oral CTN986 in mice is characterized by short t_max and t_1/2 . A similar absorption behavior has been reported for αG-rutin [19] and quercetin 4′-O-glucoside [10]. In the previous study, the plasma level of rutin reached its peak 6 - 9 h after oral administration [20], indicating that the hydrophobic compound rutin was not absorbed until it reached the terminal ileum or even the large intestine. However, for troxerutin, a trihydroxyethyl derivative of rutin with lower hydrophobic properties, the median time to peak was 1.5 h [21]. It could be postulated that the aqueous solubility of flavonoid glycosides influenced significantly their absorption behavior.

The low oral bioavailability of CTN986 may be attributed to several reasons. Firstly, it could be due to a considerable first-pass (hepatic, gastric, and intestinal) effect of CTN986 in animals. It was reported that flavonoids show considerable first-pass effects [22]. Secondly, the calculation of the oral bioavailability was based only on the concentration of the parent compound without considering its possible metabolites. Ader et al. have shown that the oral bioavailability of quercetin is only 0.5 % for the parent compound, whereas the bioavailability is about 35-fold higher (17 %) when the metabolites are also taken into account [23]. Thirdly, CTN986 contains multiple hydroxy groups, which are liable to form hydrogen bonds with the surface molecules of the gut wall and therefore limit the absorption and systemic distribution of CTN986 from the small intestine. As noted by Carbonaro et al. [24], both quercetin and rutin can attach to the small intestine of the rat. The speculation that a considerable amount of CTN986 was bound to the gut wall was also suggested by the fact that the highest levels of CTN986 were found in the stomach and small intestine wall.

Investigation on the tissue distribution of flavonoid glycosides in animals has rarely been reported. In the experiment of tissue distribution, it was observed that CTN986 could be quickly absorbed and distributed in the bodies of the experimental animals after oral ingestion. This result is consistent with the above-mentioned short t_max and t_1/2 of CTN986 in the serum. Moreover, much higher amounts of CTN986 were found in the liver, kidney and lung, suggesting that it might be mainly metabolized and eliminated in these organs. A methylated derivative of CTN986 has been found in the tissues, and investigations on metabolite identity are in progress.

In the present study, we analyzed the pharmacokinetic characters, absolute bioavailability and tissue distribution of CTN986. The results obtained should be helpful for the formulation and dosage regimens design, and for the better understanding of its pharmacological activities. In addition, it provided more information about the pharmacokinetics of flavonol triglycosides.

Acknowledgements

The authors thank Shanqin Yuan for supplying CTN986, CTN987 and hirsutin, Lihan Zhang for assistance in the animal experiments and Lei Tian for assistance with the pharmacokinetic data analysis. This work was supported by the National Natural Science Foundation of China (Grant 30672508), Beijing Natural Science Foundation (Grant 7072061) and the National High Technology Research and Development Program of China (Grant 2002AA2Z3131).

References

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Prof. Dr. Yimin Zhao

Beijing Institute of Pharmacology and Toxicology

27 Taiping Road

Haidian District

Beijing 100850

People’s Republic of China

Phone: +86-10-6693-1648

Fax: +86-10-6821-1656

Email: zhaoym@nic.bmi.ac.cn

References

• 1 Mattila P, Astola J, Kumpulainen J. Determination of flavonoids in plant material by HPLC with diode-array and electro-array detections.  J Agric Food Chem. 2000;  48 5834-41
  CrossrefPubMedSearch in Google Scholar
• 2 Rice-Evans C A, Miller N J, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids.  Free Radic Biol Med. 1996;  20 933-56
  CrossrefPubMedSearch in Google Scholar
• 3 Butterweck V, Jurgenliemk G, Nahrstedt A, Winterhoff H. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.  Planta Med. 2000;  66 3-6
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans.  Am J Clin Nutr. 2005;  81 230S-42S
  CrossrefPubMedSearch in Google Scholar
• 5 Manach C, Donovan J L. Pharmacokinetics and metabolism of dietary flavonoids in humans.  Free Radic Res. 2004;  38 771-85
  CrossrefPubMedSearch in Google Scholar
• 6 Walle T. Absorption and metabolism of flavonoids.  Free Radic Biol Med. 2004;  36 829-37
  CrossrefPubMedSearch in Google Scholar
• 7 Scalbert A, Morand C, Manach C, Remesy C. Absorption and metabolism of polyphenols in the gut and impact on health.  Biomed Pharmacother. 2002;  56 276-82
  CrossrefPubMedSearch in Google Scholar
• 8 Duthie G G, Gardner P T, Kyle J AM. Plant polyphenols: are they the new magic bullet?.  Proc Nutr Soc. 2003;  62 599-603
  CrossrefPubMedSearch in Google Scholar
• 9 Hollman P CH, Bijsman M NCP, van Gameren Y, Cnossen E PJ, de Vries J HM, Katan M B. The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man.  Free Radic Res. 1999;  31 569-73
  CrossrefPubMedSearch in Google Scholar
• 10 Graefe E U, Witting J, Mueller S, Riethling A K, Uehleke B, Drewelow B. et al . Pharmacokinetics and bioavailability of quercetin glycosides in humans.  J Clin Pharmacol. 2001;  41 492-9
  CrossrefPubMedSearch in Google Scholar
• 11 Cermak R, Landgraf S, Wolffram S. The bioavailability of quercetin in pigs depends on the glycoside moiety and dietary factors.  J Nutr. 2003;  133 2802-7
  PubMedSearch in Google Scholar
• 12 Zhang Q J, Yang M, Zhao Y M, Luan X H, Ke Y G. Isolation and sturcture identification of flavonol glycosides from glandless cotton seeds.  Acta Pharm Sin. 2001;  36 827-31
  PubMedSearch in Google Scholar
• 13 Li Y F, Yang M, Yuan L, Zhao Y M, Luan X H, Luo Z P. Antidepressant effect of quercetin 3-O-apiosyl (1→2)-[rhamnosyl(1→6)]-glucoside in mice.  Chin J Pharmacol Toxicol. 2000;  14 125-7
  PubMedSearch in Google Scholar
• 14 Guo J F, Zhao Y M, Zhao L, Zhang W Q, Zhang A J, Xu B. Simultaneous quantification of CTN986 and its deglycosylation products in rat serum using liquid chromatography/tandem mass spectrometry.  Rapid Commun Mass Spectrom. 2006;  20 1701-8
  CrossrefPubMedSearch in Google Scholar
• 15 Matsumoto M, Matsukawa N, Mineo H, Chiji H, Hara H. A soluble flavonoid-glycoside, αG-rutin, is absorbed as glycosides in the isolated gastric and intestinal mucosa.  Biosci Biotechnol Biochem. 2004;  68 1929-34
  CrossrefPubMedSearch in Google Scholar
• 16 Wolffram S, Block M, Ader P. Quercetin-3-glucoside is transported by the glucose carrier SGLT1 across the brush border membrane of rat small intestine.  J Nutr. 2002;  132 630-5
  PubMedSearch in Google Scholar
• 17 Gee J M, DuPont M S, Rhodes M J, Jhonson I T. Quercetin glucosides interact with the intestinal glucose transport pathway.  Free Radic Biol Med. 1998;  25 19-25
  CrossrefPubMedSearch in Google Scholar
• 18 Hidalgo I J, Hillgren K M, Grass G M, Borchardt R T. Characterization of the unstirred water layer in Caco-2 cell monolayers using a novel diffusion apparatus.  Pharm Res. 1991;  8 222-7
  CrossrefPubMedSearch in Google Scholar
• 19 Matsumoto M, Chiji H, Hara H. Intestinal absorption and metabolism of a soluble flavonoid, αG-rutin, in portal cannulated rats.  Free Radic Res. 2005;  39 1139-46
  CrossrefPubMedSearch in Google Scholar
• 20 Erlund I, Kosonen T, Alfthan G, Mäenpää J, Perttunen K, Kenraali J. et al . Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers.  Eur J Clin Pharmacol. 2000;  56 545-53
  CrossrefPubMedSearch in Google Scholar
• 21 Liu F, Xu Y, Rui L, Gao S, Dong H J, Guo Q X. Liquid chromatography/tandem mass spectrometry assay for the quantification of troxerutin in human plasma.  Rapid Commun Mass Spectrom. 2006;  20 3522-6
  CrossrefPubMedSearch in Google Scholar
• 22 Chung H J, Choi Y H, Choi H D, Jang J M, Shim H J, Yoo M. et al . Pharmacokinetics of DA-6034, an agent for inflammatory bowel disease, in rats and dogs: contribution of intestinal first-pass effect to low bioavailability in rats.  Eur J Pharm Sci. 2006;  27 363-74
  PubMedSearch in Google Scholar
• 23 Ader P, Wessmann A, Wolffram S. Bioavailability and metabolism of the flavonol quercetin in the pig.  Free Radic Biol Med. 2000;  28 1056-67
  CrossrefPubMedSearch in Google Scholar
• 24 Carbonaro M, Grant G. Absorption of quercetin and rutin in rat smalll intestine.  Ann Nutr Metab. 2005;  49 178-82
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Yimin Zhao

Beijing Institute of Pharmacology and Toxicology

27 Taiping Road

Haidian District

Beijing 100850

People’s Republic of China

Phone: +86-10-6693-1648

Fax: +86-10-6821-1656

Email: zhaoym@nic.bmi.ac.cn