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Examination of lymphatic transport of puerarin in unconscious lymph duct-cannulated rats after administration in microemulsion drug delivery systems
European Journal of Pharmaceutical Sciences 42 (2011) 348–353
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Examination of lymphatic transport of puerarin in unconscious lymph duct-cannulated rats after administration in microemulsion drug delivery systems Hongfei Wu a,c,1 , An Zhou b,1 , Chuanhua Lu a,c,∗ , Lei Wang a,c a b c
Department of Pharmaceutics, Anhui University of Traditional Chinese Medicine, Anhui Province, China Experimental Research Center, Anhui University of Traditional Chinese Medicine, Anhui Province, China Key Laboratory of R&D of Chinese Medicine, Hefei, China
a r t i c l e
i n f o
Article history: Received 5 September 2010 Received in revised form 20 November 2010 Accepted 23 December 2010 Available online 7 January 2011 Keywords: Puerarin Microemulsion Lymph duct-cannulated rats Lymph transport
a b s t r a c t The potential for microemulsion drug delivery systems to improve the lymphatic transport and the portal absorption of a poorly water-soluble drug, puerarin, were investigated in lymph-cannulated rats. SD rats were operated for lymph duct cannulation and were orally dosed with 3 ml puerarin microemulsion (0.6 mg/g, n = 6). The lymph and plasma were collected over 8 h and the concentrations of puerarin and triglyceride were measured. Similarly, control rats (non-lymph-cannulated, n = 6) were dosed orally with puerarin microemulsion and subsequently with puerarin injection intravenously. Plasma and lymph samples were analysed by HPLC. Lymph triglyceride was measured using an enzymatic colorimetric technique. The extent of lymphatic transport via the thoracic duct was 0.06% of the dose for the animals dosed with puerarin microemulsion. The systemic bioavailability of oral puerarin co-administered with lipid was only 16% in the lymph duct-cannulated rats compared with 40% in the controls. These data clearly indicate that the lymphatic transport process contributes significantly to intestinal absorption of puerarin and subsequently to its systemic bioavailability. The results imply that the pharmaceutical scientist may use microemulsion formulations to optimize lymph-targeting drug delivery systems, by improving the extent of lymphatic transport. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years, much attention has focused on lipid-based formulations to improve the oral bioavailability of poorly watersoluble drug compounds. As a promising drug delivery system, microemulsion, which was a thermodynamically stable system composed of at least water, oil, and surfactants, can be used to improve the bioavailability of poorly soluble drugs (Moulik and Paul, 1998; Schwuger and Stickdorn, 1995). Puerarin is a poorly water-soluble drug with low bioavailability in animals and humans upon oral administration. Orally administered puerarin is absorbed rapidly with a tmax of about 1–2 h and a t1/2 of about 1.5–2 h. The clearance of puerarin is 73.8%. It is excreted via dejecta as its prototype, and only 0.78% is excreted in the urine (Zhang et al., 1997;
∗ Corresponding author at: Department of Pharmaceutics, Anhui University of Traditional Chinese Medicine, 103 Mei Shan Road, Hefei, Anhui Province, China. Tel.: +86 0551 5169146; fax: +86 0551 5169222. E-mail addresses: [email protected], [email protected] (C. Lu). 1 Equal contribution to this article. 0928-0987/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2010.12.010
Zhu, 1979). In our previous study, we have pursued the potential for microemulsion to improve the oral bioavailability of puerarin, and showed a trend towards a higher bioavailability when dosed in microemulsion (Wu et al., 2009). Microemulsion, associated with the nano-sized droplets, would influence the transport properties of the drug due to enormous interfacial areas, which is an important factor in sustained and targeted drug delivery (Eccleston, 1994; Lawrence and Rees, 2000). The development of oral formulations remains a challenge which requires a better knowledge of the mechanisms involved in the intestinal absorption of such drugs. Studies of the uptake and transport of drugs into the body via the intestinal lymphatic system have received increasing attention in recent years. Drugs transported by way of the gastrointestinal lymphatic system bypass the liver and avoid potential hepatic first-pass metabolism. The intestinal lymph is a specialized absorption which highly lipophilic xenobiotics and drugs can gain access to the systemic circulation (Thomson et al., 1993; Porter, 1997; Porter and Charman, 2001; Holm et al., 2002). The transport of drug by way of the intestinal lymphatic system may increase the percentage of drug that can gain access to the systemic circulation. We confer that the high
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bioavailability observed in control rats in our previous experiment was partly due to the influence of lymph transport on the drug absorption. There have been a number of animal models described in the literature for the investigation of intestinal lymphatic drug transport, and the assessment of the effect of different formulations and experimental factors in this process. The majority of studies, until recently, have predominantly been performed in anaesthetized rats (Edwards et al., 2001; Porter et al., 1996a,b; Charman and Stella, 1986). Because their intestinal characteristics are similar to those found in humans and they are not limited by considerable logistical and economic constraints (Fagerholm et al., 1996; Pahl et al., 1998). Rats are considered to be the appropriate experimental animal to study oral absorption and lymphatic transport. The present study was conducted to evaluate the contribution of the lymphatic route to the intestinal absorption and transport of puerarin into the systemic circulation. Different surgical methods have been proposed, all based on an indwelling cannula in a lymphatic vessel, providing the ability to collect the lymph fluid. We used a developed model of unconscious lymph duct-cannulated rats to estimate the intestinal lymphatic transport of puerarin microemulsion. The developed model consists in diverting, collecting and analyzing lymph after oral puerarin microemulsion administration. The rate of puerarin transport into the lymph and compared the pharmacokinetics of puerarin in plasma after oral administration of puerarin microemulsion were determined in lymph duct-cannulated rats and control rats. 2. Materials and methods 2.1. Chemicals and reagents Puerarin was purchased from Nanjing Sorun Herbal Technology Company (Nanjing, China) with the purity of 98%. Puerarin standard with the purity of 99.5% was provided by the National Institute for the Control of Pharmaceutic and Biological Products (Beijing, China). Methanol with chromatography reagent grade was purchased from Shanghai Chemical Reagent Company (Shanghai, China). All other chemicals and solvents were analytical reagent grade. Atropine sulfate injection (Anhui BBCA Pharmaceuticals Co., Ltd., China), ketamine hydrochloride injection (Jiangsu Hengrui Medicine Co., Ltd., China), heparin (Nanjing Xinbai Pharmaceuticals, China), and polydioxinone sutures (Teleflex Medical, USA) were used during the surgery.
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2.3. Animal and surgery Male Sprague–Dawley rats, weighed between 240 and 260 g, were provided by the Medical Animal Test Center of Anhui Medical University. Before treatment, the animals were fasted overnight. The surgical procedure was performed as previously described (Feng et al., 2007; Boyd et al., 2004; Ionac, 2003). One hour preoperatively, the rat was given 1 ml of soybean oil by oral gavage to facilitate the identification of the thoracic duct. The rat was given 0.03–0.05 mg/kg atropine and 700 U/kg heparin subcutaneously 20 min prior to surgery. The rats were anaesthetized with an intraperitoneal injection of ketamine (4–6 mg/kg). Intravenous access was achieved using a 25-g butterfly into the right tail vein. To maintain body hydration and intestinal lymph flow, the butterfly was attached to a 60 drops/ml i.v. solution set and a 50 ml 0.09% saline bag. The thoracic lymph duct, lying parallel and partially underneath the abdominal aorta, was separated away from the vessel by applying slight pressure to the aorta and gently teasing away connective tissue. The cannula was heparin-coated sterile tubing. The rat was positioned in dorsal recumbency, and the tube was cannulated at thoracic lymph duct where the lymph flows into the systemic circulation. All surgical and experimental procedures complied with the requirements of the National Act on the use of experimental animals (People’s Republic of China).
2.4. Pharmacokinetics of oral puerarin microemulsion in lymph duct-cannulated rats The lymph duct-cannulated rats were orally administered by intragastric gavage at a dose of 0.6 mg/g of puerarin corresponding to 3.0 ml of puerarin microemulsion. The lymph cannula tubing was placed in heparinised polypropylene plastic vials for lymph collection for 1 h collections over a 8 h period after commencement of drug administration. Lymph samples were stored at 5–8 ◦ C prior to analysis (within 24 h). The blood samples were collected from the eye socket vein at specified time intervals of 1, 2, 3, 4, 5, 6 and 8 h after commencement of drug administration. Each blood sample was immediately centrifuged at 3000 rpm for 10 min to obtain plasma. Then the plasma was transferred to a polypropylene plastic vial and stored in a deep freezer (MDF-382E, Sanyo, Japan) until used.
2.2. Preparation of puerarin microemulsion
2.5. Pharmacokinetics of oral puerarin microemulsion and i.v. puerarin injection in standard rats
The composition of the formulations of puerarin loaded microemulsion was reported in previous paper (Wu et al., 2009). In brief, microemulsion formulation of puerarin contained soybean oil (38%, wt/wt), soybean lecithin (22%, wt/wt) and ethyl lactate (22%, wt/wt) as oil phase, surfactant, and co-surfactant, respectively. Because puerarin was practically insoluble in water, the aqueous solution was prepared by dissolving puerarin in 1,2propanediol first diluted to a definite volume of deionized water. The puerarin solution was added drop-wise to bean oil and surfactant mixture, under magnetic stirring at 37 ◦ C. The particles size of the microemulsion was determined by photo correlation spectroscopy using a Zetasizer 3000 (Malvern Instruments, UK). The mean size of puerarin microemulsion was 40.2 ± 5.9 nm. The concentration of puerarin in the formulations was determined prior to administration by HPLC, thereby determining the exact dose of puerarin administered.
Sprague–Dawley rats (250 ± 10 g, n = 12) were used for the study of both intravenously and orally administered puerarin formulation. Before treatment, the animals were fasted overnight. Six rats were orally administered by gavage as described in the previous section with a dose of 0.6 mg/g of puerarin corresponding to 3.0 ml of puerarin microemulsion. The blood samples were collected from the eye socket vein at specified time intervals of 1, 2, 3, 4, 5, 6 and 8 h after dosing. Puerarin injection was intravenously administrated to the other rats at a dose of 0.1 mg/g of puerarin. The blood samples were collected from the eye socket vein at specified time intervals of 0.08, 0.17, 0.33, 0.5, 0.67, 0.83, 1, 1.33, and 1.66 h after dosing. Each blood sample was immediately centrifuged at 3000 rpm for 10 min to obtain plasma. Then the plasma was transferred to a polypropylene plastic vial and stored in a deep freezer until used.
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2.6. Lymph distribution of puerarin
3. Results and discussions
Lymph fractions collected during the 2 h period were separated by ultracentrifugation in a similar manner to that previously described (Holm et al., 2003) using a rotor (JS-15R, Beckman, USA). Chylomicrons (CMs) were firstly separated by layering lymph under a sodium chloride solution (d = 1.0063 g/ml) followed by centrifugation at 44,100 rpm (15 ◦ C) for 1 h and 20 min (262,000 g) at 15 ◦ C. The bottom of the tube was then pierced with a needle to remove the remaining lymph, leaving the chylomicrons fraction, which had formed a white semisolid plug at the top of the tube. After fractionation, the amount of puerarin associated with each lipoprotein fraction was determined by HPLC.
It is commonly assumed that the co-administration or ingestion of significant quantities of lipid is a prerequisite for substantial intestinal lymphatic drug transport (Khoo et al., 2003). Different lipid vehicles produced different levels and rates of lymphatic drug transport. In this paper, the extent of lymphatic transport of formulated long-chain lipids on the intestinal lymphatic transport of puerarin after fasted oral administration to unconscious thoracic lymph duct-cannulated rats was described.
2.7. Analysis of puerarin in blood and lymph Puerarin was analysed by high performance liquid chromatography (HPLC, Agilent1100, Milan, Italy) according to the method previously described for plasma (Wu et al., 2009) and was adapted for analysis in lymph. Liquid phase extraction procedure was as follows: 15 l methanol was added to 30 l of samples and vortexed for 5 min to form the precipitate. Then 30 l of perchloric acid solution (5%) was added to precipitated protein and vortexed mixture for another 2 min and centrifuged at 12,000 rpm for 15 min. The supernatant was evaporated to dryness in a 40 ◦ C water bath in the presence of nitrogen. After that, the residue was dissolved in 50 l of mobile phase and 30 l was injected for HPLC analysis. Recovery of spiked puerarin from blank plasma or lymph was greater than 95%. The concentration of puerarin in the samples was determined by HPLC with the wavelength 250 nm at a constant temperature of 40 ◦ C. Mobile phase consisted of methanol–citric acid–water mixture (30:0.07:70, by volume) with a flow rate of 1.0 ml/min. The injection volume was 30 l. The validity of the HPLC assay for both puerarin and external standard were established examination of the linearity of response, reproducibility of standard curve and extraction recovery. The analysis of puerarin in plasma and lymph exhibited excellent linearity (r2 > 0.99) over the concentration range of 0.0521–66.6667 g/ml. The lower limit of quantification was determined to be 0.0261 g/ml. 2.8. Analysis of lymph triglyceride
3.1. Intestinal lymphatic transport of puerarin The mass of lymphatic transport of puerarin was determined of 3 ml after oral administration of puerarin microemulsion to lymph duct-cannulated rats. The extent of lymphatic transport was determined by multiplying the concentration of puerarin in lymph by the corresponding mass of lymph collected during each time period. Then these data were expressed as a cumulative fraction of the administered dose recovered in thoracic lymph for each rat over 8 h. The lymphatic transport of puerarin, expressed as the cumulative percentage of the administered dose, is presented in Fig. 1. Puerarin readily accumulated in the lymph 4 h post-administration. The total amount of the administered dose collected in the thoracic lymph after 8 h for puerarin microemulsion systems was 83.07 ± 7.53 g, equivalent to 0.06% of the administered dose. In a previous study, lymphatic transport of drug in unconscious lymph duct-cannulated rats after oral administration of lipid-based formulations was 0.025–0.05% of the dose administered (Griffin and O’Driscoll, 2006). There is no significant difference could be found between the two studies. Soybean oil was administered before the surgery in order to facilitate the identification and cannulation of the thoracic lymph duct. A fatty meal oral administration before surgery could not influence the content of drug found in the lymph (Khoo et al., 2001; Holm et al., 2003). The lymph concentration versus time profiles of the lymphatic transport of puerarin for the microemulsion vehicles are presented in Fig. 2. The microemulsion had a peak rate at 4–5 h. The microemulsion formulation may appear to prolong the period of intestinal lymphatic transport. Lipid-based formulation such as oil solutions, microemulsion, self-emulsifying drug delivery systems, may confer therapeutic advantages in terms of prolonged and higher concentrations in the intestinal lymph (Humberstone and Charman, 1997; Pouton, 2000; Griffin and O’Driscoll, 2006).
The triglyceride concentration in lymph was measured using automated clinical chemistry analyser (Hitachi, Japan) and a commercial enzyme-based colorimetric assay (Triglycerides Reagent, Jiancheng, China). 2.9. Data analysis Plasma concentrations versus time data for puerarin for individual rats were analysed by standard non-compartment analysis using the computer program 3P97. The area under the plasma concentration–time curves (AUC0–t ) was calculated using the linear trapezoidal rule from time zero to the last measured plasma concentration. The peak plasma concentration (Cmax ) and the time for this occurrence (Tmax ) were noted directly from the individual profiles. The arithmetic means and standard deviations (SDs) of the different parameters were calculated. Statistical differences were assessed using a two-sample t-test (Excel 2002, Microsoft Corporation, WA).
Fig. 1. Cumulative intestinal lymphatic transport of puerarin in thoracic lymph duct-cannulated rats after oral administration of 150 mg puerarin microemulsion formulation (mean ± SD, n = 6).
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ated. Thereby, the surgical procedures ensure that the lymphatic duct can completely transfer the lymph and refrain the lymph from entering the systemic circulation. In this study, thoracic lymph flow rates during the 8 h period immediately following surgery averaged 2.5 ± 0.5 ml/h. During the experiment, the cumulative triglyceride transport in the 8 h period post-dosing was 9.78 ± 0.9 g (mean ± SD, n = 6), which accounted for approximately 89% of lipid present in the ingested formulation. Triglyceride concentration in lymph duct-cannulated rat plasma was much lower at 0.2 mM (mean ± SD, n = 3), that is a typical fasted plasma triglyceride concentration. The triglyceride plasma concentration measured in fasted control non-cannulated rats was 0.15 ± 0.03 mM (mean ± SD, n = 3). 3.3. Lymph distribution of puerarin Fig. 2. Concentration of puerarin in lymph versus time. Puerarin (150 mg) was administered orally to thoracic lymph duct-cannulated rats in microemulsion formulation (mean ± SD, n = 6).
In addition to the stimulation of lymphatic transport, administration of lipophilic drugs with lipids may enhance drug absorption into the portal blood. Therefore, considerable efforts might be made an effort to develop pharmaceutical formulations enhancing the oral bioavailability of these lipophilic compounds which has shown low bioavailability. 3.2. Intestinal lymphatic transport of triglycerides and lymph flow As lipophilic compounds are believed to be transported in association with triglyceride-rich lipoproteins, such as chylomicrons, the triglyceride levels in the intestinal lymph samples were evaluated. The rates of lymphatic transport of triglyceride are presented in Fig. 3. The maximal rate of triglyceride transport following administration of the microemulsion formulation occurred at 4–5 h. After 8 h, the rate of triglyceride transport decreased rapidly. The integrity of the lymph duct cannulation was evaluated by determining the lymph flow rate, the systemic plasma triglyceride concentration profile and the cumulative quantity of triglyceride transported in the lymph (Lespine et al., 2006). Cannulation of individual rats was deemed successful if greater than 85% (by mass) of the ingested triglyceride present in food was recovered in thoracic lymph, and if systemic plasma triglyceride concentrations remained low (Khoo et al., 2001). Using these criteria, the rat is a logical model whose collateral lymph duct was isolated and lig-
Fig. 3. Concentration of puerarin in intestinal lymph versus time (mean ± SD, n = 6).
Highly lipophilic compounds transported by the intestinal lymph are typically associated with the triglyceride core of the lipoproteins, primarily the chylomicrons. Most of the puerarin present in lymph (85.6 ± 2.4%) was measured in the cumulated floating fractions containing triglyceride-rich particles. This result was consistent with previous study in dogs in terms of extent of the drug associated with CM (approximately 85%) (Holm et al., 2003). 3.4. Pharmacokinetics of puerarin Fig. 4 shows mean plasma concentration–time profile over a period of 8 h in control and lymph duct-cannulated rats after oral puerarin microemulsion and in control rats after puerarin injection i.v. administration. Pharmacokinetic parameters of puerarin microemulsion and injection were calculated, which are presented in Table 1. After intravenous administration of puerarin injection, plasma level of puerarin eliminated rapidly, most of which (after 1.66 h) were under limit of detection, with AUC0→T of only 38.94 g h/mL and MRT of 0.34 h. Mean pharmacokinetic parameters for puerarin microemulsion in lymph duct-cannulated rats and non-lymph-cannulated rats were as follows: Cmax (11.33 ± 3.41) versus (26.93 ± 4.42) g/mL; AUC0→∞ (45.52 ± 3.11) versus (129.23 ± 8.97) g h/mL; AUC0→T (37.66 ± 2.73) versus (92.26 ± 5.01) g h/ml, respectively.
Fig. 4. Puerarin plasma concentration–time profile in lymph duct-cannulated and control rats: puerarin was measured in the plasma of thoracic lymph ductcannulated rats () and of control non-lymph-cannulated rats () after oral administration of puerarin microemulsion or in control rats after i.v. injection of puerarin injection () (means ± SD, n = 6).
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Table 1 Puerarin bioavailability in rats after oral administration of puerarin microemulsion (mean ± SD, n = 6).
Cmax (g/mL) AUC0→∞ (g h/ml) AUC0→8 h (g h/ml) AUC0→8 h ratio (%)
i.v. injection
Lymph duct-cannulated
38.94 ± 3.15 38.57 ± 2.99
11.33 129.23 37.66 16
± ± ± ±
3.41 8.97 2.73 1
Non-lymph-cannulated 26.93 45.52 92.26 40
± ± ± ±
4.42 3.11 5.01 2
AUC0–8 h ratio was estimated based on the plasma AUC (both in lymph duct-cannulated rats and non-lymph-cannulated rats) relative to an intravenous control (F = (AUCoral /Doral )/(AUCi.v. /Di.v. )).
In lymph-cannulated rats, systemic plasma concentrations do not reflect contributions from puerarin lymphatic transport, however, plasma levels in the non-lymph-cannulated rats result from puerarin absorption directly into the blood and indirectly into the blood via the lymph. Plasma concentrations measured in lymph duct-cannulated rats represent drug absorption via the portal blood route because the lymphatic contribution has been removed via cannulation of the thoracic lymph duct. The puerarin concentrations in plasma were lower after lymph duct cannulation when compared with the non-lymph-cannulated rats while the Tmax was unchanged (5 h). Subsequently, the AUC0–8 h for puerarin was 2.5-fold higher in the non-lymphcannulated rats when compared to lymph duct-cannulated rats after oral puerarin microemulsion administration. The AUC0–8 h ratio expressed as percentage, which represented the oral drug bioavailability calculated from 0 to 8 h, was considerably decreased after lymph diversion (40% and 16% for lymph-cannulated rats, respectively). The result indicates that a considerable part of the drug had by-passed the systemic circulation and was recovered in the diverted lymph. Absorption into the intestinal lymph should thus contribute to around 25% of the systemic bioavailable puerarin. This study also explained our previous result, which microemulsion formulations improve the oral bioavailability of puerarin. The absorption of drug is mainly intestinal. The components of microemulsion promote the intestinal lymphatic transport of drugs. It is the general unanimous that it is the gut associated lymphoid tissue (GALT), involving isolated lymphatic follicles or Peyer’s patches, which is important in the particulate absorption process (Florence et al., 1995). Lymphatic transport and association with CM and other triglyceride-rich lipoproteins may lead to many pharmacokinetic and pharmacodynamic consequences (Brocks et al., 2006; Gershkovich et al., 2007). At present, the quantitative aspects of the process of uptake are still somewhat controversial, different laboratories producing different values for the percentage of the dose which is absorbed. Lespine et al. (2006) argued that the moxidectin bioavailability was 39% in the lymph-cannulated dogs compared with 71% in the controls (calculated with the AUC0–24 h ). Holm et al. (2003) reported that both the lymphatic transport and the absorption via the portal system of halofantrine were affected after administration of halofantrine in different self-microemulsifying drug delivery systems. Perhaps, the extent of lymphatic transport from microemulsion drug delivery is also affected by animal species, components, particle size and other factors.
4. Conclusion This study demonstrates that lipid-based formulations can surprisingly initiate and support the lymphatic transport of puerarin after fasted administration to rats. The microemulsion produced higher and prolonged lymph concentrations. The extent of lymphatic transport from microemulsion drug delivery was 0.06% of the dose administered. These results suggest that promotion
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Examination of lymphatic transport of puerarin in unconscious lymph duct-cannulated rats after administration in microemulsion drug delivery systems Hongfei Wu a,c,1 , An Zhou b,1 , Chuanhua Lu a,c,∗ , Lei Wang a,c a b c
Department of Pharmaceutics, Anhui University of Traditional Chinese Medicine, Anhui Province, China Experimental Research Center, Anhui University of Traditional Chinese Medicine, Anhui Province, China Key Laboratory of R&D of Chinese Medicine, Hefei, China
a r t i c l e
i n f o
Article history: Received 5 September 2010 Received in revised form 20 November 2010 Accepted 23 December 2010 Available online 7 January 2011 Keywords: Puerarin Microemulsion Lymph duct-cannulated rats Lymph transport
a b s t r a c t The potential for microemulsion drug delivery systems to improve the lymphatic transport and the portal absorption of a poorly water-soluble drug, puerarin, were investigated in lymph-cannulated rats. SD rats were operated for lymph duct cannulation and were orally dosed with 3 ml puerarin microemulsion (0.6 mg/g, n = 6). The lymph and plasma were collected over 8 h and the concentrations of puerarin and triglyceride were measured. Similarly, control rats (non-lymph-cannulated, n = 6) were dosed orally with puerarin microemulsion and subsequently with puerarin injection intravenously. Plasma and lymph samples were analysed by HPLC. Lymph triglyceride was measured using an enzymatic colorimetric technique. The extent of lymphatic transport via the thoracic duct was 0.06% of the dose for the animals dosed with puerarin microemulsion. The systemic bioavailability of oral puerarin co-administered with lipid was only 16% in the lymph duct-cannulated rats compared with 40% in the controls. These data clearly indicate that the lymphatic transport process contributes significantly to intestinal absorption of puerarin and subsequently to its systemic bioavailability. The results imply that the pharmaceutical scientist may use microemulsion formulations to optimize lymph-targeting drug delivery systems, by improving the extent of lymphatic transport. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years, much attention has focused on lipid-based formulations to improve the oral bioavailability of poorly watersoluble drug compounds. As a promising drug delivery system, microemulsion, which was a thermodynamically stable system composed of at least water, oil, and surfactants, can be used to improve the bioavailability of poorly soluble drugs (Moulik and Paul, 1998; Schwuger and Stickdorn, 1995). Puerarin is a poorly water-soluble drug with low bioavailability in animals and humans upon oral administration. Orally administered puerarin is absorbed rapidly with a tmax of about 1–2 h and a t1/2 of about 1.5–2 h. The clearance of puerarin is 73.8%. It is excreted via dejecta as its prototype, and only 0.78% is excreted in the urine (Zhang et al., 1997;
∗ Corresponding author at: Department of Pharmaceutics, Anhui University of Traditional Chinese Medicine, 103 Mei Shan Road, Hefei, Anhui Province, China. Tel.: +86 0551 5169146; fax: +86 0551 5169222. E-mail addresses: [email protected], [email protected] (C. Lu). 1 Equal contribution to this article. 0928-0987/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2010.12.010
Zhu, 1979). In our previous study, we have pursued the potential for microemulsion to improve the oral bioavailability of puerarin, and showed a trend towards a higher bioavailability when dosed in microemulsion (Wu et al., 2009). Microemulsion, associated with the nano-sized droplets, would influence the transport properties of the drug due to enormous interfacial areas, which is an important factor in sustained and targeted drug delivery (Eccleston, 1994; Lawrence and Rees, 2000). The development of oral formulations remains a challenge which requires a better knowledge of the mechanisms involved in the intestinal absorption of such drugs. Studies of the uptake and transport of drugs into the body via the intestinal lymphatic system have received increasing attention in recent years. Drugs transported by way of the gastrointestinal lymphatic system bypass the liver and avoid potential hepatic first-pass metabolism. The intestinal lymph is a specialized absorption which highly lipophilic xenobiotics and drugs can gain access to the systemic circulation (Thomson et al., 1993; Porter, 1997; Porter and Charman, 2001; Holm et al., 2002). The transport of drug by way of the intestinal lymphatic system may increase the percentage of drug that can gain access to the systemic circulation. We confer that the high
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bioavailability observed in control rats in our previous experiment was partly due to the influence of lymph transport on the drug absorption. There have been a number of animal models described in the literature for the investigation of intestinal lymphatic drug transport, and the assessment of the effect of different formulations and experimental factors in this process. The majority of studies, until recently, have predominantly been performed in anaesthetized rats (Edwards et al., 2001; Porter et al., 1996a,b; Charman and Stella, 1986). Because their intestinal characteristics are similar to those found in humans and they are not limited by considerable logistical and economic constraints (Fagerholm et al., 1996; Pahl et al., 1998). Rats are considered to be the appropriate experimental animal to study oral absorption and lymphatic transport. The present study was conducted to evaluate the contribution of the lymphatic route to the intestinal absorption and transport of puerarin into the systemic circulation. Different surgical methods have been proposed, all based on an indwelling cannula in a lymphatic vessel, providing the ability to collect the lymph fluid. We used a developed model of unconscious lymph duct-cannulated rats to estimate the intestinal lymphatic transport of puerarin microemulsion. The developed model consists in diverting, collecting and analyzing lymph after oral puerarin microemulsion administration. The rate of puerarin transport into the lymph and compared the pharmacokinetics of puerarin in plasma after oral administration of puerarin microemulsion were determined in lymph duct-cannulated rats and control rats. 2. Materials and methods 2.1. Chemicals and reagents Puerarin was purchased from Nanjing Sorun Herbal Technology Company (Nanjing, China) with the purity of 98%. Puerarin standard with the purity of 99.5% was provided by the National Institute for the Control of Pharmaceutic and Biological Products (Beijing, China). Methanol with chromatography reagent grade was purchased from Shanghai Chemical Reagent Company (Shanghai, China). All other chemicals and solvents were analytical reagent grade. Atropine sulfate injection (Anhui BBCA Pharmaceuticals Co., Ltd., China), ketamine hydrochloride injection (Jiangsu Hengrui Medicine Co., Ltd., China), heparin (Nanjing Xinbai Pharmaceuticals, China), and polydioxinone sutures (Teleflex Medical, USA) were used during the surgery.
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2.3. Animal and surgery Male Sprague–Dawley rats, weighed between 240 and 260 g, were provided by the Medical Animal Test Center of Anhui Medical University. Before treatment, the animals were fasted overnight. The surgical procedure was performed as previously described (Feng et al., 2007; Boyd et al., 2004; Ionac, 2003). One hour preoperatively, the rat was given 1 ml of soybean oil by oral gavage to facilitate the identification of the thoracic duct. The rat was given 0.03–0.05 mg/kg atropine and 700 U/kg heparin subcutaneously 20 min prior to surgery. The rats were anaesthetized with an intraperitoneal injection of ketamine (4–6 mg/kg). Intravenous access was achieved using a 25-g butterfly into the right tail vein. To maintain body hydration and intestinal lymph flow, the butterfly was attached to a 60 drops/ml i.v. solution set and a 50 ml 0.09% saline bag. The thoracic lymph duct, lying parallel and partially underneath the abdominal aorta, was separated away from the vessel by applying slight pressure to the aorta and gently teasing away connective tissue. The cannula was heparin-coated sterile tubing. The rat was positioned in dorsal recumbency, and the tube was cannulated at thoracic lymph duct where the lymph flows into the systemic circulation. All surgical and experimental procedures complied with the requirements of the National Act on the use of experimental animals (People’s Republic of China).
2.4. Pharmacokinetics of oral puerarin microemulsion in lymph duct-cannulated rats The lymph duct-cannulated rats were orally administered by intragastric gavage at a dose of 0.6 mg/g of puerarin corresponding to 3.0 ml of puerarin microemulsion. The lymph cannula tubing was placed in heparinised polypropylene plastic vials for lymph collection for 1 h collections over a 8 h period after commencement of drug administration. Lymph samples were stored at 5–8 ◦ C prior to analysis (within 24 h). The blood samples were collected from the eye socket vein at specified time intervals of 1, 2, 3, 4, 5, 6 and 8 h after commencement of drug administration. Each blood sample was immediately centrifuged at 3000 rpm for 10 min to obtain plasma. Then the plasma was transferred to a polypropylene plastic vial and stored in a deep freezer (MDF-382E, Sanyo, Japan) until used.
2.2. Preparation of puerarin microemulsion
2.5. Pharmacokinetics of oral puerarin microemulsion and i.v. puerarin injection in standard rats
The composition of the formulations of puerarin loaded microemulsion was reported in previous paper (Wu et al., 2009). In brief, microemulsion formulation of puerarin contained soybean oil (38%, wt/wt), soybean lecithin (22%, wt/wt) and ethyl lactate (22%, wt/wt) as oil phase, surfactant, and co-surfactant, respectively. Because puerarin was practically insoluble in water, the aqueous solution was prepared by dissolving puerarin in 1,2propanediol first diluted to a definite volume of deionized water. The puerarin solution was added drop-wise to bean oil and surfactant mixture, under magnetic stirring at 37 ◦ C. The particles size of the microemulsion was determined by photo correlation spectroscopy using a Zetasizer 3000 (Malvern Instruments, UK). The mean size of puerarin microemulsion was 40.2 ± 5.9 nm. The concentration of puerarin in the formulations was determined prior to administration by HPLC, thereby determining the exact dose of puerarin administered.
Sprague–Dawley rats (250 ± 10 g, n = 12) were used for the study of both intravenously and orally administered puerarin formulation. Before treatment, the animals were fasted overnight. Six rats were orally administered by gavage as described in the previous section with a dose of 0.6 mg/g of puerarin corresponding to 3.0 ml of puerarin microemulsion. The blood samples were collected from the eye socket vein at specified time intervals of 1, 2, 3, 4, 5, 6 and 8 h after dosing. Puerarin injection was intravenously administrated to the other rats at a dose of 0.1 mg/g of puerarin. The blood samples were collected from the eye socket vein at specified time intervals of 0.08, 0.17, 0.33, 0.5, 0.67, 0.83, 1, 1.33, and 1.66 h after dosing. Each blood sample was immediately centrifuged at 3000 rpm for 10 min to obtain plasma. Then the plasma was transferred to a polypropylene plastic vial and stored in a deep freezer until used.
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2.6. Lymph distribution of puerarin
3. Results and discussions
Lymph fractions collected during the 2 h period were separated by ultracentrifugation in a similar manner to that previously described (Holm et al., 2003) using a rotor (JS-15R, Beckman, USA). Chylomicrons (CMs) were firstly separated by layering lymph under a sodium chloride solution (d = 1.0063 g/ml) followed by centrifugation at 44,100 rpm (15 ◦ C) for 1 h and 20 min (262,000 g) at 15 ◦ C. The bottom of the tube was then pierced with a needle to remove the remaining lymph, leaving the chylomicrons fraction, which had formed a white semisolid plug at the top of the tube. After fractionation, the amount of puerarin associated with each lipoprotein fraction was determined by HPLC.
It is commonly assumed that the co-administration or ingestion of significant quantities of lipid is a prerequisite for substantial intestinal lymphatic drug transport (Khoo et al., 2003). Different lipid vehicles produced different levels and rates of lymphatic drug transport. In this paper, the extent of lymphatic transport of formulated long-chain lipids on the intestinal lymphatic transport of puerarin after fasted oral administration to unconscious thoracic lymph duct-cannulated rats was described.
2.7. Analysis of puerarin in blood and lymph Puerarin was analysed by high performance liquid chromatography (HPLC, Agilent1100, Milan, Italy) according to the method previously described for plasma (Wu et al., 2009) and was adapted for analysis in lymph. Liquid phase extraction procedure was as follows: 15 l methanol was added to 30 l of samples and vortexed for 5 min to form the precipitate. Then 30 l of perchloric acid solution (5%) was added to precipitated protein and vortexed mixture for another 2 min and centrifuged at 12,000 rpm for 15 min. The supernatant was evaporated to dryness in a 40 ◦ C water bath in the presence of nitrogen. After that, the residue was dissolved in 50 l of mobile phase and 30 l was injected for HPLC analysis. Recovery of spiked puerarin from blank plasma or lymph was greater than 95%. The concentration of puerarin in the samples was determined by HPLC with the wavelength 250 nm at a constant temperature of 40 ◦ C. Mobile phase consisted of methanol–citric acid–water mixture (30:0.07:70, by volume) with a flow rate of 1.0 ml/min. The injection volume was 30 l. The validity of the HPLC assay for both puerarin and external standard were established examination of the linearity of response, reproducibility of standard curve and extraction recovery. The analysis of puerarin in plasma and lymph exhibited excellent linearity (r2 > 0.99) over the concentration range of 0.0521–66.6667 g/ml. The lower limit of quantification was determined to be 0.0261 g/ml. 2.8. Analysis of lymph triglyceride
3.1. Intestinal lymphatic transport of puerarin The mass of lymphatic transport of puerarin was determined of 3 ml after oral administration of puerarin microemulsion to lymph duct-cannulated rats. The extent of lymphatic transport was determined by multiplying the concentration of puerarin in lymph by the corresponding mass of lymph collected during each time period. Then these data were expressed as a cumulative fraction of the administered dose recovered in thoracic lymph for each rat over 8 h. The lymphatic transport of puerarin, expressed as the cumulative percentage of the administered dose, is presented in Fig. 1. Puerarin readily accumulated in the lymph 4 h post-administration. The total amount of the administered dose collected in the thoracic lymph after 8 h for puerarin microemulsion systems was 83.07 ± 7.53 g, equivalent to 0.06% of the administered dose. In a previous study, lymphatic transport of drug in unconscious lymph duct-cannulated rats after oral administration of lipid-based formulations was 0.025–0.05% of the dose administered (Griffin and O’Driscoll, 2006). There is no significant difference could be found between the two studies. Soybean oil was administered before the surgery in order to facilitate the identification and cannulation of the thoracic lymph duct. A fatty meal oral administration before surgery could not influence the content of drug found in the lymph (Khoo et al., 2001; Holm et al., 2003). The lymph concentration versus time profiles of the lymphatic transport of puerarin for the microemulsion vehicles are presented in Fig. 2. The microemulsion had a peak rate at 4–5 h. The microemulsion formulation may appear to prolong the period of intestinal lymphatic transport. Lipid-based formulation such as oil solutions, microemulsion, self-emulsifying drug delivery systems, may confer therapeutic advantages in terms of prolonged and higher concentrations in the intestinal lymph (Humberstone and Charman, 1997; Pouton, 2000; Griffin and O’Driscoll, 2006).
The triglyceride concentration in lymph was measured using automated clinical chemistry analyser (Hitachi, Japan) and a commercial enzyme-based colorimetric assay (Triglycerides Reagent, Jiancheng, China). 2.9. Data analysis Plasma concentrations versus time data for puerarin for individual rats were analysed by standard non-compartment analysis using the computer program 3P97. The area under the plasma concentration–time curves (AUC0–t ) was calculated using the linear trapezoidal rule from time zero to the last measured plasma concentration. The peak plasma concentration (Cmax ) and the time for this occurrence (Tmax ) were noted directly from the individual profiles. The arithmetic means and standard deviations (SDs) of the different parameters were calculated. Statistical differences were assessed using a two-sample t-test (Excel 2002, Microsoft Corporation, WA).
Fig. 1. Cumulative intestinal lymphatic transport of puerarin in thoracic lymph duct-cannulated rats after oral administration of 150 mg puerarin microemulsion formulation (mean ± SD, n = 6).
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ated. Thereby, the surgical procedures ensure that the lymphatic duct can completely transfer the lymph and refrain the lymph from entering the systemic circulation. In this study, thoracic lymph flow rates during the 8 h period immediately following surgery averaged 2.5 ± 0.5 ml/h. During the experiment, the cumulative triglyceride transport in the 8 h period post-dosing was 9.78 ± 0.9 g (mean ± SD, n = 6), which accounted for approximately 89% of lipid present in the ingested formulation. Triglyceride concentration in lymph duct-cannulated rat plasma was much lower at 0.2 mM (mean ± SD, n = 3), that is a typical fasted plasma triglyceride concentration. The triglyceride plasma concentration measured in fasted control non-cannulated rats was 0.15 ± 0.03 mM (mean ± SD, n = 3). 3.3. Lymph distribution of puerarin Fig. 2. Concentration of puerarin in lymph versus time. Puerarin (150 mg) was administered orally to thoracic lymph duct-cannulated rats in microemulsion formulation (mean ± SD, n = 6).
In addition to the stimulation of lymphatic transport, administration of lipophilic drugs with lipids may enhance drug absorption into the portal blood. Therefore, considerable efforts might be made an effort to develop pharmaceutical formulations enhancing the oral bioavailability of these lipophilic compounds which has shown low bioavailability. 3.2. Intestinal lymphatic transport of triglycerides and lymph flow As lipophilic compounds are believed to be transported in association with triglyceride-rich lipoproteins, such as chylomicrons, the triglyceride levels in the intestinal lymph samples were evaluated. The rates of lymphatic transport of triglyceride are presented in Fig. 3. The maximal rate of triglyceride transport following administration of the microemulsion formulation occurred at 4–5 h. After 8 h, the rate of triglyceride transport decreased rapidly. The integrity of the lymph duct cannulation was evaluated by determining the lymph flow rate, the systemic plasma triglyceride concentration profile and the cumulative quantity of triglyceride transported in the lymph (Lespine et al., 2006). Cannulation of individual rats was deemed successful if greater than 85% (by mass) of the ingested triglyceride present in food was recovered in thoracic lymph, and if systemic plasma triglyceride concentrations remained low (Khoo et al., 2001). Using these criteria, the rat is a logical model whose collateral lymph duct was isolated and lig-
Fig. 3. Concentration of puerarin in intestinal lymph versus time (mean ± SD, n = 6).
Highly lipophilic compounds transported by the intestinal lymph are typically associated with the triglyceride core of the lipoproteins, primarily the chylomicrons. Most of the puerarin present in lymph (85.6 ± 2.4%) was measured in the cumulated floating fractions containing triglyceride-rich particles. This result was consistent with previous study in dogs in terms of extent of the drug associated with CM (approximately 85%) (Holm et al., 2003). 3.4. Pharmacokinetics of puerarin Fig. 4 shows mean plasma concentration–time profile over a period of 8 h in control and lymph duct-cannulated rats after oral puerarin microemulsion and in control rats after puerarin injection i.v. administration. Pharmacokinetic parameters of puerarin microemulsion and injection were calculated, which are presented in Table 1. After intravenous administration of puerarin injection, plasma level of puerarin eliminated rapidly, most of which (after 1.66 h) were under limit of detection, with AUC0→T of only 38.94 g h/mL and MRT of 0.34 h. Mean pharmacokinetic parameters for puerarin microemulsion in lymph duct-cannulated rats and non-lymph-cannulated rats were as follows: Cmax (11.33 ± 3.41) versus (26.93 ± 4.42) g/mL; AUC0→∞ (45.52 ± 3.11) versus (129.23 ± 8.97) g h/mL; AUC0→T (37.66 ± 2.73) versus (92.26 ± 5.01) g h/ml, respectively.
Fig. 4. Puerarin plasma concentration–time profile in lymph duct-cannulated and control rats: puerarin was measured in the plasma of thoracic lymph ductcannulated rats () and of control non-lymph-cannulated rats () after oral administration of puerarin microemulsion or in control rats after i.v. injection of puerarin injection () (means ± SD, n = 6).
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Table 1 Puerarin bioavailability in rats after oral administration of puerarin microemulsion (mean ± SD, n = 6).
Cmax (g/mL) AUC0→∞ (g h/ml) AUC0→8 h (g h/ml) AUC0→8 h ratio (%)
i.v. injection
Lymph duct-cannulated
38.94 ± 3.15 38.57 ± 2.99
11.33 129.23 37.66 16
± ± ± ±
3.41 8.97 2.73 1
Non-lymph-cannulated 26.93 45.52 92.26 40
± ± ± ±
4.42 3.11 5.01 2
AUC0–8 h ratio was estimated based on the plasma AUC (both in lymph duct-cannulated rats and non-lymph-cannulated rats) relative to an intravenous control (F = (AUCoral /Doral )/(AUCi.v. /Di.v. )).
In lymph-cannulated rats, systemic plasma concentrations do not reflect contributions from puerarin lymphatic transport, however, plasma levels in the non-lymph-cannulated rats result from puerarin absorption directly into the blood and indirectly into the blood via the lymph. Plasma concentrations measured in lymph duct-cannulated rats represent drug absorption via the portal blood route because the lymphatic contribution has been removed via cannulation of the thoracic lymph duct. The puerarin concentrations in plasma were lower after lymph duct cannulation when compared with the non-lymph-cannulated rats while the Tmax was unchanged (5 h). Subsequently, the AUC0–8 h for puerarin was 2.5-fold higher in the non-lymphcannulated rats when compared to lymph duct-cannulated rats after oral puerarin microemulsion administration. The AUC0–8 h ratio expressed as percentage, which represented the oral drug bioavailability calculated from 0 to 8 h, was considerably decreased after lymph diversion (40% and 16% for lymph-cannulated rats, respectively). The result indicates that a considerable part of the drug had by-passed the systemic circulation and was recovered in the diverted lymph. Absorption into the intestinal lymph should thus contribute to around 25% of the systemic bioavailable puerarin. This study also explained our previous result, which microemulsion formulations improve the oral bioavailability of puerarin. The absorption of drug is mainly intestinal. The components of microemulsion promote the intestinal lymphatic transport of drugs. It is the general unanimous that it is the gut associated lymphoid tissue (GALT), involving isolated lymphatic follicles or Peyer’s patches, which is important in the particulate absorption process (Florence et al., 1995). Lymphatic transport and association with CM and other triglyceride-rich lipoproteins may lead to many pharmacokinetic and pharmacodynamic consequences (Brocks et al., 2006; Gershkovich et al., 2007). At present, the quantitative aspects of the process of uptake are still somewhat controversial, different laboratories producing different values for the percentage of the dose which is absorbed. Lespine et al. (2006) argued that the moxidectin bioavailability was 39% in the lymph-cannulated dogs compared with 71% in the controls (calculated with the AUC0–24 h ). Holm et al. (2003) reported that both the lymphatic transport and the absorption via the portal system of halofantrine were affected after administration of halofantrine in different self-microemulsifying drug delivery systems. Perhaps, the extent of lymphatic transport from microemulsion drug delivery is also affected by animal species, components, particle size and other factors.
4. Conclusion This study demonstrates that lipid-based formulations can surprisingly initiate and support the lymphatic transport of puerarin after fasted administration to rats. The microemulsion produced higher and prolonged lymph concentrations. The extent of lymphatic transport from microemulsion drug delivery was 0.06% of the dose administered. These results suggest that promotion
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