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Methotrexate-loaded porous polymeric adsorbents as oral sustained release formulations
Materials Science and Engineering C 78 (2017) 598–602
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Methotrexate-loaded porous polymeric adsorbents as oral sustained release formulations Xiuyan Wang a, Husheng Yan a,b,⁎ a b
Key Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
a r t i c l e
i n f o
Article history: Received 28 November 2016 Accepted 21 April 2017 Available online 24 April 2017 Keywords: Adsorption Drug delivery Oral formulation Porous polymeric adsorbent Sustained release
a b s t r a c t Methotrexate as a model drug with poor aqueous solubility was adsorbed into porous polymeric adsorbents, which was used as oral sustained release formulations. In vitro release assay in simulated gastrointestinal fluids showed that the methotrexate-loaded adsorbents showed distinct sustained release performance. The release rate increased with increase in pore size of the adsorbents. In vivo pharmacokinetic study showed that the maximal plasma methotrexate concentrations after oral administration of free methotrexate and methotrexate-loaded DA201-H (a commercial porous polymeric adsorbent) to rats occurred at 40 min and 5 h post-dose, respectively; and the plasma concentrations decreased to 22% after 5 h for free methotrexate and 44% after 24 h for methotrexate-loaded DA201-H, respectively. The load of methotrexate into the porous polymeric adsorbents not only resulted in obvious sustained release, but also enhanced the oral bioavailability of methotrexate. The areas under the curve, AUC0–24 and AUC0-inf, for methotrexate-loaded DA201-H increased 3.3 and 7.7 times, respectively, compared to those for free methotrexate. © 2017 Elsevier B.V. All rights reserved.
1. Introduction It has been studied extensively in the development of drug delivery systems to optimize the drug therapeutics and to improve patient compliance. An ideal drug delivery system should deliver a drug to the target site for absorption or action at a controlled rate over a period of time. The controlled release is usually achieved by adding other ingredients (excipients) or loading the drug to a carrier in a formulation. Ion exchange resins have been used as functional excipients (e.g., taste masking agent, drug stabilizing agent, and sustained release agent) and also as active drug ingredients (e.g., cholestyramine for cholesterol lowering) in oral formulations and some of these formulations have been clinically used [1–8]. The limitation of using exchange resins as drug carriers is that the drugs must contain ionizable groups. In addition, polymer (e.g., Eudragit polmers) coating films on the surface of the ion exchange resins are often needed to achieve sustained release [4–7]. Porous polymeric adsorbents are comprised of crosslinked polymer beads with porous structure. They usually have the same polymeric matrix as the ion-exchange resins but without ionizable groups. Various
⁎ Corresponding author at: Key Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China. E-mail address: [email protected] (H. Yan).
http://dx.doi.org/10.1016/j.msec.2017.04.136 0928-4931/© 2017 Elsevier B.V. All rights reserved.
porous polymeric adsorbents are commercially available and they are widely used in a variety of applications such as in the adsorption of organic compounds from aqueous solutions [9–12]. Porous polymeric adsorbents have advantages such as high specific surface areas and tunable pore sizes, variable surface hydrophobicity/hydrophilicity, which can be controlled by selection of monomers in the polymerization or by post-chemical modification, and availability in low cost and in large quantity. A variety of functional groups can also be introduced onto the surfaces of porous polymeric adsorbents [13]. The driving forces for the adsorption of organic compounds from aqueous media include hydrophobic effect, π-π stacking, and hydrogen bonding [9,14–17]. Methotrexate (MTX) is an antimetabolite and antifolate drug that interferes in the formation of DNA, RNA and proteins. It is widely used for the treatment of malignancies (e.g., childhood acute lymphocytic leukemia, osteosarcoma, lung cancer, and breast cancer) and in the therapy of auto-immune diseases (e.g., rheumatoid arthritis, psoriasis, and lupus) [18,19]. MTX can be administered clinically via different routes, such as oral, intravenous, subcutaneous and/or intra-muscullar administration routes [20]. And MTX is one of a few chemotherapy agents that have been clinically applied via oral administration route [21]. The clinical efficacy of MTX is often compromised by toxic dose-related side effects, short half-life in the bloodstream, and low bioavailability due to its poor water solubility and permeability [20,22,23]. Many MTX delivery systems suitable for oral administration have been reported, including MTX-loaded mesoporous silica nanoparticles
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[24,25], solid lipid nanoparticles [26], self microemulsifying delivery system [27], proteinoid microspheres [28], gelatin microspheres [29], beta-casein nanovehicles [30], chitosan microspheres [31], and crosslinked guar gum microspheres [32]. The problems existed in these oral sustained release systems include: (1) the release is often non-ideal sustained release, i.e., either very slow release (e.g., the time needed for release of 50% of the loaded MTX in media simulating gastrointestinal fluids are several days [29], which are much longer than the residence time of the formulations in the gastrointestinal tract), or too rapid release [24–28,31], and (2) toxic crosslinking agent glutarladehyde, which can be released in gastrointestinal tract, was used [29,32]. In this paper, MTX was loaded into porous polymeric adsorbents and the MTX-loaded adsorbents were tested as oral sustained release formulations. The loading was performed by the adsorption of MTX from an aqueous medium into porous polymeric adsorbents. The process is green, reproducible and easy up-scalable. This MTX oral sustained release formulation overcomes the disadvantages of the MTX oral formulations mentioned above. To our best knowledge, there is no study on using porous polymeric adsorbents as carriers for oral drug release. 2. Materials and methods 2.1. Materials MTX (90%) was purchased from Shengbaolai Biotechnology Co., Ltd. (Hubei, China). Porous polymeric adsorbents ADS-5 and H-103 were purchased from Tianjin Nankai Hecheng Science & Technology Co., Ltd. (Tianjin, China). Porous polymeric adsorbent DA201-H was purchased from Jiangsu Suqing Water Treatment Engineering Group (Jiangyin, China). Polymeric adsorbents were sieved to a particle diameter range of 0.25–0.43 mm (40–60 mesh), soaked in ethanol for 24 h and then washed by de-ionized water before use.
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simulating the pH value of gastric fluid (0.025 M HCl, pH 1.6, 30 mL) and the mixture was shaken at 37 °C for 2 h. The release medium was then replaced by a medium simulating the pH value of intestinal fluid (30 mM phosphate buffer, pH 6.8, 30 mL) and the mixture was shaken at 37 °C for 34 h. At the predetermined time points during the release process, 3 mL of the release medium was taken for MTX concentration analysis and 3 mL of the same fresh release medium was added. MTX concentration was analyzed by spectrophotometry at 302 nm. 2.6. In vivo plasma pharmacokinetic study Sixteen Wistar rats (male, 250 ± 50 g) were divided into four groups (4 per group). All the animals were fasted but allowed free access to water for 12 h before the experiment. The test samples (free MTX, MTX-loaded H103, MTX-loaded ADS-5, and MTX-loaded DA201-H) were intragastrically administrated to the rats at a MTX dose of 60 mg/kg by gavage. At predetermined time points, 400 μL of blood samples were collected from each animal via the saphenous vein. The blood samples were centrifuged at 10,000 rpm for 2 min and the plasma samples were collected. All samples were stocked at −20 °C. To each of plasma samples (200 μL), 40 μL of trichloroacetic acid (2 M in ethanol) was added and the mixture was vortexed for 2 min, and then centrifuged at 10,000 rpm for 15 min. Finally, 20 μL of the supernatant was analyzed by HPLC to determine MTX concentration. HPLC was performed using a Shimadzu LC- 10AT HPLC system (Shimadzu, Japan) with a reverse phase column (Phenomenex Luna 5u C18(2) 100A, 250 × 4.6 mm, 5 μm). The mobile phase was comprised of acetonitrile-buffer (0.01 M KH2PO4–0.02 M tetramethylammonium chloride, pH 2.5) (10/90, v/v) and an isocratic flow rate of 1 mL/min was used. The detection wavelength was 313 nm. 3. Results and discussion 3.1. MTX loading into porous polymeric adsorbents
2.2. Porosity measurement Nitrogen adsorption/desorption isotherms of porous polymeric adsorbents were measured at 77 K on an ASAP 2020 Physisorption Analyzer (Micromeritics). Samples were degassed for 10 h at 90 °C before the measurements. Porosities of the porous polymeric adsorbents were computed using the Micromeritics software package associated with the instrument. 2.3. Preparation of MTX-loaded polymeric adsorbents. MTX-loaded polymeric adsorbents were prepared via adsorption of MTX into polymeric adsorbents from aqueous media. Typically, MTX (60 mg) and H103 (wet, 300 mg) were dispersed in water (40 mL) and the mixture was stirred at 40 °C for 72 h. The aqueous phase was poured out and the polymeric adsorbent was washed several times with water.
Poly(styrene-divinylbenzene)-based porous polymeric adsorbents were mostly applied polymeric adsorbents. They are often used to adsorb hydrophobic molecules especially those with delocalized π-electrons from aqueous solutions by hydrophobic effect/π-π stacking. MTX, having a similar chemical structure to folic acid, is a hydrophobic compound with delocalized π electrons in the pteridine and benzene rings. It is predicted that MTX can be adsorbed into poly(styrene-divinylbenzene)-based porous polymeric adsorbents from aqueous media via hydrophobic effect and π-π stacking. Thus we selected poly(styrene-divinylbenzene)-based porous polymeric adsorbents as carriers of MTX in this work. Three commercially available porous polymeric adsorbents, H103, ADS-5 and DA201-H, which have the same poly(styrene-divinylbenzene) matrix but different pore structures, were used as carriers of MTX. The different pore structures especially pore sizes are expected to have effects on the drug release rate. Table 1 presented the porosities of these adsorbents provided by the suppliers. Due to these adsorbents were from
2.4. Determination of MTX loading capacities. A certain amount of dry MTX-loaded adsorbent was dispersed in a given volume of ethanol/100 mM aqueous NaOH solution (1/2, v/v) and the mixture was shaken for 8 h. MTX concentration in the supernatant was determined by spectrophotometry at 302 nm using a standard calibration curve experimentally obtained with the same solvent. MTX loading capacity was obtained by dividing the total quantity of MTX in the supernatant by the quantity of the adsorbent used. 2.5. In vitro release assay The in vitro release process was performed as described below. A MTX-loaded adsorbent sample (30 mg) was dispersed in a medium
Table 1 Parameters of porous polymeric adsorbents.a Adsorbent
H103 ADS-5 DA201-H a
Parameters provided by the suppliers
Parameters measured
S (m2/g)
D (nm)
Sb (m2/g)
Dc (nm)
900–1000 520–600 N800
8.4–9.4 25–30 6–8
1371 558 897
5.4 8.6 7.1
S, specific surface area, D, average pore diameter. Brunauer-Emmett-Teller (BET) specific surface area obtained from nitrogen adsorption isotherms. c Barrett-Joyner-Halenda (BJH) average pore diameter obtained from nitrogen adsorption isotherms. b
600
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different suppliers, the porosities of these adsorbents were measured by nitrogen adsorption isotherms under the same conditions in this work. The results were listed in Table 1. The specific surface areas of these three adsorbents measured in this work were consistent with the data provided by the suppliers. The measured average pore diameter of DA201-H was in good agreement with that provided by the supplier. However, the average pore diameters of H103 and ADS-5 measured in this work were lower than those provided by the supplier. Since the solubility of MTX in water is very low (0.01 mg/mL at 20 °C [18]), the adsorption was carried out by stirring of a dispersion of MTX and adsorbents in aqueous media for a long time to get high adsorption capacities. We expected that, during the adsorption, dissolved MTX would be adsorbed into the adsorbents, and subsequently the concentration of MTX would decease to a level lower than the saturation concentration. This would result in further dissolution of suspended MTX powder to approach saturation. Therefore MTX dissolution and adsorption progresses till equilibrium obtained. Adsorption of MTX into H103 at 25 °C, as an example, was performed and the adsorption capacities at different times are shown in Fig. 1a. The adsorption rate was quite high during the first 24 h, with the adsorption capacity reaching 101 mg MTX per gram of adsorbent over 24 h; while the adsorption rate became very slow after 24 h, with the adsorption capacity being only 139 mg MTX per gram of adsorbent after 96 h. The slow adsorption rate is presumably due to very low concentration of MTX in water and low diffusion rate of MTX in the small pores in the adsorbent. In order to increase the solubility and diffusion rate of MTX and subsequently to increase the adsorption rate, the adsorption was performed at higher temperatures (40 °C and 50 °C) and the plots of the adsorption capacities at different temperatures against time are shown in Fig. 1a. The higher adsorption rates and capacities were obtained at higher temperatures. The adsorption rates were enhanced markedly at the early stage at 40 °C and 50 °C compared to that at 25 °C. After 3 days of stirring, the adsorption capacities approached a plateau. The adsorption capacities at 40 °C and 50 °C were almost the same after stirring for 3 and 4 days. The loads of MTX into ADS-5 and DA201-H were performed by adsorption of MTX into the adsorbents at 40 °C in the same way and the results, together with the adsorption results of H103, are shown in Fig. 1b. ADS-5 and DA201-H had similar MTX adsorption profiles to H103. The adsorption capacities of H103, ADS-5 and DA201-H were 223, 159 and 189 mg/g, respectively, after adsorption for 3 days at 40 °C. The adsorption capacities were well correlated with the specific surface areas of these adsorbents (Table 1). 3.2. In vitro release MTX contains two carboxylate groups with pKa values of 4.8 and 5.5 [20]. Therefore the release (desorption) of loaded MTX from the porous polymeric adsorbents should be affected by the pH of the release medium. In vitro release of loaded MTX from the adsorbents was performed in media simulating the pH values of gastrointestinal tract and the
Fig. 2. MTX release profiles from adsorbents in simulated gastric fluid (pH 1.6) for 2 h, followed by in simulated intestinal fluid (pH 6.8).
release profiles are shown in Fig. 2. The cumulative release from H103 was low: only about 50% of the loaded MTX released after 36 h. In contrast, the release from ADS-5 was too rapid, with the cumulative release reaching 90% over 5 h. The release from DA201-H showed both high cumulative release (80% and 90% cumulative releases over 24 and 36 h, respectively) and ideal sustained release performance. The release profiles for the three MTX-loaded adsorbents (Fig. 2) indicated that the release rate increased with increase in pore sizes of the adsorbents. This can be attributed to the factor that the diffusion rate of free MTX in larger pores is higher than in smaller pores, and thus the desorbed MTX could diffuse more quickly from larger pores to bulk release media than from smaller pores. MTX is an ionizable compound and its solubility in an aqueous medium should be affected by ionic strength. Salts (mainly NaCl) should exist in gastrointestinal fluids and their concentrations vary individually and depending on the fasted and fed states. The effect of salt concentrations on MTX release from DA201-H was investigated and the results are shown in Fig. 3. The release rate slightly deceased in the presence of salt. The higher the salt concentration, the lower the release rate was. The release rate decrease extent was smaller upon increase in NaCl concentrations from 75 mM to 150 mM than from 0 to 75 mM. Salt concentrations had similar effects on the MTX release from H103 and ADS-5 (data not shown). 3.3. In vivo plasma pharmacokinetic study MTX-loaded H103, ADS-5 and DA201-H adsorbents, as well as free MTX at the same MTX dose (60 mg MTX per kg rat body weight) were orally administered to rats and the plasma concentrations of
Fig. 1. (a) MTX adsorption capacity-time profile for H103 at different temperature and (b) MTX adsorption capacities in various polymeric adsorbents at 40 °C.
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Fig. 3. The effect of salt concentrations on the MTX release from DA201-H in simulated gastric fluid (pH 1.6) for 2 h, followed by in simulated intestinal fluid (pH 6.8).
MTX at different time points were determined. The MTX concentration in plasma versus time curves are shown in Fig. 4. The plasma concentration-time profiles of all the three MTX-loaded adsorbents exhibited double peak curves. The first peaks, which appeared at the same position with the peak in the profile of free MTX, presumably resulted from the immediate release of MTX that was adsorbed on the outer surface and in the very large pores of the adsorbents; while the second peaks should result from the release of MTX that was adsorbed in the small pores of the adsorbents. Fig. 4 shows that, after oral administration of free MTX, the plasma MTX concentration increased rapidly, with maximal plasma concentration occurring around 40 min after dosing. Thereafter, the plasma concentrations decreased quickly. The plasma concentrations at 1.5 h, 3 h, 5 h and 7 h points after dosing were 49%, 27%, 22% and 0% of the maximal plasma concentration. In contrast, after administration of the MTX-loaded polymeric adsorbents, it took a much longer time to reach maximal blood concentrations for the second peaks and the plasma MTX concentrations also decreased in a much slower rate. For example, the maximal plasma concentration for MTXloaded DA201-H occurred 5 h after dosing, and 44% of the maximal plasma concentration remained even after 24 h. These results indicated that a distinct sustained release of MTX from the polymeric adsorbents was observed in comparison with free MTX. Especially for MTX-loaded DA201-H, a smooth plasma concentration–time profile was exhibited and a high level of plasma MTX concentrations still remained after 24 h post-administration.
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To obtain the pharmacokinetic parameters, the plasma concentration-time profiles shown in Fig. 4 were analyzed using PK Solver (version 2.0). The data of the first peaks in the profiles of the MTX-loaded adsorbents were omitted in the pharmacokinetic analysis. The fitting calculation using PK Solver indicated that the data from the MTX-loaded adsorbents fitted better to the one compartment model than to the two compartment model. This is consistent with the observation that the pharmacokinetic profiles tend to follow a one-compartment model if the release rate is much smaller than the intrinsic absorption rate and the distribution rate [33]. The pharmacokinetic parameters using one compartment model fitting are shown in Table 2. The pharmacokinetic data showed that the absorption rate constants (ka) for MTX-loaded adsorbents were much smaller than that for free MTX. The load of MTX into H103, ADS-5 and DA201-H resulted in 14-, 19- and 8-fold increases, respectively, in the absorption half-lives (t1/2ka) with respect to free MTX. The elimination half-lives (t1/2k10) of MTX for MTX-loaded H103, ADS-5 and DA201-H were 20, 3 and 26 times longer than that of free MTX, respectively. The mean residence times (MRT) of MTX for the MTX-loaded H103, ADS-5 and DA201-H were 19, 4 and 24 times longer than that of free MTX, respectively. The load of MTX into the porous polymeric adsorbents not only resulted in obvious sustained release, which resulted in a low plasma concentration fluctuation and a smooth plasma concentration–time profile, but also enhanced the oral bioavailability of MTX. The apparent clearance (CL/F, as a function of bioavailability) of MTX decreased from 66 ± 12 mL/kg ⋅ h for free MTX to 12 ± 6, 4.2 ± 3 and 12 ± 6 mL/kg ⋅ h for MTX-loaded H103, ADS-5 and DA201-H, respectively, and the apparent distribution volume (V/F, as a function of bioavailability) increased from 107.7 ± 28.5 mL/kg for free MTX to 343.0 ± 57.6, 142.0 ± 28.2 and 356.3 ± 38.1 mL/kg for MTX-loaded H103, ADS-5 and DA201-H, respectively. Both AUC0–24 (area under the curve from zero to 24 h) values and AUC0-inf (area under the curve from zero to infinity) values increased upon loading into the porous polymeric adsorbents. Especially for MTX-loaded DA201-H, the AUC0–24 and AUC0-inf increased 3.3 and 7.4 times, respectively, compared to that of free MTX. These results indicated that the load of MTX on the porous polymeric adsorbents increased greatly the oral bioavailability of MTX compared to free MTX. Compared to MTX delivery systems suitable for oral administration reported in literature [24–32], MTX-loaded porous polymeric adsorbents especially MTX-loaded DA201-H showed ideal sustained release performance in both in vitro and in vivo assays. The disadvantages in the literature works mentioned above including too slow release [29], or too rapid release [24–28,31], or toxic crosslinking agent being used [29,32] (MTX release was not reported in in Ref [30]) were overcome in this work. 4. Conclusions
Fig. 4. Plasma MTX concentration–time profiles after oral administration to rats at a MTX dose of 60 mg/kg.
MTX was adsorbed into polymeric adsorbents from aqueous media. Raising adsorption temperature accelerated the adsorption rate. The adsorption capacities were proportional to the specific areas of the adsorbents. In vitro sustained release of MTX from MTX-adsorbed polymeric adsorbents in simulated gastrointestinal fluids was observed, with higher release rates from adsorbents with larger pores. The release from DA201-H that has an average pore diameter of 7.1 nm, the intermediate pore size of the 3 studied polymeric adsorbents, showed both high cumulative release (80% and 90% cumulative releases over 24 and 36 h, respectively) and ideal sustained release performance. In vivo pharmacokinetic study showed that much more smooth pharmacokinetic profiles for MTX-loaded adsorbents were exhibited compared to that for free MTX. In particular, 44% of the maximal plasma methotrexate concentration still remained after 24 h of oral administration of methotrexate-loaded DA201-H. The loading MTX into the adsorbents also significantly increased the bioavailability of MTX. Both AUC values and t1/2 values for MTX-loaded adsorbents increased markedly
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Table 2 Pharmacokinetic parameters (mean ± SD) by one compartment model analysis of MTX in rats after oral administration at a single dose of 60 mg/kg. Parameter
MTX
H103
ADS-5
DA201-H
A (μg/mL) ka (1/h) k10 (1/h) t1/2ka (h) t1/2k10 (h) V/F (mL/kg) CL/F (mL/kg⋅h) Tmax (h) Cmax (μg/mL) AUC0–24 (μg⋅h/mL) AUC0-inf (μg⋅h/mL) MRT (h)
700.4 ± 250.0 5.66 ± 2.19 0.654 ± 0.187 0.137 ± 0.052 1.123 ± 0.297 107.7 ± 28.5 66 ± 12 0.454 ± 0.084 428.2 ± 85.1 867.1 ± 190.2 919.4 ± 203.3 1.82 ± 0.36
194.5 ± 57.9 3.08 ± 2.69 0.0356 ± 0.0158 1.92 ± 3.06 22.23 ± 9.51 343.0 ± 57.6 12 ± 6 6.31 ± 9.09 151.9 ± 2.3 2581 ± 392 6002 ± 3637 34.84 ± 17.79
4105 ± 1412 0.315 ± 0.181 0.280 ± 0.157 2.63 ± 1.51 2.94 ± 1.64 142.0 ± 28.2 4.2 ± 3 4.01 ± 2.28 167.5 ± 31.8 1164 ± 306 1927 ± 1384 8.03 ± 4.55
177.4 ± 21.0 0.626 ± 0.070 0.027 ± 0.011 1.12 ± 0.12 29.34 ± 13.65 356.3 ± 38.1 12 ± 6 5.41 ± 1.03 147.7 ± 14.4 2848 ± 278 7074 ± 3109 43.94 ± 19.82
compared to those for free methotrexate. The MTX-loaded adsorbents as oral formulations show simultaneously the advantages of high loading capacity, high bioavailability, sustained release, low toxicity, low cost, and easy scale-up compared with the MTX-loaded oral formulations reported in literature, each of which lacks at least one of the advantages mentioned above.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Methotrexate-loaded porous polymeric adsorbents as oral sustained release formulations Xiuyan Wang a, Husheng Yan a,b,⁎ a b
Key Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
a r t i c l e
i n f o
Article history: Received 28 November 2016 Accepted 21 April 2017 Available online 24 April 2017 Keywords: Adsorption Drug delivery Oral formulation Porous polymeric adsorbent Sustained release
a b s t r a c t Methotrexate as a model drug with poor aqueous solubility was adsorbed into porous polymeric adsorbents, which was used as oral sustained release formulations. In vitro release assay in simulated gastrointestinal fluids showed that the methotrexate-loaded adsorbents showed distinct sustained release performance. The release rate increased with increase in pore size of the adsorbents. In vivo pharmacokinetic study showed that the maximal plasma methotrexate concentrations after oral administration of free methotrexate and methotrexate-loaded DA201-H (a commercial porous polymeric adsorbent) to rats occurred at 40 min and 5 h post-dose, respectively; and the plasma concentrations decreased to 22% after 5 h for free methotrexate and 44% after 24 h for methotrexate-loaded DA201-H, respectively. The load of methotrexate into the porous polymeric adsorbents not only resulted in obvious sustained release, but also enhanced the oral bioavailability of methotrexate. The areas under the curve, AUC0–24 and AUC0-inf, for methotrexate-loaded DA201-H increased 3.3 and 7.7 times, respectively, compared to those for free methotrexate. © 2017 Elsevier B.V. All rights reserved.
1. Introduction It has been studied extensively in the development of drug delivery systems to optimize the drug therapeutics and to improve patient compliance. An ideal drug delivery system should deliver a drug to the target site for absorption or action at a controlled rate over a period of time. The controlled release is usually achieved by adding other ingredients (excipients) or loading the drug to a carrier in a formulation. Ion exchange resins have been used as functional excipients (e.g., taste masking agent, drug stabilizing agent, and sustained release agent) and also as active drug ingredients (e.g., cholestyramine for cholesterol lowering) in oral formulations and some of these formulations have been clinically used [1–8]. The limitation of using exchange resins as drug carriers is that the drugs must contain ionizable groups. In addition, polymer (e.g., Eudragit polmers) coating films on the surface of the ion exchange resins are often needed to achieve sustained release [4–7]. Porous polymeric adsorbents are comprised of crosslinked polymer beads with porous structure. They usually have the same polymeric matrix as the ion-exchange resins but without ionizable groups. Various
⁎ Corresponding author at: Key Laboratory of Functional Polymer Materials (Ministry of Education), Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China. E-mail address: [email protected] (H. Yan).
http://dx.doi.org/10.1016/j.msec.2017.04.136 0928-4931/© 2017 Elsevier B.V. All rights reserved.
porous polymeric adsorbents are commercially available and they are widely used in a variety of applications such as in the adsorption of organic compounds from aqueous solutions [9–12]. Porous polymeric adsorbents have advantages such as high specific surface areas and tunable pore sizes, variable surface hydrophobicity/hydrophilicity, which can be controlled by selection of monomers in the polymerization or by post-chemical modification, and availability in low cost and in large quantity. A variety of functional groups can also be introduced onto the surfaces of porous polymeric adsorbents [13]. The driving forces for the adsorption of organic compounds from aqueous media include hydrophobic effect, π-π stacking, and hydrogen bonding [9,14–17]. Methotrexate (MTX) is an antimetabolite and antifolate drug that interferes in the formation of DNA, RNA and proteins. It is widely used for the treatment of malignancies (e.g., childhood acute lymphocytic leukemia, osteosarcoma, lung cancer, and breast cancer) and in the therapy of auto-immune diseases (e.g., rheumatoid arthritis, psoriasis, and lupus) [18,19]. MTX can be administered clinically via different routes, such as oral, intravenous, subcutaneous and/or intra-muscullar administration routes [20]. And MTX is one of a few chemotherapy agents that have been clinically applied via oral administration route [21]. The clinical efficacy of MTX is often compromised by toxic dose-related side effects, short half-life in the bloodstream, and low bioavailability due to its poor water solubility and permeability [20,22,23]. Many MTX delivery systems suitable for oral administration have been reported, including MTX-loaded mesoporous silica nanoparticles
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[24,25], solid lipid nanoparticles [26], self microemulsifying delivery system [27], proteinoid microspheres [28], gelatin microspheres [29], beta-casein nanovehicles [30], chitosan microspheres [31], and crosslinked guar gum microspheres [32]. The problems existed in these oral sustained release systems include: (1) the release is often non-ideal sustained release, i.e., either very slow release (e.g., the time needed for release of 50% of the loaded MTX in media simulating gastrointestinal fluids are several days [29], which are much longer than the residence time of the formulations in the gastrointestinal tract), or too rapid release [24–28,31], and (2) toxic crosslinking agent glutarladehyde, which can be released in gastrointestinal tract, was used [29,32]. In this paper, MTX was loaded into porous polymeric adsorbents and the MTX-loaded adsorbents were tested as oral sustained release formulations. The loading was performed by the adsorption of MTX from an aqueous medium into porous polymeric adsorbents. The process is green, reproducible and easy up-scalable. This MTX oral sustained release formulation overcomes the disadvantages of the MTX oral formulations mentioned above. To our best knowledge, there is no study on using porous polymeric adsorbents as carriers for oral drug release. 2. Materials and methods 2.1. Materials MTX (90%) was purchased from Shengbaolai Biotechnology Co., Ltd. (Hubei, China). Porous polymeric adsorbents ADS-5 and H-103 were purchased from Tianjin Nankai Hecheng Science & Technology Co., Ltd. (Tianjin, China). Porous polymeric adsorbent DA201-H was purchased from Jiangsu Suqing Water Treatment Engineering Group (Jiangyin, China). Polymeric adsorbents were sieved to a particle diameter range of 0.25–0.43 mm (40–60 mesh), soaked in ethanol for 24 h and then washed by de-ionized water before use.
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simulating the pH value of gastric fluid (0.025 M HCl, pH 1.6, 30 mL) and the mixture was shaken at 37 °C for 2 h. The release medium was then replaced by a medium simulating the pH value of intestinal fluid (30 mM phosphate buffer, pH 6.8, 30 mL) and the mixture was shaken at 37 °C for 34 h. At the predetermined time points during the release process, 3 mL of the release medium was taken for MTX concentration analysis and 3 mL of the same fresh release medium was added. MTX concentration was analyzed by spectrophotometry at 302 nm. 2.6. In vivo plasma pharmacokinetic study Sixteen Wistar rats (male, 250 ± 50 g) were divided into four groups (4 per group). All the animals were fasted but allowed free access to water for 12 h before the experiment. The test samples (free MTX, MTX-loaded H103, MTX-loaded ADS-5, and MTX-loaded DA201-H) were intragastrically administrated to the rats at a MTX dose of 60 mg/kg by gavage. At predetermined time points, 400 μL of blood samples were collected from each animal via the saphenous vein. The blood samples were centrifuged at 10,000 rpm for 2 min and the plasma samples were collected. All samples were stocked at −20 °C. To each of plasma samples (200 μL), 40 μL of trichloroacetic acid (2 M in ethanol) was added and the mixture was vortexed for 2 min, and then centrifuged at 10,000 rpm for 15 min. Finally, 20 μL of the supernatant was analyzed by HPLC to determine MTX concentration. HPLC was performed using a Shimadzu LC- 10AT HPLC system (Shimadzu, Japan) with a reverse phase column (Phenomenex Luna 5u C18(2) 100A, 250 × 4.6 mm, 5 μm). The mobile phase was comprised of acetonitrile-buffer (0.01 M KH2PO4–0.02 M tetramethylammonium chloride, pH 2.5) (10/90, v/v) and an isocratic flow rate of 1 mL/min was used. The detection wavelength was 313 nm. 3. Results and discussion 3.1. MTX loading into porous polymeric adsorbents
2.2. Porosity measurement Nitrogen adsorption/desorption isotherms of porous polymeric adsorbents were measured at 77 K on an ASAP 2020 Physisorption Analyzer (Micromeritics). Samples were degassed for 10 h at 90 °C before the measurements. Porosities of the porous polymeric adsorbents were computed using the Micromeritics software package associated with the instrument. 2.3. Preparation of MTX-loaded polymeric adsorbents. MTX-loaded polymeric adsorbents were prepared via adsorption of MTX into polymeric adsorbents from aqueous media. Typically, MTX (60 mg) and H103 (wet, 300 mg) were dispersed in water (40 mL) and the mixture was stirred at 40 °C for 72 h. The aqueous phase was poured out and the polymeric adsorbent was washed several times with water.
Poly(styrene-divinylbenzene)-based porous polymeric adsorbents were mostly applied polymeric adsorbents. They are often used to adsorb hydrophobic molecules especially those with delocalized π-electrons from aqueous solutions by hydrophobic effect/π-π stacking. MTX, having a similar chemical structure to folic acid, is a hydrophobic compound with delocalized π electrons in the pteridine and benzene rings. It is predicted that MTX can be adsorbed into poly(styrene-divinylbenzene)-based porous polymeric adsorbents from aqueous media via hydrophobic effect and π-π stacking. Thus we selected poly(styrene-divinylbenzene)-based porous polymeric adsorbents as carriers of MTX in this work. Three commercially available porous polymeric adsorbents, H103, ADS-5 and DA201-H, which have the same poly(styrene-divinylbenzene) matrix but different pore structures, were used as carriers of MTX. The different pore structures especially pore sizes are expected to have effects on the drug release rate. Table 1 presented the porosities of these adsorbents provided by the suppliers. Due to these adsorbents were from
2.4. Determination of MTX loading capacities. A certain amount of dry MTX-loaded adsorbent was dispersed in a given volume of ethanol/100 mM aqueous NaOH solution (1/2, v/v) and the mixture was shaken for 8 h. MTX concentration in the supernatant was determined by spectrophotometry at 302 nm using a standard calibration curve experimentally obtained with the same solvent. MTX loading capacity was obtained by dividing the total quantity of MTX in the supernatant by the quantity of the adsorbent used. 2.5. In vitro release assay The in vitro release process was performed as described below. A MTX-loaded adsorbent sample (30 mg) was dispersed in a medium
Table 1 Parameters of porous polymeric adsorbents.a Adsorbent
H103 ADS-5 DA201-H a
Parameters provided by the suppliers
Parameters measured
S (m2/g)
D (nm)
Sb (m2/g)
Dc (nm)
900–1000 520–600 N800
8.4–9.4 25–30 6–8
1371 558 897
5.4 8.6 7.1
S, specific surface area, D, average pore diameter. Brunauer-Emmett-Teller (BET) specific surface area obtained from nitrogen adsorption isotherms. c Barrett-Joyner-Halenda (BJH) average pore diameter obtained from nitrogen adsorption isotherms. b
600
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different suppliers, the porosities of these adsorbents were measured by nitrogen adsorption isotherms under the same conditions in this work. The results were listed in Table 1. The specific surface areas of these three adsorbents measured in this work were consistent with the data provided by the suppliers. The measured average pore diameter of DA201-H was in good agreement with that provided by the supplier. However, the average pore diameters of H103 and ADS-5 measured in this work were lower than those provided by the supplier. Since the solubility of MTX in water is very low (0.01 mg/mL at 20 °C [18]), the adsorption was carried out by stirring of a dispersion of MTX and adsorbents in aqueous media for a long time to get high adsorption capacities. We expected that, during the adsorption, dissolved MTX would be adsorbed into the adsorbents, and subsequently the concentration of MTX would decease to a level lower than the saturation concentration. This would result in further dissolution of suspended MTX powder to approach saturation. Therefore MTX dissolution and adsorption progresses till equilibrium obtained. Adsorption of MTX into H103 at 25 °C, as an example, was performed and the adsorption capacities at different times are shown in Fig. 1a. The adsorption rate was quite high during the first 24 h, with the adsorption capacity reaching 101 mg MTX per gram of adsorbent over 24 h; while the adsorption rate became very slow after 24 h, with the adsorption capacity being only 139 mg MTX per gram of adsorbent after 96 h. The slow adsorption rate is presumably due to very low concentration of MTX in water and low diffusion rate of MTX in the small pores in the adsorbent. In order to increase the solubility and diffusion rate of MTX and subsequently to increase the adsorption rate, the adsorption was performed at higher temperatures (40 °C and 50 °C) and the plots of the adsorption capacities at different temperatures against time are shown in Fig. 1a. The higher adsorption rates and capacities were obtained at higher temperatures. The adsorption rates were enhanced markedly at the early stage at 40 °C and 50 °C compared to that at 25 °C. After 3 days of stirring, the adsorption capacities approached a plateau. The adsorption capacities at 40 °C and 50 °C were almost the same after stirring for 3 and 4 days. The loads of MTX into ADS-5 and DA201-H were performed by adsorption of MTX into the adsorbents at 40 °C in the same way and the results, together with the adsorption results of H103, are shown in Fig. 1b. ADS-5 and DA201-H had similar MTX adsorption profiles to H103. The adsorption capacities of H103, ADS-5 and DA201-H were 223, 159 and 189 mg/g, respectively, after adsorption for 3 days at 40 °C. The adsorption capacities were well correlated with the specific surface areas of these adsorbents (Table 1). 3.2. In vitro release MTX contains two carboxylate groups with pKa values of 4.8 and 5.5 [20]. Therefore the release (desorption) of loaded MTX from the porous polymeric adsorbents should be affected by the pH of the release medium. In vitro release of loaded MTX from the adsorbents was performed in media simulating the pH values of gastrointestinal tract and the
Fig. 2. MTX release profiles from adsorbents in simulated gastric fluid (pH 1.6) for 2 h, followed by in simulated intestinal fluid (pH 6.8).
release profiles are shown in Fig. 2. The cumulative release from H103 was low: only about 50% of the loaded MTX released after 36 h. In contrast, the release from ADS-5 was too rapid, with the cumulative release reaching 90% over 5 h. The release from DA201-H showed both high cumulative release (80% and 90% cumulative releases over 24 and 36 h, respectively) and ideal sustained release performance. The release profiles for the three MTX-loaded adsorbents (Fig. 2) indicated that the release rate increased with increase in pore sizes of the adsorbents. This can be attributed to the factor that the diffusion rate of free MTX in larger pores is higher than in smaller pores, and thus the desorbed MTX could diffuse more quickly from larger pores to bulk release media than from smaller pores. MTX is an ionizable compound and its solubility in an aqueous medium should be affected by ionic strength. Salts (mainly NaCl) should exist in gastrointestinal fluids and their concentrations vary individually and depending on the fasted and fed states. The effect of salt concentrations on MTX release from DA201-H was investigated and the results are shown in Fig. 3. The release rate slightly deceased in the presence of salt. The higher the salt concentration, the lower the release rate was. The release rate decrease extent was smaller upon increase in NaCl concentrations from 75 mM to 150 mM than from 0 to 75 mM. Salt concentrations had similar effects on the MTX release from H103 and ADS-5 (data not shown). 3.3. In vivo plasma pharmacokinetic study MTX-loaded H103, ADS-5 and DA201-H adsorbents, as well as free MTX at the same MTX dose (60 mg MTX per kg rat body weight) were orally administered to rats and the plasma concentrations of
Fig. 1. (a) MTX adsorption capacity-time profile for H103 at different temperature and (b) MTX adsorption capacities in various polymeric adsorbents at 40 °C.
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Fig. 3. The effect of salt concentrations on the MTX release from DA201-H in simulated gastric fluid (pH 1.6) for 2 h, followed by in simulated intestinal fluid (pH 6.8).
MTX at different time points were determined. The MTX concentration in plasma versus time curves are shown in Fig. 4. The plasma concentration-time profiles of all the three MTX-loaded adsorbents exhibited double peak curves. The first peaks, which appeared at the same position with the peak in the profile of free MTX, presumably resulted from the immediate release of MTX that was adsorbed on the outer surface and in the very large pores of the adsorbents; while the second peaks should result from the release of MTX that was adsorbed in the small pores of the adsorbents. Fig. 4 shows that, after oral administration of free MTX, the plasma MTX concentration increased rapidly, with maximal plasma concentration occurring around 40 min after dosing. Thereafter, the plasma concentrations decreased quickly. The plasma concentrations at 1.5 h, 3 h, 5 h and 7 h points after dosing were 49%, 27%, 22% and 0% of the maximal plasma concentration. In contrast, after administration of the MTX-loaded polymeric adsorbents, it took a much longer time to reach maximal blood concentrations for the second peaks and the plasma MTX concentrations also decreased in a much slower rate. For example, the maximal plasma concentration for MTXloaded DA201-H occurred 5 h after dosing, and 44% of the maximal plasma concentration remained even after 24 h. These results indicated that a distinct sustained release of MTX from the polymeric adsorbents was observed in comparison with free MTX. Especially for MTX-loaded DA201-H, a smooth plasma concentration–time profile was exhibited and a high level of plasma MTX concentrations still remained after 24 h post-administration.
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To obtain the pharmacokinetic parameters, the plasma concentration-time profiles shown in Fig. 4 were analyzed using PK Solver (version 2.0). The data of the first peaks in the profiles of the MTX-loaded adsorbents were omitted in the pharmacokinetic analysis. The fitting calculation using PK Solver indicated that the data from the MTX-loaded adsorbents fitted better to the one compartment model than to the two compartment model. This is consistent with the observation that the pharmacokinetic profiles tend to follow a one-compartment model if the release rate is much smaller than the intrinsic absorption rate and the distribution rate [33]. The pharmacokinetic parameters using one compartment model fitting are shown in Table 2. The pharmacokinetic data showed that the absorption rate constants (ka) for MTX-loaded adsorbents were much smaller than that for free MTX. The load of MTX into H103, ADS-5 and DA201-H resulted in 14-, 19- and 8-fold increases, respectively, in the absorption half-lives (t1/2ka) with respect to free MTX. The elimination half-lives (t1/2k10) of MTX for MTX-loaded H103, ADS-5 and DA201-H were 20, 3 and 26 times longer than that of free MTX, respectively. The mean residence times (MRT) of MTX for the MTX-loaded H103, ADS-5 and DA201-H were 19, 4 and 24 times longer than that of free MTX, respectively. The load of MTX into the porous polymeric adsorbents not only resulted in obvious sustained release, which resulted in a low plasma concentration fluctuation and a smooth plasma concentration–time profile, but also enhanced the oral bioavailability of MTX. The apparent clearance (CL/F, as a function of bioavailability) of MTX decreased from 66 ± 12 mL/kg ⋅ h for free MTX to 12 ± 6, 4.2 ± 3 and 12 ± 6 mL/kg ⋅ h for MTX-loaded H103, ADS-5 and DA201-H, respectively, and the apparent distribution volume (V/F, as a function of bioavailability) increased from 107.7 ± 28.5 mL/kg for free MTX to 343.0 ± 57.6, 142.0 ± 28.2 and 356.3 ± 38.1 mL/kg for MTX-loaded H103, ADS-5 and DA201-H, respectively. Both AUC0–24 (area under the curve from zero to 24 h) values and AUC0-inf (area under the curve from zero to infinity) values increased upon loading into the porous polymeric adsorbents. Especially for MTX-loaded DA201-H, the AUC0–24 and AUC0-inf increased 3.3 and 7.4 times, respectively, compared to that of free MTX. These results indicated that the load of MTX on the porous polymeric adsorbents increased greatly the oral bioavailability of MTX compared to free MTX. Compared to MTX delivery systems suitable for oral administration reported in literature [24–32], MTX-loaded porous polymeric adsorbents especially MTX-loaded DA201-H showed ideal sustained release performance in both in vitro and in vivo assays. The disadvantages in the literature works mentioned above including too slow release [29], or too rapid release [24–28,31], or toxic crosslinking agent being used [29,32] (MTX release was not reported in in Ref [30]) were overcome in this work. 4. Conclusions
Fig. 4. Plasma MTX concentration–time profiles after oral administration to rats at a MTX dose of 60 mg/kg.
MTX was adsorbed into polymeric adsorbents from aqueous media. Raising adsorption temperature accelerated the adsorption rate. The adsorption capacities were proportional to the specific areas of the adsorbents. In vitro sustained release of MTX from MTX-adsorbed polymeric adsorbents in simulated gastrointestinal fluids was observed, with higher release rates from adsorbents with larger pores. The release from DA201-H that has an average pore diameter of 7.1 nm, the intermediate pore size of the 3 studied polymeric adsorbents, showed both high cumulative release (80% and 90% cumulative releases over 24 and 36 h, respectively) and ideal sustained release performance. In vivo pharmacokinetic study showed that much more smooth pharmacokinetic profiles for MTX-loaded adsorbents were exhibited compared to that for free MTX. In particular, 44% of the maximal plasma methotrexate concentration still remained after 24 h of oral administration of methotrexate-loaded DA201-H. The loading MTX into the adsorbents also significantly increased the bioavailability of MTX. Both AUC values and t1/2 values for MTX-loaded adsorbents increased markedly
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Table 2 Pharmacokinetic parameters (mean ± SD) by one compartment model analysis of MTX in rats after oral administration at a single dose of 60 mg/kg. Parameter
MTX
H103
ADS-5
DA201-H
A (μg/mL) ka (1/h) k10 (1/h) t1/2ka (h) t1/2k10 (h) V/F (mL/kg) CL/F (mL/kg⋅h) Tmax (h) Cmax (μg/mL) AUC0–24 (μg⋅h/mL) AUC0-inf (μg⋅h/mL) MRT (h)
700.4 ± 250.0 5.66 ± 2.19 0.654 ± 0.187 0.137 ± 0.052 1.123 ± 0.297 107.7 ± 28.5 66 ± 12 0.454 ± 0.084 428.2 ± 85.1 867.1 ± 190.2 919.4 ± 203.3 1.82 ± 0.36
194.5 ± 57.9 3.08 ± 2.69 0.0356 ± 0.0158 1.92 ± 3.06 22.23 ± 9.51 343.0 ± 57.6 12 ± 6 6.31 ± 9.09 151.9 ± 2.3 2581 ± 392 6002 ± 3637 34.84 ± 17.79
4105 ± 1412 0.315 ± 0.181 0.280 ± 0.157 2.63 ± 1.51 2.94 ± 1.64 142.0 ± 28.2 4.2 ± 3 4.01 ± 2.28 167.5 ± 31.8 1164 ± 306 1927 ± 1384 8.03 ± 4.55
177.4 ± 21.0 0.626 ± 0.070 0.027 ± 0.011 1.12 ± 0.12 29.34 ± 13.65 356.3 ± 38.1 12 ± 6 5.41 ± 1.03 147.7 ± 14.4 2848 ± 278 7074 ± 3109 43.94 ± 19.82
compared to those for free methotrexate. The MTX-loaded adsorbents as oral formulations show simultaneously the advantages of high loading capacity, high bioavailability, sustained release, low toxicity, low cost, and easy scale-up compared with the MTX-loaded oral formulations reported in literature, each of which lacks at least one of the advantages mentioned above.
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