Drug Metab. Pharmacokinet. 20 (6): 435–442 (2005).
Regular Article Pharmacokinetics and Biodisposition of Poly(vinyl alcohol) in Rats and Mice Yoshiharu KANEO, Shiori HASHIHAMA, Atsufumi KAKINOKI, Tetsuro TANAKA, Takayuki NAKANO and Yuka IKEDA Laboratory of Biopharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Poly(vinyl alcohol) (PVA) of various molecular weight (MW=10,560–116,600) was successfully labeled with ‰uorescein isothiocyanate isomer I (FITC) according to the method of de Belder and Granath. A high-performance size-exclusion chromatographic procedure was developed for the quantitative analysis of FITC-labeled poly(vinyl alcohol) (F-PVA) in biological samples. F-PVA (80 K) disappeared slowly from the blood circulation according to the ˆrst-order kinetics (t1 W2=7 h) after intravenous injection to rats. A dose-independent behavior of F-PVA (80 K) was observed in the blood circulation, in the tissue distribution and in the urinary and fecal excretions. This suggested that PVAs are eliminated exclusively by the mechanisms that do not involve saturable transport processes. Furthermore, it was found that PVAs are very stable in the body because no degradation product was detected in the urine and feces. 125I-labeled poly(vinyl alcohol) (125I-PVA) was prepared by introducing tyramine residues to the hydroxyl groups of PVA molecules by the 1,1?-cabonyldiimidazole (CDI) activation method. 125I-PVA (80 K) was retained in the blood circulation for several days after intravenous injection to mice. Although the tissue distribution of PVAs was small, a signiˆcant accumulation into the liver and the spleen was observed. Fluorescence microscopic examination of para‹n section of the liver revealed that F-PVA (80 K) was endocytosed by the liver parenchymal cells. 125I-PVA (80 K) captured by liver was slowly transported via the bile canaliculi and gall bladder to the intestine and excreted in the feces. It was suggested, therefore, a long time is necessary for 125I-PVA (80 K) to be excreted perfectly from the body.
Key words: poly(vinyl alcohol); FITC; radioiodination; biodisposition; pharmacokinetics; high performance size exclusion chromatography; ‰uorescence microscopy; rat; mouse is polymerized in to poly(vinyl acetate) and then hydrolyzed to produce PVA. Today it has become one of the most important polymers in industry and used in a variety of applications, including textile ˆbers, papercoating agents, emulsion stabilizers, and biomedical applications.6) PVA's biocompatibility makes it an excellent material for use in medical applications such as soft contact lenses. Recently, PVA has been used for long-term implants, including a bioartiˆcial pancreas, artiˆcial cartilage, nonadhesive ˆlm, and esophagus or scleral buckling material.7) PVA also provide a potential targetable drug delivery system. Kojima and Maeda8) modiˆed superoxide
Introduction The idea of using drug carriers to improve the therapeutic e‹cacy of pharmacological agents is receiving increasing attention. The consequence of attachment of low molecular weight drugs to macromolecular carriers alters their rate of excretion from the body, changes their toxicity and immunogenicity and limits their uptake by cells via endocytosis, thus providing the opportunity to direct the drug to the particular cell type where its activity is needed.1–5) Poly(vinyl alcohol) (PVA) is a polymer which is synthesized by polymerizing not a vinyl alcohol monomer but a vinyl acetate monomer. This monomer
Received; July 19, 2005, Accepted; October 14, 2005 To whom correspondence should be addressed : Prof. Yoshiharu KANEO, Laboratory of Biopharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan. Tel. +81-84-936-2111, Fax. +81-84-936-2024, E-mail: kaneo@ fupharm.fukuyama-u.ac.jp Abbreviations used are: FITC, ‰uorescein isothiocyanate isomer I; PVA, poly(vinyl alcohol); F-PVA, FITC-labeled poly(vinyl alcohol); DMSO, dimethyl sulphoxide; CDI, 1,1?-carbonyldiimidazole; HPSEC, high-performance size-exclusion chromatography; PVP, poly(vinylpyrrolidone)
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dismutase (SOD) by conjugating with PVA. The PVASOD exhibited a lower antigenicity and immunogenicity and enhanced therapeutic eŠect against ischemic edema of mouse foot pad. Davis et al.9) studied the photosensitizing and biodistribution characteristics of a photosensitizer conjugated to modiˆed poly(vinyl alcohol). Arranz et al.10) synthesized a poly(vinyl alcohol)-nalidixic acid adduct and studied its controlled release behaviour. Yamaoka et al.11) has ˆrst examined the body distribution of PVAs with various molecular weights. They have demonstrated that PVA was located in most organs but very small accumulation in the relatively short period of time. Tabata et al.12) examined the tumor accumulation of PVA with diŠerent molecular sizes after intravenous injection to the tumor-bearing mice. The use of natural macromolecules as drug carriers has received considerable attention. However, the use of soluble synthetic polymers has not been extensively studied so far. The aim of this paper is to investigate the basic biodisposition of PVA in experimental animals such as biodegradability, dose-dependency and the body distribution especially in the long period of time, as one of the most expected synthetic polymers for the soluble drug carrier system. Materials and Methods Materials: PVA samples with various molecular weights (10 K, MW=10,560; 27 K, MW=27,280; 46 K, MW=46,200; 80 K, MW=80,520; 116 K, MW= 116,600) were kindly supplied by Japan Vam&Poval Co., Ltd., Osaka, Japan. Fluorescein isothiocyanate isomer I (FITC) was obtained from Wako Pure Chemical, Osaka, Japan. Animals were purchased from Shimizu laboratory supplies. All other chemicals and reagents were of the highest grade commercially available. Preparation of FITC-labeled PVA: FITC-labeled PVA (F-PVA) was prepared by the modiˆed method of deBelder and Granath.13) PVA (300 mg) was dissolved in dimethyl sulphoxide (DMSO) (8 mL) containing 50 mL of pyridine. FITC (50 mg) was added, followed by dibutyltin dilaurate (20 mg), and the mixture was heated C. After several precipitations in butanol for 2 h at 959 to remove free dye, the fraction of F-PVA was dried in vacuo at 809 C. The F-PVA was further puriˆed by size-exclusion chromatography on Sephadex G-25, and then freeze-dried. The F-PVA sample was dissolved in 25 mM borate buŠer (pH 9.0), and the absorbance at 495 nm was measured. The ‰uorescein content of F-PVA was estimated using the calibration curve of ‰uorescein sodium (uranine). Preparation of 125I-labeled poly(vinyl alcohol) 125 ( I-PVA): Tyramine residues were introduced to the
hydroxyl groups of PVA molecules by the 1,1?-cabonyldiimidazole (CDI) activation method.11,14,15) Two milliliters of DMSO containing CDI (7.4 mg) was added to 200 mg of PVA (80 K) dissolved in 50 mL of DMSO, followed by stirring for 30 min at room temperature. The reaction mixture was dialyzed against distilled C and further against 10 mM sodium water for 48 h at 49 borate buŠer (pH 8.5) for 24 h. Then, tyramine (62.3 mg) was added to the CDI-activated PVA (80 K) and stirred for 48 h at 259 C. The resulting tyramine-bound PVA (80 K) was dialyzed against distilled water for 48 h at 49C and the macromolecular fraction was lyophilized. Tyramine-bound PVA (80 K) was yielded as a white powder (180 mg) which was found to contain wz of tyramine by the measurement of the absor1 wW bance at 280 nm. The tyramine-bound PVA (80 K) (200 mg) was labeled with 0.25 mCi of 125I iodine (Amersham Pharmasia Biotech, Tokyo, Japan) by the chloramine T method.16) Unreacted 125I was removed by chromatography on a PD-10 column (Amersham Pharmacia Biotech). Plasma level: Male Wistar rats weighing 200–300 g were fasted for 16 h prior to the experiments. Rats were anesthetized by intraperitoneal injection of sodium kg) and were kept on a 409C pentobarbital (50 mg W plate. Rats were injected with a single dose of F-PVA (80 K) in 0.2 mL of saline through the jugular vein. Blood samples (0.3 mL) were obtained periodically through a cannulated femoral artery after the drug administration. The samples were centrifuged in a microcentrifuge for 5 min, and the resultant serum was harvested. At the end of the experiment, liver was excised and treated as described later. Tissue distribution: Rats were injected with a single dose of F-PVA (80 K) in 0.2 mL of saline through the tail vein. At 6 h after the administration, blood was collected from the vena cava under ether anesthesia and various organs such as liver, kidney, spleen, lung, heart, stomach, large intestine, small intestine and brain were excised and weighed. Each organ was homogenized on ice with a Potter-Elvehjem-type te‰on homogenizer using a 3-fold volume of 0.1 M phosphate buŠer (pH 7.4). F-PVA (80 K) in biological samples was analyzed by high-performance size-exclusion chromatography described later. kg) in Mice were injected with 125I-PVA (80 K) (6 mg W 0.2 mL of saline through the tail vein. Aliquots of the sample solution were stored for the calibration of the disintegration of radioiodine. At appropriate times after the administration, blood was collected from the vena cava under ether anesthesia and various organs such as liver, kidney, spleen, and lung were excised and weighed. The radioactivity was determined with a gamma counter (Aloka 301). Urinary and fecal excretions: Urine and feces sam-
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ples were collected for 72 h in rats after the administration of various doses of F-PVA (80 K) through the tail vein. Urine samples were ˆltered and diluted appropriately. Feces were homogenized with a Waring Blendertype homogenizer using 9-fold volume of the buŠer. The homogenate was centrifuged at 2500 rpm and the supernatant was ˆltered. Urine and feces samples were also collected for 8 weeks in mice after the administration of 125I-PVA (80 K) (6 mg W kg) through the tail vein. Fluorescence microscopic examination: Mice were injected with F-PVA (80 K) (120 mg W kg) in 0.2 mL of saline through the tail vein. The liver was excised periodically under ether anesthesia. The liver tissues were ˆxed overnight in 10z formaldehyde in neutral phosphate buŠer in the dark at room temperature. After dehydration in EtOH, the tissues were embedded in para‹n for the preparation of thin sections. Specimens were examined with a Hitachi transmitted light ‰uorescence microscope. High-performance size-exclusion chromatography (HPSEC): F-PVA (80 K) in biological samples was analyzed by HPSEC. A hundred microliters of H2 O and 80 mL of 30z (w W v) trichloroacetic acid were added to 100 mL of plasma. The mixture was vortexed and then centrifuged at 14000 rpm for 5 min. The supernatant (100 mL) was neutralized by addition of 15 mL of 11z (w W v) of NaOH and was diluted with 85 mL of a mobile phase to make 200 mL. After ˆltration, 50 mL of the sample solution was injected into the HPLC system. In biological samples other than plasma, 200 mL of the sample solution was used without addition of 100 mL of H2 O. HPSEC was carried out using a Tosoh HPLC system (CCPD; Tokyo, Japan) equipped with a variablewavelength ‰uorescent detector (RF-530, Shimadzu, Kyoto, Japan). The excitation and emission wavelengths were set at 495 nm and 520 nm, respectively. A 7.8×300 mm, TSKgel G4000PWXL column (Tosoh) was used at ambient temperature. The mobile phase was 0.2 M NaCl in 0.05 M phosphate buŠer, pH min. Data analysis 7.0 and the ‰ow rate was 1.0 mL W was done using a Tosoh super system controller SC-8010. Fluorometric determination: The amount of F-PVA (80 K) in the urine and feces samples was also determined ‰uorometrically. The ˆltrate (100 mL) of the homogenate supernatant was added to 1.9 mL of a 0.5 M Tris-HCl buŠer (pH 8.0) containing 0.1z of sodium dodecylsulfate. After thorough mixing, the ‰uorescence at 520 nm was determined by the excitation at 495 nm. Results FITC-labeling and chromatographic analysis of
Fig. 1.
Chemical structure of F-PVA.
Fig. 2. HPSEC chromatograms of F-PVA with diŠerent molecular weight. A. 116 K, B. 80 K, C. 46 K, D. 27 K, E. 10 K. HPSEC was carried out using a HPLC system equipped with a variable-wavelength ‰uorescence detector. The excitation and emission wavelengths were set at 495 nm and 520 nm, respectively. A 7.8×300 mm, TSKgel G4000PWXL column was used at ambient temperature. The mobile phase was 0.2 M NaCl in 0.05 M phosphate buŠer, pH 7.0 and the ‰ow rate was 1.0 mL W min.
F-PVAs: PVAs with ˆve diŠerent molecular weights of 10 K, 27 K, 46 K, 80 K, and 116 K were labeled with FITC by the modiˆed method of deBelder and Granath.13) The proposed structure of F-PVA is depicted in Fig. 1. The degree of substitution by FITC was OH mol; on estimated to be 1.37–2.07×10-3 FITC mol W the average 480–730 viniyl alcohol groups were found to have one FITC molecule. Size-exclusion chromatography of each F-PVA which was monitored by ‰uorescence detection (lex=495 nm, lem=520 nm) yielded a single peak as shown in Fig. 2. A strong linear relationship was observed between the retention time of F-PVAs and the logarithm of their molecular weight in the range 10,000–116,000 (Fig. 3). The same retention time was observed in the case of
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Fig. 3. Weight average molecular weight calibration curve for PVA () and the F-PVA (). HPSEC was carried out using a HPLC system equipped with a diŠerential refractometer.
Fig. 5. Tissue distribution of F-PVA (80 K) at 6 h after intravenous injection to rats at various doses. Values are given as mean±S.D. for groups of three rats.
Fig. 4. Plasma level of F-PVA (80 K) after intravenous injection to rats at various doses. () 6 mg W kg, () 12 mg W kg, ($) 24 mg W kg, () 48 mg W kg.
non-FITC-labeled PVAs which were determined by HPSEC with a diŠerential refractometer, suggesting that the FITC-substitution has no in‰uence on the molecular weight estimation. On the basis of a 50 mL-injection volume, a linear relationship was observed between the peak area of F-PVA (80 K) and their concentrations in serum or other biological specimen. The recovery ratios of F-PVA (80 K) from the serum and the liver homogenate were 91z and 92z, respectively. The sensitivity of the assay was close to 1.5 mg W mL (1.9×10-8 M). Tissue distribution of F-PVA (80 K) in rats: Figure 4 shows the plasma levels of F-PVA (80 K) after intravenous injection to rats at various doses. The plasma concentration-time curves were linear on a
semilogarithmic scale and the blood persistence of F-PVA (80 K) was independent of the dose. An apparent biological half life and volume of distribution estimated from each linear regression line were almost identical, being 7 h and 11 mL, respectively. Figure 5 shows the tissue distribution of F-PVA (80 K) at 6 h after intravenous injection to rats at various doses. Although the relatively high levels of F-PVA (80 K) were observed in the plasma as shown in Fig. 4, it was slightly distributed into each organ but not detected in the kidney, muscle and brain (Fig. 5). No dosedependency was found in the tissue distribution. Excretion of F-PVA (80 K) into urine and feces in rats: Time proˆles of cumulative urinary and fecal excretions of F-PVA (80 K) after intravenous injection to rats at various doses are illustrated in Fig. 6. The urinary excretion was almost terminated in 72 h and 25z of the dose was excreted totally in the urine. The amount of F-PVA (80 K) excreted in the feces was 2–3z of the dose at 72 h after injection, but continued to increase gradually. No dose-dependency was also found in the urinary and fecal excretions. F-PVA (80 K) was excreted as an intact form both in the urine and feces. As shown in Fig. 7, the two diŠerent measurements both by the HPSEC method and the ‰uorometric method gave the same results. Biodisposition of 125I-PVA (80 K) in mice: Mice were injected intravenously with 125I-PVA (80 K) (6 mg W kg). At appropriate times until 56 d (8 w), the radioactivities in a variety of organs were determined with a gamma counter. Figure 8 shows the time proˆle of the
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Fig. 6. Cumulative excretions of F-PVA (80 K) after intravenous injection to rats at various doses. () urine 6 mg W kg, () urine 12 mg W kg, (#) urine 24 mg W kg, () urine 48 mg W kg, () feces 6 mg W kg, () feces 12 mg W kg, ($) feces 24 mg W kg, () feces 48 mg W kg.
Fig. 8. Time proˆle of tissue distribution of intravenous injection (6 mg W kg) to mice.
125
I-PVA (80 K) after
Fig. 7. Cumulative excretions of F-PVA (80 K) after intravenous injection (24 mg W kg) to rats. () urinary and () fecal excretions measured by the ‰uorometric method. (#) urinary and () fecal excretions measured by the HPSEC method.
tissue distribution of 125I-PVA (80 K) after intravenous injection to mice. It was found that 125I-PVA (80 K) was retained in the blood circulation for several days and was gradually distributed into the liver and the spleen. Time proˆles of cumulative urinary and fecal excretions of 125I-PVA (80 K) after intravenous injection to mice are illustrated in Fig. 9. The urinary excretion was almost terminated in 28 d and 40z of the dose was excreted totally in the urine. The amount of 125I-PVA excreted in the feces was 18z of the dose at 56 d after injection, and was continued to increase slightly. Figure 10 shows the comprehensive biodistribution of 125 I-PVA (80 K) after intravenous injection to mice. Totally 58z of 125I-PVA (80 K) was excreted in the urine and feces, and 3z of the dose was still remained in the liver even 56 d after injection. Furthermore, 10z
Fig. 9. Cumulative urinary () and fecal () excretions of 125I-PVA (80 K) after intravenous injection (6 mg W kg) to mice. Values are given as mean for groups of three mice.
of the radioactivity was detected in the carcass at the end of the experiment (56 d). Therefore, over 70z of the dose was recovered in this experiment. Fluorescence microscopic examination: Fluorescence microscopic examination of para‹n section of the
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Fig. 10. Time proˆle of biodisposition of 125I-PVA (80 K) after intravenous injection (6 mg W kg) to mice. () urine, () feces, ($) liver, (&) spleen, () plasma.
Fig. 11. Fluorescence microscopic examination of para‹n section of mouse liver at 1 week after intravenous injection of F-PVA(80 K)(120 mg W kg). The bar represents the length of 50 mm.
liver at 1 w after intravenous injection to mice revealed that F-PVA (80 K) was endocytosed by the liver parenchymal cells (Fig. 11). Figure 12 shows that FITClabeled arabinogalactan as a positive control was eŠectively endocytosed by the liver parenchymal cells via the asialoglycoprotein receptor.17,18) Discussion A high-performance size-exclusion chromatographic procedure for the quantitative analysis of FITC-labeled dextrans in biological media was ˆrst developed by Kurtzhals et al.19) Mehver and Shepard have reported a systematic kinetic analysis of various molecular weights of FITC-labeled dextrans in rats by the HPSEC
Fig. 12. Fluorescence microscopic examination of para‹n section of mouse liver at 30 min after intravenous injection of FITC-labeled arabinogalactan (120 mg W kg). The bar represents the length of 50 mm.
method.20) We have demonstrated the pharmacokinetics and biodisposition of ‰uorescein-labeled arabinogalactan in rats by the same method.18) In this study, we have successfully labeled PVAs by FITC according to the method of deBelder and Granath. Then, a speciˆc and sensitive HPSEC method has been established to measure the concentrations of F-PVAs in serial tissues, urine and feces. No signiˆcant release of ‰uorescein was seen in the biological specimen or in the treatment with trichloroacetic acid, indicating that the thiocarbamoyl linkage between the ‰uorescein and the PVA molecule is stable under these experimental conditions. It was reported that no signiˆcant change was seen in either the retention time or the peak area of the FITC-labeled dextrans for more than three days at C in all biological media.19,21) Furthermore, a good 379 linear relationship was observed between the retention time of F-PVAs and the logarithm of their molecular weights. Therefore, the method is useful to determine the molecular weight change of F-PVAs in the body based on the retention time analysis. In the previous work, we reported that the blood persistence of polysaccharides such as dextrans and pullulans was increased with an increase in dose.22,23) It was found that the marked dose-dependency in the non-linear pharmacokinetics of dextrans and pullulans was mainly due to the saturation of hepatic uptake of these polysaccharides. As can be seen in Figs. 4–6, a dose-dependent behavior of F-PVA (80 K) was found neither in the blood circulation, nor in the tissue distribution, nor in the urinary and fecal excretions. Therefore, it was suggested that PVAs are eliminated exclusively by the mechanisms that do not involve saturable transport processes. Furthermore, it was found that PVAs are very stable in the body because no degradation product was detected in the urine and feces
Pharmacokinetics and Biodisposition of PVA
(Fig. 7). Ravin et al. used 131I-labelled and [14C]poly(vinylpyrrolidone) (PVP) to determine whether or not PVP can be metabolized by the body and the long-term fate of the polymer.24) It has been shown that PVP is not metabolized to any signiˆcant degree by the rat, dog or man. The reticuloendothelial system retains PVP with a molecular weight in excess of 110,000 to 120,000 for a long time, probably years. The half-life of 125I-PVA in the blood circulation largely depends on the molecular weight. The cut-oŠ molecular weight of PVAs, which is consists of nonionic, random-coiled chain molecules, for the glomerular permeability was reported to be –30,000.11) Mean residence time of 125I-PVA (80 K) for the elimination phase in the mice plasma was estimated to be 12.9 d (Fig. 10). Although the tissue distribution of PVAs was small, a signiˆcant accumulation into liver and spleen was observed (Fig. 8). It was reported that 125I-PVP was taken up by non-speciˆc ‰uid-phase endocytosis in rat liver and spleen.25,26) The uptake clearances of 125I-PVA (80 K) by liver and spleen were estimated to be 0.005–0.018 mL W hW g organ and 0.006–0.009 mL W hW g organ, respectively, which were almost similar to those of 125I-PVP reported. The participation of parenchymal, KupŠer, and endothelial liver cells was suggested in the clearance of PVAs by the reticuloendotherial system after intravenous injection in mice. Althogh the speciˆc rate of uptake by parenchymal cells was always smaller than that shown by both KupŠer and endothelial cells, the total contribution of parenchymal cells to the clearance of PVAs was suggested to be even greater than that of the other cell classes.26) This was conˆrmed by the ‰uorescence microscopic examination of para‹n section of the liver at 1 w after intravenous injection to mice, showing that F-PVA (80 K) was endocytosed by the liver parenchymal cells (Fig. 11). Mean residence times of 125I-PVA (80 K) in the liver and spleen were estimated to be 72.9 d and 64.3 d, respectively (Fig. 10). The amount of 125I-PVA excreted in the feces was 18z of the dose at 56 d after injection, and was continued to increase slightly (Fig. 10). 125 I-PVA (80 K) captured by the liver was slowly transported via the bile canaliculi and gall bladder to the intestine and excreted in the feces. It was suggested, therefore, a long time is necessary for 125I-PVA (80 K) to be excreted perfectly from the body.
dependent on their molecular weight. The use of polymers displaying a molecular mass above the renal threshold limit permits the polymers to escape renal clearance, shifting the primary route of blood clearance to elimination through extravasation into tissues.11,27,28) If very large polymer molecules are needed for drug delivery, one can produce such molecules by crosslinking several smaller nondegradable polymers via biodegradable links. The cleavage of those linkages in the body will result in the release of the smaller polymers, which can be eventually removed through the renal clearance route.4) Although PVAs are candidate for a useful drug carrier, our results demonstrated here must be kept in mind in the future design of drug delivery system with such synthetic polymers. Acknowledgement: This work was supported in part by Grants in Aid (No 13672406) for Scientiˆc Research (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to YK). Technical assistance by Kaori Imai is gratefully acknowledged. The authors are also grateful to Mr. Hiroshi Noguchi of Japan Vam&Poval Co., Ltd., Osaka, Japan for the gift of PVA samples with various molecular weights. References 1)
2)
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Conclusion Water-soluble nonionic polymers such as PVAs are usually characterized by relatively low toxicity. When administered systemically these polymers are often removed from the body without degradation. The rates and mechanisms of clearance of the polymers are
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