Pharmacokinetic Study of Nicotine and Its Metabolite Cotinine to Clarify Possible Association Between Smoking and Voiding Dysfunction in Rats Using UPLC/ESI-MS

Drug Metab. Pharmacokinet. 26 (4): 416422 (2011). Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Pharmacokinetic Study of Nicotine and Its Metabolite Cotinine to Clarify Possible Association between Smoking and Voiding Dysfunction in Rats Using UPLC/ESI-MS

Satomi ONOUE*,NoriyukiYAMAMOTO,YoshikiSETO and Shizuo YAMADA Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The present study was undertaken to clarify the possible association between nicotine intake/ cigarette smoking and detrusor instability. For pharmacokinetic characterization of nicotine and cotinine (a major and pharmacologically less active metabolite of nicotine), a rapid ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS) method was developed that requires only a small amount of sample and simple pretreatment. The UPLC/ESI-MS method was validated with a focus on speciﬁcity, sensitivity (limit of detection, 2.5 ng/mL; limit of quantiﬁcation, 5 ng/mL), linearity (r > 0.998), accuracy (97.2102.8%), precision (relative standard deviation <8%) and robustness in accordance with ICH guidelines (Q2B Validation of Analytical Procedures: Methodology). The developed method was successfully applied to determine nicotine and cotinine levels in rat biological samples such as plasma, urine and several tissues. After subcutaneous administration of nicotine ditartrate (2 mg/kg of body weight) in rats, the absorbed nicotine was rapidly and extensively metabolized into cotinine. However, nicotine was found to be predominant in cortex and bladder, where nicotinic acetylcholine receptors were expressed for neuronal control of voiding function. Repeated administration of nicotine led to a ca. 3-fold higher accumulation of nicotine than that of cotinine in rat urine. The results of the pharmacokinetic study using the UPLC/ESI-MS method further support the possible involvement of nicotine in increased risk of urinary dysfunction in smokers.

Keywords: nicotine; cotinine; UPLC; urinary excretion; cigarette smoking

eases6¥ and bladder dysfunctions.7¥ In particular, recent Introduction attention has been drawn to the increased risk of urinary Cigarette smoke is a complex aerosol consisting of 92% dysfunction in female smokers.8¥ Previous population-based gaseous components and 8% particulates, and side stream studies also demonstrated cigarette smoking to be associated and direct stream smoke are similar in composition.1¥ The with both urinary incontinence7¥ and urgency in elderly inhalation of cigarette smoke either directly by cigarette people,9¥ although the evidence was conflicting. In general, smokers or indirectly by people in the same enclosed areas several factors such as weakening of the pelvic floor muscles represents an environmental health problem for millions of and collagen tissue alterations through aging and childbirth people.2¥ Cigarette smoking has long been identified as a may contribute to both conditions, and these urinary major causative factor in the development of inflammatory dysfunctions have a substantial influence on quality of life and lung diseases such as chronic bronchitis, pulmonary fibrosis, activities of daily living. emphysema and chronic obstructive pulmonary disease.3¥ Although little is known about the pathogenesis of In addition to these respiratory diseases, cigarette smoking smoking-related voiding dysfunction, detrusor instability in has also been causally linked with obesity, osteoporotic female smokers may be related to an anti-estrogenic fractures,4¥ cardiovascular diseases,5¥ neurodegenerative dis- hormonal effect on the bladder or urethra, an adverse effect

Received; March 8, 2011, Accepted; April 26, 2011 J-STAGE Advance Published Date: May 12, 2011, doi:10.2133/dmpk.DMPK-11-RG-019 *To whom correspondence should be addressed: Dr. Satomi ONOUE, Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Tel. +81-54-264-5633, Fax. +81-54-264-5635, E-mail: [email protected]

416 Pharmacokinetic Study of Nicotine in Rats Using UPLC/ESI-MS 417 on collagen synthesis10¥ and an increased turnover of vitamin detected at retention times of 1.90, 2.30 and 3.26 min, C.11¥ It is also well established that nicotine plays an respectively. important role in the control of bladder function, mediated Method validation: The newly developed UPLC/ by nicotinic acetylcholine receptors in both central and ESI-MS method was validated in terms of linearity, accuracy, peripheral nervous systems.12®14¥ In this context, nicotine in precision and assay recovery according to International cigarette smoke might also lead to detrusor instability via Conference on Harmonization of Technical Requirements neuronal control of urinary bladder responses.15¥ Better for Registration of Pharmaceuticals for Human Use ¤ICH¥ understanding of the pharmacokinetic behavior of nicotine guidelines úQ2B Validation of Analytical Procedures: may identify the involvement of nicotine in detrusor Methodologyû. Assay method precision was investigated instability in smokers; however, it has never been fully using six independent test solutions and a standard elucidated. preparation. The intermediate precision of the assay was Many analytical methods have been developed for also evaluated using different analysts on three different the analysis of nicotine and its metabolites, including days. The accuracy of the assay was evaluated in triplicate gas chromatography,16¥ high-performance liquid chromatog- using three concentration levels: 10, 300 and 600 ng/mL. raphy ¤HPLC¥,17,18¥ enzyme-linked immunosorbent assay The limit of detection ¤LOD¥ and limit of quantification ¤ELISA¥19¥ and radio-immunoassay ¤RIA¥.20¥ The present ¤LOQ¥ for nicotine and cotinine were estimated by injecting study was undertaken to develop a more rapid analytical a series of dilute solutions with known concentration. The method for pharmacokinetic characterization of nicotine LOD and LOQ were estimated from standard deviation and cotinine, a major metabolite of nicotine, using ultra- values of replicate responses of tested chemicals ¤signal-to- performance liquid chromatography ¤UPLC¥/electrospray noise ratios, 3:1 for LOD and 10:1 for LOQ¥. To determine ionization ¤ESI¥-MS. The UPLC/ESI-MS method for nicotine the robustness of the method, experimental conditions were was validated with a focus on linearity, accuracy, precision, purposely altered and the peak response for each tested assay recovery and robustness. Plasma and urine concen- chemicals was examined by injecting system suitability trations of nicotine and cotinine after subcutaneous injection solution. The flow rate was changed to 0.20 and 0.30 were monitored using the developed UPLC/ESI-MS mL/min. The column temperature was varied by ¤+¥3ôC, method, and the tissue distribution of these chemicals was and the organic strength was varied by ¤+¥2%. also assessed. Pharmacokinetic study: Animals Materials and Methods Male Sprague®Dawley rats, weighing ca. 650 g ¤n © 4 for Chemicals: Ammonium acetate, nicotine ditartrate each in vivo experiment, 25®30 weeks of age; Japan SLC, and diethyl ether were purchased from Wako Pure Chemical Shizuoka, Japan¥, were housed two per cage in the laboratory Industries ¤Osaka, Japan¥. Cotinine, antipyrine and sodium with free access to food and water, and maintained on a 12-h pentobarbital were purchased from Sigma ¤St. Louis, dark/light cycle in a room with controlled temperature MO, USA¥. Acetonitrile ¤HPLC grade¥ and Millex-LG ¤24 + 1ôC¥ and humidity ¤55 + 5%¥. All procedures used in membrane filters ¤pore size: 0.2 µm¥ were bought from the present study were conducted in accordance with the Kanto Chemical ¤Tokyo, Japan¥ and Millipore ¤Bedford, guidelines approved by the Institutional Animal Care and MA, USA¥, respectively. Ethical Committee of the University of Shizuoka. UPLC/ESI-MS analysis: The amounts of nicotine and Plasma concentration cotinine in biological samples from rats were determined by Rats were anesthetized using pentobarbital ¤50 mg/kg an internal standard method using a Waters Acquity UPLC body weight¥ and then a guide cannula ¤PUC-40, EICOM, system ¤Waters, Milford, MA¥, which included a binary Kyoto, Japan¥ was inserted into the jugular vein on the solvent manager, sample manager, column compartment day before nicotine ditartrate ¤2 mg/kg body weight¥ was and SQD connected with MassLynx software. An Acquity administered subcutaneously. Blood samples ¤approximately UPLC BEH C 18 column ¤particle size: 1.7 µm, column 500 µL¥ were collected from the cannulated jugular vein at size: 2.1 mm ' 50 mm; Waters¥ was used, and the column the indicated times ¤0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and temperature was maintained at 40ôC. The standard and 12 h¥ after administration of nicotine ditartrate and mixed samples were separated using a gradient mobile phase with 1,250 µL of acetonitrile containing 0.053 nM antipyrine. consisting of acetonitrile ¤A¥ and 5 mM ammonium acetate The mixture was centrifuged ¤10,000 ' g, 10 min, 4ôC¥ and ¤B¥ with a flow rate of 0.25 mL/min. The gradient condition the supernatant was dried with a stream of nitrogen gas. Each of the mobile phase was 0®0.5 min, 5% A; 0.5®3.5 min, 5® dried sample was redissolved in 100 µL of 50% acetonitrile 25% A; 3.51®4.5 min, 95% A; and 4.51®5.5 min, 5% A. solution and filtered with Millex-LG membrane filter Analysis was carried out using selected ion recording ¤SIR¥ ¤Millipore, Bedford, MA, USA¥ for UPLC/ESI-MS analysis. for m/z 163 for nicotine ªM ¦ H«¦, 177 for cotinine Urine concentration ªM ¦ H«¦ and 189 for antipyrine ªM ¦ H«¦, an internal After single subcutaneous administration of nicotine standard. Peaks for nicotine, cotinine and antipyrine were ditartrate ¤2 mg/kg body weight¥, urine samples were

Table 1. Accuracy and precision of nicotine and cotinine measurement in rat plasma

Intra-day Inter-day Spiked concentration ¤ng/mL¥ Measured concentration Accuracy Precision Measured concentration Accuracy Precision ¤ng/mL¥a ¤%¥ ¤RSD, %¥b ¤ng/mL¥a ¤%¥ ¤RSD, %¥b Nicotine 10 9.7 + 0.7 97.2 7.2 9.8 + 0.6 98.1 6.1 300 308.4 + 12.7 102.8 4.1 295.6 + 10.4 98.5 3.5 600 607.8 + 16.4 101.3 2.7 609.7 + 18.9 101.6 3.1 Cotinine 10 9.6 + 0.5 96.4 5.6 10.2 + 0.7 102.0 6.9 300 306.4 + 11.2 102.1 3.7 303.4 + 14.3 101.1 4.7 600 612.2 + 18.1 102.0 3.0 592.2 + 20.2 98.7 3.4 aData represent mean + SD ¤n © 6¥. bPercentage relative standard deviation. collected every hour. To monitor urine excretion after were adjusted to provide optimal performance of the assay, repeated subcutaneous administration of nicotine ditartrate and the mobile phase for the analysis was selected on the ª2 mg/kg body weight, bis in die ¤b.i.d.¥, 10 days«, urine basis of its polarity. Nicotine and cotinine were separated samples were collected every 12 h. Urine samples ¤30 µL¥ on a UPLC BEH C18 column using a linear gradient of were mixed with 1,470 µL of acetonitrile, and the mixture acetonitrile and 5 mM ammonium acetate, which has the was centrifuged ¤10,000 ' g, 10 min, 4ôC¥. Twenty micro- advantage of lower solvent consumption and increased mass liters of 0.53 nM antipyrine was added to 180 µL of each sensitivity. Various sample treatment procedures were supernatant, and the mixture was filtered with a Millex-LG evaluated, including solid-phase extraction, protein precip- membrane filter ¤Millipore¥ for UPLC/ESI-MS analysis. itation and liquid-liquid extraction. Considering recovery for Tissue distribution all analytes, a simple protein precipitation procedure was At 0.5 and 2 h after oral subcutaneous administration of developed with acetonitrile, yielding over 80% extraction nicotine ditartrate ¤2 mg/kg body weight¥, rats were recovery for nicotine and cotinine. Nicotine and cotinine sacrificed by taking blood from the descending aorta under standard solutions were found to be stable in the mobile temporary anesthesia with diethyl ether, and the tissues phase for at least 24 h at room temperature. The recovery were then perfused with cold saline from the aorta. The proportions of these standard solutions stored for 24 h at bladder, salivary gland, heart and cortex were dissected, and room temperature were calculated to be 99.4% and 100.5% then fat and blood vessels were removed by trimming. Each for nicotine and cotinine, respectively, when compared with tissue was minced with scissors and homogenized in a freshly prepared solutions. Physcotron ¤Microtech, Chiba, Japan¥ in 9 times its weight The specificity of the analytical method was demonstrated of ice-cold saline. A total of 400 µL of acetonitrile was added by comparing selected ion recording ¤SIR¥ chromatograms of to 100 µL of homogenate, and the mixture was centrifuged nicotine, cotinine and antipyrine, an internal standard, for a ¤10,000 ' g, 10 min, 4ôC¥. Twenty microliters of 0.53 nM blank plasma sample and a spiked plasma sample. All the antipyrine was added to 180 µL of each supernatant, and the analytes and the internal standard could be detected on their mixtures were filtered with a Millex-LG membrane filter own selected ion chromatograms without any significant ¤Millipore¥ for UPLC/ESI-MS analysis. The tissue-to-plasma interference. Calibration graphs were constructed at five ¤ ¥ concentration ratio Kp value was calculated as the ratio of concentration levels ranging from 10 to 600 ng/mL, and the tissue concentration of unchanged drug to the plasma both nicotine and cotinine showed good linearity with a concentration. correlation coefficient ¤r¥ value greater than 0.998 ¤data not Statistical analysis shown¥. The precision of the method was determined by For statistical comparisons, one-way analysis of variance repeatability ¤intra-day¥, and the intermediate precision ¤ANOVA¥ with pairwise comparison by Fisherös least ¤inter-day¥ is expressed as relative standard deviation ¤RSD, significant difference procedure was used. A P value of less %¥ of a series of measurements. The experimental values than 0.05 was considered significant for all analyses. obtained for the determination of nicotine and cotinine in plasma are presented in Table 1. The results show an RSD Results and Discussion of 7.2% at 10 ng/mL nicotine, and the intra-day variability Method development and validation: A UPLC/ was calculated from assays on 3 days and shows a mean RSD ESI-MS method that can be conveniently employed for of 6.1%. The accuracy of the method was determined and pharmacokinetic profiling was developed for determination the mean recovery was found to be over 98%, indicating of nicotine and cotinine. The chromatographic conditions an agreement between the true value and the value found.

300 Table 2. Pharmacokinetic parameters of nicotine and cotinine following subcutaneous administration of nicotine ditartrate (2.0 mg/kg body weight of rats)

AUC ®W 200 C ¤ng/mL¥ t ¤h¥ 0 max 1/2 ¤ng&h/mL¥ Nicotine 122 + 17.2 1.05 + 0.190 103 + 4.50 Cotinine 226 + 17.6 7.08 + 0.400 1940 + 88.1 100 Cmax, maximum concentration; T1/2, half-life; and AUC0®W, area under the curve of plasma concentration vs. time from t © 0tot©W after administration. Data represent mean + SE ¤n © 3or4¥. Plasma concentration (ng/mL) 0 0 2 4 6 8 10 12 rapidly and extensively metabolized to inactive cotinine by Time (h) CYP2A6 in human livers, which had a major impact on ¥ Fig. 1. Plasma concentrations of nicotine and cotinine in rats nicotine clearance.21 The accumulation of cotinine might be after subcutaneous administration of nicotine ditartrate (2.0 associated with the adverse effects of cigarette smoking; mg/kg body weight) however, cotinine has over two orders of magnitude lower ,nicotine; , cotinine. Data represent mean + SE (n ¦ 4). afﬁnity with nicotinic acetylcholine receptors than nicotine itself.22¥ Tissue distribution of nicotine and cotinine after Fine accuracy and precision were also observed in the subcutaneous administration of nicotine in rats: determination of cotinine. LOD and LOQ of nicotine in rat Furthermore, the tissue distributions of nicotine and cotinine plasma were estimated to be 2.5 and 5 ng/mL, respectively. at 0.5 and 2 h after subcutaneous administration of nicotine With respect to robustness, the chromatogram for the tested ditartrate solution ¤2 mg/kg¥ were examined using the chemicals showed reproducible peak responses in all the developed UPLC/ESI-MS method ¤Fig. 2¥. In addition to deliberately varied chromatographic conditions ¤data not the concentrations in cortex and bladder, where nicotinic shown¥. Thus, the proposed UPLC/ESI-MS method allows acetylcholine receptors are expressed for neuronal control of rapid and economical microanalysis of nicotine and cotinine detrusor function,14,23¥ those in heart and salivary gland were in biological samples without any time-consuming sample also determined because cardiovascular disorders and dry preparation. mouth have been recognized as major adverse effects of Plasma concentration of nicotine and cotinine nicotine. As shown in Figure 2A, the tissue concentrations after subcutaneous administration of nicotine in of cotinine at 0.5 h after administration were found to be ca. rats: The newly developed UPLC/ESI-MS method was 4- to 9-fold lower than those of nicotine. In particular, applied for pharmacokinetic characterization of nicotine nicotine tended to accumulate in the bladder and salivary in rats following subcutaneous administration of nicotine gland. At 2 h after administration, the tissue-to-plasma ¤ ¥ ¤ ¥ ditartrate 2 mg/kg body weight of rat . Figure 1 shows concentration ratios Kp values of nicotine in the bladder, the plasma concentration®time proﬁles of nicotine and salivary gland, heart and cortex were estimated to be 57, 74, cotinine in rats after subcutaneous administration; the 9.2 and 21 mL/g tissue, respectively ¤Fig. 2B¥. In contrast, relevant pharmacokinetic parameters including Cmax,T1/2 the Kp values of cotinine in these tissues were calculated to and AUC0®W are listed in Table 2. Subcutaneous admin- be around 1 mL/g tissue, and much lower than this in heart. istration of nicotine ditartrate solution ¤2 mg/kg¥ resulted Thus, the tissue concentration of nicotine highly exceeded in rapid elevation of plasma nicotine levels up to Cmax that of cotinine during the early period after administration, 122 + 17.2 ng/mL within 15 min, and the plasma concen- and the much lower blood concentration of nicotine might tration of nicotine decreased rapidly with an elimination be responsible for the huge difference of Kp values between half-life of 1.05 + 0.19 h. In contrast, there appeared to be nicotine and cotinine. gradual increase of cotinine, reaching its maximum level From these data, taken together with weak biological ¤226 + 17.6 ng/mL¥ at ca. 2 h after subcutaneous admin- activities of cotinine, it is unlikely that nicotine intake or istration. Elimination of cotinine from the blood was found cigarette smoking would yield a cotinine concentration high to be ca. 7-fold slower than that of nicotine ¤elimination enough to activate nicotinic receptors in the central and rate for nicotine, 0.66 h%1; cotinine, 9.7 ' 10%2 h%1¥. The peripheral tissues, whereas cotinine seemed to be predom- plasma concentration of cotinine exceeded that of nicotine inant in the blood. The deliverable nicotine in the central in all tested periods, and the AUC0®W values for nicotine nervous system and bladder might be responsible for and cotinine were calculated to be 103 + 4.5 and detrusor instability in smokers. However, further inves- 1,940 + 88.1 ng.h/mL, respectively. This was consistent tigations such as pharmacodynamic characterization and with previous observations that absorbed nicotine was analysis of receptor occupancy in each tissue are required to

A B 10 10

1 1 ## ## #

## 0.1 0.1 Concentration (ng/mg tissue) Concentration (ng/mg tissue) 27 1.0 24 1.1 4.2 0.3 11 0.9 57 0.9 74 1.1 9.2 0.2 21 1.1 0.01 0.01 Bladder Salivary gland Heart Cortex Bladder Salivary gland Heart Cortex

Fig. 2. Tissue distribution of nicotine and cotinine in rats at 0.5 h (A) and 2.0 h (B) after subcutaneous administration of nicotine ditartrate (2.0 mg/kg body weight)

Open bars, nicotine; ﬁlled bars, cotinine. The mean Kp value (mL/g tissue) is indicated in each column. Data represent mean + SE (n ¦ 4). ##P g 0.01; #P g 0.05, signiﬁcantdifferenceinnicotineconcentrationfromthebladder.

A 8 B 40

6 g/mL) 30 µ µ

4 20

2 10 Urine concentration ( Urine concentration ( g/mL)

0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 Time (h) Time (day)

Fig. 3. Urine concentrations of nicotine and cotinine in rats after single (A) and repeated (B; b.i.d., 10 days) subcutaneous administration of nicotine ditartrate (2.0 mg/kg body weight) ,nicotine; , cotinine. Data represent mean + SE (n ¦ 4). provide more reliable conclusions since cotinine could affect and the urine concentration of nicotine reached its maximal the afﬁnity states of nicotinic receptors and thus counteract level ¤5.52 + 0.58 µg/mL, corresponding to 34 µM¥ at 4 h some of the effects of nicotinic agonists at the concentrations after administration. Unlike the pharmacokinetic behavior in reached upon cigarette smoking, even without detectable blood, the urine concentration of cotinine was much lower stimulation.22¥ than that of nicotine up to ca. 6 h after administration, Urine concentration of nicotine and cotinine possibly due to the limited C-oxidation of nicotine in the after subcutaneous administration of nicotine in bladder and/or low urinary excretion of cotinine. There was rats: Urinary excretion and nicotinic C-oxidation in the gradual elimination of both nicotine and cotinine in urine liver are major routes of elimination for nicotine.24¥ Since with elimination rates of 0.31 and 0.23 h%1, respectively. nicotinic acetylcholine receptors are expressed in the urinary Previously, our group showed that the IC50 value of nicotine bladder epithelium,14¥ urine concentration of nicotine and for brain ª3H«nicotine binding was 17 nM,25¥ and Vainio ª3 « metabolite should not be neglected for better understanding demonstrated that the EC50 for brain H epibatidine binding of the relationship between cigarette smoking and voiding was 300 nM for nicotine and 130,000 nM for cotinine.22¥ dysfunction. In the present study, after single and repeated Thus, the IC50 and EC50 values for nicotinic receptor binding subcutaneous administration of nicotine ditartrate solution of nicotine were much lower than the urine concentration ¤2 mg/kg¥, urinary excretions of nicotine and cotinine were of nicotine in the current study, so nicotine in the urine monitored using the UPLC/ESI-MS method ¤Fig. 3¥. Rapid might activate nicotinic receptors in the urinary bladder and intense urinary excretion of nicotine was observed after epithelium, thereby signiﬁcantly affecting bladder function. subcutaneous administration of nicotine ditartrate ¤Fig. 3A¥, Interestingly, when nicotine ditartrate ¤2 mg/kg¥ was

Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Pharmacokinetic Study of Nicotine in Rats Using UPLC/ESI-MS 421 administered repeatedly ¤b.i.d., 10 days¥, nicotine was cotinine, support the hypothesis that nicotine, rather than always predominant in urine, with the ratio of urinary cotinine, plays an important role in detrusor instability in excreted amount between nicotine and cotinine in the range cigarette smokers. 2.3®3.7 ¤Fig. 3B¥. Previously, Nakajima and co-workers demonstrated in real-time RT-PCR experiments on human Acknowledgements: The authors are grateful to Mr. tissues that mRNA for CYP2A6 was highly expressed in Tatsuya Ando for excellent technical assistance throughout liver and breast and moderately expressed in lung.26¥ In the work. This work was supported in part by a Grant-in- contrast, the copy number of CYP2A6 mRNA in the bladder Aid for Young Scientists ¤B¥¤No. 22790043; S. Onoue¥ was found to be over two orders of magnitude lower than from the Ministry of Education, Culture, Sports, Science and that in the liver. Although there might be some species Technology, and by a grant from the Smoking Research differences in the expression level of CYP2A6 mRNA, it can Foundation Japan. be hypothesized that the metabolizing enzyme for nicotine, CYP2A6, in the bladder might be saturated by chronic References treatment with nicotine. 1¥ Hoffmann, D. and Wynder, L.: Chemical constituents and Thus, pharmacokinetic studies using the UPLC/ESI-MS bioactivity of tobacco smoke. In Zaridge D. 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