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Serum and tissue concentrations of doxycycline in broilers after the sub-cutaneous injection of a long-acting formulation

This article was downloaded by: [UNAM Ciudad Universitaria]On: 14 February 2013, At: 11:09Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK British Poultry Science
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Serum and tissue concentrations of doxycycline in
broilers after the sub-cutaneous injection of a long-
acting formulation
L. Gutiérrez a , D. Vargas-Estrada a , C. Rosario b & H. Sumano a
a Departamento de Fisiología y Farmacología, Facultad de Medicina Veterinaria y
Zootecnia, Universidad Nacional Autónoma de México, Av. Universidad 3000, Delegación
Coyoacán, Ciudad de México C.P. 04510
b Departamento de Producción Animal, Aves, Universidad Nacional Autónoma de México,
Av. Universidad 3000, Delegación Coyoacán, Ciudad de México C.P. 04510, México
To cite this article: L. Gutiérrez , D. Vargas-Estrada , C. Rosario & H. Sumano (2012): Serum and tissue concentrations
of doxycycline in broilers after the sub-cutaneous injection of a long-acting formulation, British Poultry Science, 53:3,
366-373
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British Poultry Science Volume 53, Number 3 (June 2012), pp. 366—373 Serum and tissue concentrations of doxycycline in broilers after thesub-cutaneous injection of a long-acting formulation ´RREZ, D. VARGAS-ESTRADA, C. ROSARIO1 AND H. SUMANO Departamento de Fisiologı´a y Farmacologı´a, Facultad de Medicina Veterinaria y Zootecnia, UniversidadNacional Auto´noma de Me´xico, Av. Universidad 3000, Delegacio´n Coyoaca and 1Departamento de Produccio´n Animal, Aves, Universidad Nacional Auto´noma de Me´xico, Av. Universidad3000, Delegacio´n Coyoaca ´n, Ciudad de Me´xico C.P. 04510, Me´xico 1. The antibacterial agent doxycycline hyclate (Dox) is usually administered to broilers in drinking water or as a feed supplement. Parenteral injection is not the usual route for administration, soa long-acting formulation (Dox-LA) was tested to evaluate if serum concentrations can achieve thepharmacokinetic/pharmacodynamic (PK/PD) ratios regarded as adequate for the drug.
2. A poloxamer-based matrix was used to provide Dox-LA. Serum and tissue concentrations of Dox vstime were determined in two day-old broilers after subcutaneous (SC) injection of Dox-LA or oraladministration of a single bolus of aqueous Dox (Dox-PO), at a dose of 20 mg/kg. Weight gain, feedconversion rate, haematological variables, aspartate aminotransferase and alanine aminotransferaseactivities, blood urea and creatinine were determined and compared for Dox-LA with Dox-PO and non-medicated controls.
3. Dox-LA had a high relative bioavailability (1200%). Maximum serum concentrations were notstatistically different (5Á1 Æ 1Á1 mg/ml for Dox-LA and 6Á1 Æ 1.4 mg/ml for Dox-PO), but half-life of Dox-LAwas much greater than the value obtained for Dox-PO (73Á0 Æ 0Á9 h and 2Á0 Æ 0Á02 h, respectively). Tissueconcentrations were higher, and stayed higher for longer periods in the Dox-LA group.
4. In conclusion, considering the minimum effective serum concentration against Mycoplasma spp is0Á5 mg/ml, a dose-interval of 180 h can be achieved with Dox-LA, but only for 24 h after Dox-PO. BetterPK/PD ratios for Dox-LA should result in improved clinical outcomes compared with Dox-PO.
Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 therapeutic agent. Doxycycline is a tetracyclinederivative with broad spectrum activity against Parenteral administration of drugs to newly- Gram-positive and Gram-negative aerobic and hatched broilers is not a common practice, this Chlamydia and Rickettsia species (Shaw and (Vermeulen et al., 2002). When necessary, meta- Rubin, 1986; Dorrestein et al., 1990; Goren phylactic and therapeutic doses of antibacterial drugs are administered to broilers with the Doxycycline has some advantages over older drinking water or as in-feed medication during tetracycline derivatives including higher lipid the first days after hatching; for example, to solubility, better bioavailability and tissue distri- control Mycoplasma spp and associated infections bution, longer elimination half-life, and lower (Ismail and El-Kattan, 2004; Stipkovits et al., affinity for calcium ions (Aronson, 1980).
´rez et al., 2006). Among such antibacter- ial drugs, doxycycline ( -6-deoxy-5-hydroxytetra- oral administration have been studied in healthy cycline) has gained a reputation as a valuable chickens (Anadon et al., 1994; Laczay et al., 2001) ´ctor Sumano, Departamento de Fisiologı´a y Farmacologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional ´noma de Me´xico, Av. Universidad 3000, Delegacio ´n Coyoaca´n, Ciudad de Me´xico C.P. 04510, Me´xico. E-mail: [email protected] Accepted for publication 8th November 2011.
ISSN 0007–1668(print)/ISSN 1466–1799 (online)/12/030366—8 ß 2012 British Poultry Science Ltdhttp://dx.doi.org/10.1080/00071668.2012.701004 CONCENTRATIONS OF A LONG-ACTING DOXYCYCLINE IN BROILERS and in Mycoplasma gallisepticum-affected broilers included in a reverse gel copolymer polyoxypro- pyl-polyoxyethylene (poloxamer) (BASF, Mexico Bioavailability has been determined as approxi- City, Mexico), adjusting pH to 7Á0 with a phos- mately 65—70% but there is no parenteral formu- phate buffer solution with constant stirring at 4C lation available due to causing severe tissue to produce a final concentration of 10% Dox in irritation. A possible exception is an experimen- 15% poloxamer. The preparation was completed (Dox-LA) preparation that causes minimum irri- mixture clarified. Quantities (10 ml) were dis- tation and exhibits high bioavailability values pensed into vials, stored at 4C and utilised (4600%) in goats (Vargas et al., 2008), calves (Vargas-Estrada et al., 2008a) and rats (Vargas-Estrada et al., 2008b). Considering the above, itwas considered useful to evaluate and compare the pharmacokinetic parameters of this prepara- A total of 5 groups were formed as follows.
tion with two-day-old broilers compared with Group 1 containing 400 broilers was dosed with those obtained after oral administration of an the experimental preparation (Dox-LA) at approx- aqueous doxycyclin solution (Dox-PO).
imately 1 mg/broiler ($ 20 mg/kg) in a totalvolume of 200 ml using a precision syringe (syringe for oily vaccine, Broiler ND KÕ,CEVAC; Barcelona, Spain) for pharmacokinetic determination. Group 2 (200 broilers) received asimilar Dox-LA dose and these broilers were used The study was approved by the Institutional to investigate any side effects such as tissue damage. Production variables were determined Nacional Autonoma de Mexico, according to during the following 6-week period. In both these the Mexican Official Regulation NOM-062-ZOO- groups, birds were observed for up to 20 min 1999. The experiment was carried out in isola- after injection to detect any changes in behav- tion units at the Universidad Nacional Autonoma iour. Groups 3 (400 broilers) and 4 (200 broilers) ´xico in Mexico City, with a total of 1,400 were given oral doxycyclin (Dox PO) to compare two-day-old broiler chickens having a mean the pharmacokinetics and production variables, (Æ SD) weight of 48 Æ 2Á4 g as determined by respectively. These two groups were treated with weighing 10% of the population. Upon arrival, a single oral dose of a freshly diluted 0Á5% broilers were allowed to settle and water was aqueous solution of doxycycline hyclate by means made available ad libitum through nipple-type of rigid tubing into the proventriculus in a dispensers at 20 birds per cup (Marks, 1981; volume of 200 ml. Group 5 was the untreated Quintana, 1988). House temperature was main- control group (CG with 200 broilers) and dosed tained at 30 Æ 1Á5C using gas heaters and a orally with 200 ml of saline solution.
Sampling times were selected to provide the least amount of error in the estimate of thepharmacokinetic parameters. To analyse concen- trations of doxycycline in tissues, 15 broilers Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 A subcutaneous Dox-LA preparation (100 mg/ml) of from each group were killed, as specified by doxycycline hyclate (PARFARM Pharmaceuticals, Mexican regulations (NOM-009-Z00-1994), 6 h Mexico City, Mexico) was produced under sterile after dosing and then daily for 6 d. The small conditions. Inclusion complexes of doxycyclin intestine, large intestine plus caecum-rectum, 10% (w/v) with -cyclodextrin (1:0Á1 M) (Cerestar liver, lungs and yolk sac from each animal were Pharmaceutical Excipients, Hammond IN, USA) dissected and stored in Eppendorf tubes at were first formed by the kneading method which À20C until analysed. Because of the small can be described as follows: -cyclodextrin (0Á1 M) amount of small intestine and yolk sac collected and distilled water were mixed together in a from each broiler, three separate samples, each mortar to produce a homogeneous paste.
made up of tissue from 5 broilers, were needed Doxycycline (1 M) was added slowly. The mixture to obtain sufficient material for determination of was ground for 30 min and an appropriate quan- doxycycline concentration. Yolk sac was only tity of water added to produce a paste-like determined for 5 d (7 day-old broilers) until this consistency. This was dried at 40—50C for 24 h.
tissue was no longer detectable. Tissue reactions The dried complex was pulverised into a fine at the site of injection were investigated in all powder (Bekers et al., 1991). The resulting powder broilers from the Dox-LA-treated groups during was diluted with a solution of 15% propylenegly- an 8 d follow-up period. For groups Dox-LA col — 10% ethyl alcohol in water. This mixture was (Group 1) and Dox-PO (Group 3), 5 broilers were sampled for blood by direct jugular punc- Percent recovery achieved was 86Á5%, with intra- ture using a one ml syringe and 30 gauge needle and inter-assay coefficients of variation of 5% and at the following times: before treatment and at 1, 2, 4, 6, 8, 12, 24, 26, 28, 30, 32, 36, 48, 50, 52, 54, 56, 60, 72, 74, 76, 78, 80, 84, 96, 98, 100, 102, 104, PKAnalyst (MicroMathÕ, Saint Louis, Missouri, 108, 120, 122, 124, 126, 128, 132, 144, 146, 148, USA), was used to fit and analyse the concentra- 150, 152, 156, 168, 170, 172, 174, 176, 180 and tion-versus-time patterns for each group. Models 196 h. In Group 2, Group 3 and Group 5, blood of best fit (r ! 0Á99) were chosen after analysis by samples were obtained from 10 broilers ran- use of residual sum of squares and the minimal domly chosen before the start of the experiment, Akaike’s information criterion (Welling, 1997).
and at weekly intervals for 6 weeks for haemato- Best fit for the SC route was obtained by use of logical analysis (cell volume, red blood cell counts, total white blood cell count and haemo-globin concentration). Aspartate aminotransfer- activities were determined using an autoanalyser (CELLY 70 Autoanalyzer, Chronolab SA de CV,Mexico City). Finally, blood urea nitrogen and Variables obtained were: AUCt ¼ area under the creatinine were determined as described by curve; AUMC0—1 ¼ area under the first moment Boisness and Taussky (1985). Production vari- curve from 0 to 1 with extrapolation of the ables (weekly weight gain, feed conversion rate terminal phase; MRT ¼ mean residence time; and cumulative mortality) were assessed in these ¼ rate constant for the elimination phase; T½ ¼ elimination half life; T½ab ¼ absorption Within the next three weeks from the end of the experiment, samples were thawed and doxy- cycline concentrations in serum and tissue sam- For Dox-PO, the following general formula ples determined by the quantitative/qualitative agar plate diffusion method (Bennet et al., 1966) Laboratories, Detroit, MI, USA) and Bacillus ¼ AeÀ ÁTime þ BeÀ ÁTime þ CeÀKabÁTime cereus var. mycoides (ATTCC, 11778) as testorganism. This method measured concentration Variables obtained were as above. Relative bioavailability was calculated as follows: chosen bacterium was seeded at an approximate inoculum of 5 Â 105 CFU/ml in Mueller-Hinton City, Mexico) and using chicken serum as dilu- Data are presented as mean Æ standard devi- ent. No baseline activity with blank serum was ation for three sets of observations for each detected. Percentage recovery achieved with this parameter; and for statistical comparisons of technique was 92Á5 Æ 3Á5%. The limit of quanti- Cmax, Tmax, AUCt, MRT and T½ among groups fication (LOQ) was 0Á01 mg/ml identified by the ANOVA was followed by a Bonferroni-test.
means of doxycycline enriched serum samples, Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 which correspond to 8Á0 mm of inhibition halo asmeasured Correlation coefficient of the standard solutionof doxycycline was 0Á95. Intra-assay and inter- Broilers did not manifest signs of pain or assay coefficients of variation were 55% and discomfort after injection of the Dox-LA formu- lation, and no inflammatory response was evi- dent at the injection site. A small painless (T25 Top Ultra Turrax, Staufen, Germany) in swelling was detectable in some broilers. These 2Á0 ml potassium phosphate buffer (0Á1 M, pH 4Á5) swellings had an initial size of approximately and centrifuged at 3000 g for 15 min. The super- 2—4 mm in diameter and gradually disappeared natant layer was transferred to a clean tube.
Triplicates of each tissue sample as well asstandard concentrations in different matrices Haematological, as well as blood urea and creat- (100 ml) were pipetted into individual wells inine and liver enzyme activities remained within made in agar plates and were incubated for the accepted limits for the species and a distin- 18 h at 37C. The limit of quantification was guishable pattern or change between groups was 0Á01 mg/ml, with 8 mm diameter as the lowest not detected (data not shown). Table 1 shows calibration standard on linear standard curves.
CONCENTRATIONS OF A LONG-ACTING DOXYCYCLINE IN BROILERS cumulative mortality in groups 2, 4 and 5. Mean and T½ , showed that all these variables were significantly greater in the Dox-LA treated group doxycycline for group 1 (Dox-LA) and group 3 than in the Dox-PO group (P50Á01). The differ- (Dox-PO) are shown in Figures 1 and 2 and the ence between mean elimination half-lives of determined pharmacokinetic variables are sum- 73 Æ 0Á9 h for Dox-LA and 2 Æ 0Á02 h for Dox-PO marised in Table 2. Comparisons for AUCt, MRT was highly significant (P50Á001). In contrast,Cmax was greater in the Dox-PO group (6Á1 mg/mlin Dox-PO vs. 5Á1 mg/ml in Dox-LA) (P50Á05).
Table 1. Mean Æ SD weight gain, feed conversion rate and A flip-flop pharmacokinetics of this prepara- cumulative mortality in broilers after a single dose of tion is postulated applying Boxenbaum (1998) doxycycline (20 mg/kg) administered either by subcutaneous and Laczay et al. (2001) criteria with the following injection (Dox-LA, 200 broilers) or orally (Dox-PO, 200 broilers). Values of an untreated control group (200 broilers) where Vz is the terminal exponential volume ofdistribution; K is the terminal disposition rate constant once drug absorption is complete (best determined from IV dosing); C is the plasma concentration at time t; and ÁC is the change in plasma concentration over the time interval Át.
For Dox-LA plasma concentration—time data at 24 and 48 h, ÁC/Át ¼ 0Á03 mg/ml/h. At the mid- point of this time period (36 h), (K)(C) ¼ 4Á1 mg/ ml/h. Since KC ) ÁC/Át, and rate of absorp- tion % rate of elimination, a ‘‘flip-flop’’ condition exists and the Dox-LA here described can be The agar diffusion technique used here to deter- a—iDifferent letters within a week-row are statistically significant (P50Á05) Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 90 100 110 120 130 140 150 160 170 180 190 Mean and SD serum concentrations of doxycycline after the subcutaneous injection of a single dose of 20 mg/kg of a doxycycline-LA experimental preparation (Dox-LA), and after administering the same dose orally with a semi-rigid tube (Dox-PO).
Mean Æ 1 SD concentrations of doxycycline in lungs, small intestine, vitellin sac, large intestine plus caecum, and liver after the subcutaneous injection of a single dose of 20 mg/kg of a doxycycline-LA experimental preparation (Dox-LA, n ¼ 15) in a totalvolume of 200 ml on the second day after hatching; or after administering an aqueous solution of the drug by directly depositing it intothe crop with a semi-rigid tube (Dox-PO, n ¼ 15) using the same dose and volume.
doxycycline is a dependable method, allowing Table 2. Mean Æ SD pharmacokinetic parameters in broilers pharmacokinetic data to be extrapolated to after a single dose of doxycycline (20 mg/kg) administered either antibacterial activity with certainty (Santos et al., by subcutaneous injection (Dox-LA) or orally (Dox-PO).
1996; Vargas et al., 2008; Vargas-Estrada et al., Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 been reported in chickens following oral, intra- venous (IV) and intramuscular (IM) administra- et al., 2001; Atef et al., 2002; Ismail and El-Kattan, 2004; El-Gendi et al., 2010). The elimination half- life of doxycycline following oral administration varies with age between 10 and 12 h (Pashov and Kamelov, 1994; Hantash et al., 2008). However, these values are notably different from the T½ values of 3Á64 to 4Á75 h reported by Anado AUCt ¼ area under the curve; AUC0—1 ¼ area under the curve to infinity; (1994), Atef et al. (2002) and El-Gendy et al.
MRT ¼ mean residence time; T½ ¼ half life of the elimination; (2010). In the present study a T½ of 2Á0 Æ 0Á02 T½ab ¼ half life of the absorption; Kel ¼ constant for drug elimination; Tmax ¼ peak time; VdAUC ¼ apparent volume of distribution based on explained by the use of an oral bolus dose.
trapezoid AUC; Fr ¼ relative bioavailability, n ¼ 400 Elimination half life after IM and SC administra- 1 Calculated for flip-flop pharmacokinetics (Concordet, 2010).
tion of a large dose of 100 mg/kg were reported CONCENTRATIONS OF A LONG-ACTING DOXYCYCLINE IN BROILERS to be 86 and 63 h, respectively (Greth et al., 1993). In contrast, Atef et al. (2002) report a half- (Yoshida et al., 1990; Uekama et al., 1998).
life of only 2Á5 h after the IM administration of Because injection of Dox-LA failed to induce 15 mg/kg. In the present study, T½ in the group significant changes in haematological variables, receiving Dox-LA was 73 h. These differences may or a reduction in production values, lack of be explained by the dose (Greth et al., 1993) toxicity of this drug preparation is envisaged.
causing an inflammatory reaction and a depot- It has been postulated that maximum effi- like effect, whereas a real depot effect is likely to cacy in a clinical setting with Dox is achieved have been obtained in this study without an when serum concentrations of the drug are barely at or above the MIC level for the pathogen in question, for as long as possible within the LA and Dox-PO groups (5—6 mg/ml) were higher dosing interval (Craig, 1998; Prescott et al., 2000).
than values obtained after dosing fasted or fed Values of MIC that can be adopted in this broilers with this drug (3Á07 mg/ml to 4Á47 mg/ml, experiment can be categorised as susceptible respectively; Laczay et al., 2001), but they were similar to those reported by Atef et al. (2002) Takahashi and Yoshida, 1989; Stipkovits et al., after IM administration of 15 mg/kg (6Á33 mg/ 2004) and less susceptible for Escherichia coli ml) and Hantash et al. (2008) after oral admin- (1—4 mg/ml: Cunha et al., 1982). Additionally, Notari (1987) suggested a MTC from 0Á5 to 1 mg/ Predictably, for a highly lipid soluble drug ml for this antibacterial drug and a similar value such as doxycycline, a high value of the apparent has been advanced for broilers (Hantash et al., volume of distribution AUC was achieved with 2008). Considering the above, a MTC range for Dox-LA, with a very small total body clearance.
susceptible bacteria can be set from 0Á5 mg/ml to The apparent VdAUC value derived from Dox-PO 1Á5 mg/ml. Hence, the length of time in which is similar to the one reported by Hantash et al.
MTC can be achieved with Dox-PO varies from (2008) and almost double that reported by Atef 10 h to 24 h, and for Dox-LA this interval is et al. (2002). However, no studies are available on extended from 150 to 180 h. If lung concentra- the pharmacokinetics following SC injection of a tions of doxycycline are considered as key values to design a dosing scheme, then SC administra- Relative Dox bioavailability for the Dox-LA tion of Dox-LA is considerably superior as com- group was 1200% as compared with the Dox-PO pared with administering doxycycline through group. Absolute bioavailability in other studies the drinking water. Concentrations in lung was never greater than 70% when comparing the higher than 0Á4 mg/ml are extended until d 6 in the Dox-LA group with a single dose of 20 mg/kg, Although these values of F cannot be directly injected SC. This is consistent with the unusually compared, differences appear substantial. This is not uncommon for formulations with prolonged dictor of therapeutic efficacy for tetracyclines in humans, is the ratio AUC at steady state (AUCss)/ ´lou, 2004). A recycling phenomenon due to the noticeably high lipid solubility of Dox may was not evaluated in this study, if a pathogen is have contributed to these differences (Aronson, susceptible at 1Á5 mg/ml, the AUCDox-LA/MIC Downloaded by [UNAM Ciudad Universitaria] at 11:09 14 February 2013 ratio achieves a value of 407. In contrast, the The rationale behind the design of the Dox- AUCDox-PO/MIC ratio is only 32. Differences are LA preparation was to use the poly (ethylene considerable and clinical efficacy should be higher. Additionally, Dox-LA provides reasonably oxide) block copolymer (poloxamer) as delivery steady serum concentrations for 5 d, while oral vehicle-matrix, because it improves solubility, administration of the drug over such a period will reduces hydrolytic degradation, achieves con- vary according to water consumption with daily trolled release and often results in improved Doxycycline is effective against respiratory Kabanov et al., 1992). The poloxamer exhibits pathogens in avian medicine, as well as in other low viscosity at room temperature (28—32C) and domestic species, because it penetrates well into becomes a gel at body temperature (37—40C) respiratory tissues (Anadon et al., 1994; Atef et al., (Schmolka, 1991), thus allowing the long-acting 2002; Ismail and El-Kattan, 2004). By injecting effect observed. Additionally, reduced irritation Dox-LA, this feature seems to be boosted, and is is postulated to be due to -cyclodextrin by longer-lasting compared with Dox-PO. This is reducing the local concentration of free drug likely to be beneficial when treating susceptible below the irritancy threshold (Szejtli, 1985; pathogens such as Mycoplasma spp, E. coli and Yoshida et al., 1990; Uekama et al., 1998).
others. Additionally, the gastrointestinal system is one of the main routes for elimination of infection. Deutsche Veterinaar Medizinische Gesellschaft, doxycycline, which is therefore effective against colibacillosis and other enterobacterial infec- L-GENDI, A.Y.I., ATEF, A., AZIZA, M.M. & KAMEL, G.M. (2010) Pharmacokinetic and tissue distribution of doxycycline in tions, as the same reasoning applies.
broiler chickens pretreated with either: diclazuril or In conclusion, Dox-LA is a preparation that halofuginone. Food and Chemical Toxicology, 48: 3209–3214.
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Adres van het centrum waar

Informatie voor deelnemers aan het onderzoek “Losartan en Marfan syndroom” Coördinerend onderzoeker: Dr. M. Groenink, cardioloog AMC Amsterdam Artsonderzoekers: F. S. van Dijk, artsonderzoeker VUmc Amsterdam T. Radonic, artsonderzoeker AMC Amsterdam Academisch Medisch Centrum Meibergdreef 9 1105 AZ Amsterdam VU Medisch Centrum Amsterdam De Boelelaan 1117 1081 HV Ams

Eftba_eu_juni 2012

EFTBA UPDATE – June 2012 EFTBA Activity The EFTBA AGM on the 13th May was well attended in the magnificent location of Longchamp racecourse in Paris. Many issues of current concern and relevance were debated and the packed AGM agenda was followed by racing in the afternoon including a prize giving to Dietrich von Boetticher from Germany as the 2012 EFTBA award winner. Following the AGM m

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