Villa

World Rabbit Sci. 2003, 11: 87 - 100 WRSA, UPV, 2003 TISSUE DISTRIBUTION AND RESIDUE DEPLETION OF
FLUMEQUINE IN THE RABBIT
VILLA R., CAGNARDI P., BACCHETTA S., SONZOGNI O., ARIOLI F., CARLI S.
Department of Veterinary Sciences and Technologies for Food Safety University of Milan, Via Celoria, 10. 20133 MILAN, Italy.
Abstract: Flumequine is a fluoroquinolone derivative used in food-producing species to control systemic
infections caused by susceptible microorganisms, in particular Gram negative species such as
Escherichia coli, Salmonella spp. and Pasteurella spp. Our study was carried out in order to evaluate
the distribution and residue depletion of flumequine in rabbits. Tissue distribution was defined
administering a single oral dose of 15 mg of flumequine per kg body weight. Residue depletion was
determined administering the drug via drinking water at the ranging dose of 15 mg per kg body weight
for 5 days. The tissue concentrations were quantified using a HPLC method, with a quantification limit
of 25 mg.kg-1 for muscles, fat and lungs and of 50 mg.kg-1 for livers and kidneys. The experimental results
show that in rabbits flumequine reaches effective tissue concentrations rapidly after oral treatment. At
the moment of sacrifice (withdrawal time 0 hours) the residue depletion study showed the highest
concentrations in the kidney and the liver (2064 with SD 1571 and 388 with SD 25 mg.kg-1, respectively),
while in the other tissues analysed (muscles, fat and lungs) the residues were much lower (27 with SD
30, 38 with SD 12, 60 with SD 34 mg.kg-1 in muscles, fat and lungs, respectively). The residue
concentrations decrease quickly and fall below the maximum residual limits, as defined by the European
Authorities (200, 250, 500 and 1000 mg.kg-1 for muscles, fat, livers and kidneys, respectively), within 24
hours from the cessation of medication. Considering the tissue concentrations observed after the repeated
administration it can be concluded that at the dose employed (15 mg.kg-1) potentially effective drug
concentrations are recorded only in the liver and the kidney.
Key words: flumequine, rabbit, residues, tissue distribution.
As reported in a previous paper (VILLA et al., 2001), in all food-producing species, it is very important to guarantee the correct use of drugs in order to ensurethe production of safe foodstuff for human consumption. A proper pharmacological Correspondence: R. Villa.
E-mail: [email protected] VILLA et al.
therapy is based on various aspects; however, there are two main problems thatmust be considered. The definition of dosing regimens must be to achieve prefixedtherapeutic objectives in the target species, while also protecting the environmentand reducing the cost of therapy. Besides, the determination of the withdrawal timesmust be adequate in order to ensure the absence of tissue residues higher than themaximum residual limits (MRLs) in food intended for human consumption.
As stated in the Report by the Committee for Veterinary Medicinal Products (CVMP) (EMEA, 1997), these aspects are investigated principally in the so-calledmajor species and scarcely in the minor animal species (equine, ovine-caprine, poultry[nonbroiler], rabbits, fishes [non-salmonidae]). In relation to the world-wide food-stuff production at world-wide, minor species, have a reduced importance; however,locally, in well-defined geographical areas, they are very important.
Few experimental studies on tissue distribution and residue depletion of the drugs used in this animal species have been undertaken and published. In this studythe distributive and excretive behaviour of flumequine, a synthetic antibacterial drugthat is increasingly being used on rabbits, was investigated. Structurally related tonalidixic acid (MEVIUS et al., 1990a; REYNOLDS, 1996; DELMAS et al., 1997),Flumequine (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid) is a second generation quinolone developed for use in veterinarymedicine. Flumequine (FLU) is never administered on humans because of theavailability of more active compounds with broader antimicrobial spectrum and abetter tissue distribution (LEMELAND et al., 1981; CRUMPLIN, 1988). Conversely, inseveral animal species (ruminants, swine, fowl, fish) the drug is diffusely used tocontrol systemic infections caused by Gram negative microorganisms (e.g.,Escherichia coli, Salmonella spp. and Pasteurella spp.) (DORRESTEIN et al., 1983;PIJPERS et al., 1989; MEVIUS et al., 1990b; WERKMAN, 1996). The minimal inhibitoryconcentrations, such as MIC (the least amount of antibiotic that will inhibit the growth of 90% of the test organisms) for the above reported strains, ranges fromabout 0.1 to 0.8 mg.mL-1, (CHEVALIER et al., 1982; ZIV et al., 1986; ATEV et al.,1987; HANNAN et al., 1989; PIJPERS et al., 1989).
RESIDUE STUDY OF FLUMEQUINE IN RABBITS FLU is slightly metabolised by the liver, and its principal metabolite, identified in faeces and urine, is hydroxylated. The extent of this metabolism ranges from 10to 20%. For this reason only flumequine as parent compound is considered to be theMarker Residue in the definition of the MRL.
Several studies are available on the kinetics of FLU in ruminants and fowl when administered at doses ranging from 5 to 15 mg.kg-1 body weight (b.w.) (GOREN etal., 1982; ZIV et al., 1986; MEVIUS et al., 1990a; DELMAS et al., 1997). Conversely,like the information pertaining to most antimicrobials administered to rabbits, FLUinformation for this species is scarce. Consequently, the clinical protocols and thewithdrawal times for FLU are often defined on the basis of experimental studiesperformed on other species.
The objective of this study is to define the tissue distribution and the residue depletion of FLU in rabbits orally treated. This was done in order to verify theusefulness of the drug in controlling systemic infections by susceptible pathogensand to determine its distributive ability in the most important tissues while calculatingan appropriate withdrawal time considering the MRL values (200, 250, 500 and1000 mg.kg-1 for muscles, fat, livers and kidneys, respectively) as established by theEuropean Authorities (EMEA, 2002).
MATERIALS AND METHODS
The experiment was carried out on 52 "French crossbreed" food-producing rabbits from the Perego farm in Milano, Italy.
Animals: 24 rabbits of both gender, with a mean weight of 2.7 kg and clinically healthy, were used. The animals were caged individually and submitted to a 12-hlight/dark cycle in accordance with European requirements (EEC, 1986). They werehoused for an acclimatisation period of seven days before the start of the experiment.
VILLA et al.
During periods of acclimatisation and experimentation, the animals were fed onlycommercial pellets with no active ingredient potentially interfering with the FLUtitration. The pelletted feed was produced by Martini & Co. in Forlì, Italy. Theanimals had free access to food and water.
Treatment: Four animals out of 24 were not treated in order to obtain negative controls. The other rabbits were treated individually by gavage feeding at a dose of15 mg.kg-1 b.w., a commercial formulation (Flumexil granulato idrosolubile 10%,A.T.I., Italy) being administered. During the gavage feeding process, a mouth gagprepared from the plastic case of a 5 ml syringe and a urinary catheter (Rusch Nelaton40 CH.14) were used.
Sacrifice and Sample collection: the 20 animals treated were allocated ad random to 5 groups (4 animals per group, two males and two females). The treated groupswere sacrificed at 1, 3, 6, 9 and 12 hours post-treatment; the control animals weredivided ad random into 2 groups of 2 subjects (1 male and 1 female) and sacrificedat the time-point of 3 and 6 hours post-treatment. All the animals were euthanised ina CO chamber in accordance with the requirements set out in Recommendations for Euthanasia of Experimental Animals (Commission of the European Communities,1993). From each animal, samples of muscles, livers, kidneys, fat and lungs werecollected and stored at – 80°C pending assay.
Residue depletion study
Animals: 28 clinically healthy rabbits of both genders, with a mean weight of 2.5, were used. As reported in the distribution study, the animals were cagedindividually, acclimatised, maintained and fed. During the whole experimental periodfood and water consumption was registered daily.
Treatment: 4 animals were not treated in order to obtain negative controls. The other 24 animals were treated for 5 days with medicated drinking water (150 ppm ofFLU) prepared daily using a commercial-type formula (Flumexil granulatoidrosolubile 10%, A.T.I., Italy). In order to ensure a daily intake of about 15 mg of RESIDUE STUDY OF FLUMEQUINE IN RABBITS FLU per kg b.w., the amount of the drug dissolved in drinking water was calculatedin accordance with the recorded water consumption. Medicated water wasadministered to rabbits from individual bottles and the individual water consumptionand the consequent drug assumption were recorded daily.
Sacrifice and Sample collection: after the administration, the animals were randomly allocated in 4 groups of 6 animals (3 males and 3 females) and sacrificedat 0, 24, 48 and 72 hours; the control animals were randomly divided in 2 groups of2 animals (1 male and 1 female) and sacrificed at time-point 0 and 24 hours post-treatment. The animals were euthanised as reported in the distribution study.
Furthermore, the samples collected and the storage conditions were the same as inthe previous trial.
Method of analysis
HPLC was used for the analyses. Samples (5 g) were homogenised with 20 ml of extraction buffer (A: an aqueous solution of metaphosphoric acid 1%; B: methanol[A 60%: B 40%]). After sonication in a water-bath (30 min at 50°C) and centrifugation(10000 g per 10 min at 4°C), the liquid phase was cleaned up using a SPE columnSep-Pak VAC C18 (Waters, Millipore, Italy). Elution was carried out using 10 mlof methanol, which was dried under nitrogen flow and then added with 1 ml ofortophosphoric solution 0.02M. The eluates were assayed under the followingchromatographic conditions: ortophosphoric acid 0.02M, acetonitrile andtetrahydrofuran (69:18:13) during the mobile phase; 1 ml.min-1 flow rate; 325 nmexcitation wavelength; 365 nm emission wavelength; chromatographic column: 5ODS (3) Prodigy, 5 mm, 250 x 4.6 mm (Phenomenex, Torrance, USA). The limitsof quantification (LOQ), which are the lowest concentrations at which the methodof analysis is able to quantify the substance with sufficient linearity, accuracy andreproducibility, were 25 mg.kg-1 for muscles, fat and lungs and 50 mg.kg-1 for liversand kidneys. The limits of detection (LOD), which are the lowest concentrations atwhich the method is able to recognize, though without quantifying the substance,were 5.9, 5.9, 4.7, 4.5 and 4.1 mg.kg-1 for muscles, livers, kidneys, fat and lungs,respectively. The method, validated intra-laboratory, was specific, linear,reproducible and accurate. Mean recoveries were 99.5, 94.6, 100.9, 92.7, 90.1 % VILLA et al.
for muscles, livers, kidneys, fat and lungs, respectively.
The statistical analysis of the groups was undertaken using the Kruskal-Wallis test (non-parametric ANOVA) with Dunn's post test, performed with GraphPad InStatVersion 3.00 for Windows 95/NT, GraphPad Software (San Diego, CA, USA).
The LOQs were set taking into account the suggestions of the CVMP (EMEA, 1996) for a LOQ having at least half the value of the established MRL. Our methodwas also linear at concentrations far below half the MRL, and therefore we decidedto set the LOQs at the lowest tissue concentrations where the validation criteriawere satisfied (25 mg.kg-1 for muscles, fat and lungs and 50 mg.kg-1 for livers andkidneys). The LOD values were calculated as mean of the noise threshold of 20chromatograms of blank samples plus 3 times the standard deviations. In Tables 1and 2 the residue values, presented as mean ± standard deviation, are sometimesbelow the LOQ defined for that tissue. The reason for this is that in the calculationof the mean values the results below LOQ (<LOQ) were considered as the LOQvalue and the ND (not detected) as 0.
The experimental data obtained following individual administration by gavage feeding is reported in Table 1. In all the tissues investigated, mean peak concentrationswere achieved about 1 hour after treatment. Thereafter the kinetic profiles of liverand kidneys showed a constant decrease until the 12th hour post-treatment (200 ±8mg.kg-1 and 632±173 mg.kg-1, respectively). For muscles, FLU was measured onlyuntil the 6th hour post-treatment (27±21 mg.kg-1); then, in all the other samples FLUconcentrations were not detectable or below LOQ. For lungs, after the peak levelsrecorded at the first collection time, the FLU concentrations resulted quite constantat the 3h and 6h time-points, and then decreased below the LOQ as mean value atthe 9h collection time. Finally, for fat FLU concentrations also resulted low at the RESIDUE STUDY OF FLUMEQUINE IN RABBITS Table 1: FLU concentrations (mkg-1) after gavage administration at 15 mg.kg-1.
LOQ muscles, fat and lungs= 25 mg.kg-1; LOQ liver and kidney = 50 mg.kg--1; *ND = not detected, considered as 0 in the statistical analyses; ** <LOQ = residues below the LOQ were considered as LOQ in statistical analyses; (§)= significantly different fromconcentrations at 12h (P<0.05); (#)= significantly different from concentrations at 9h (P<0.05); SD = standard deviation.
VILLA et al.
first collection time (41±12 mg.kg-1), decreasing rapidly until reaching values eitherbelow LOQ or undetectable between 3h and 6h time-points.
The relevant variations of the rabbits recorded in this trial could not be attributable to differences in the individual drug assumptions, which werehomogeneous. However, the substantial individual variability could be attributed tophysiological and metabolic differences among the animals and by the presence ofdifferent quantities of food in the digestive tract.
With regard to the 5-day administration of medicated water (residue study), the higher tissue concentrations are recorded at the suspension of treatment, as reportedin Table 2. The decrease of FLU concentrations was very rapid, arriving at valueslower than the LOQs already at 24 hours of withdrawal time in 5 out of 6 rabbits inlivers and in 2 out of 6 rabbits in kidneys. FLU was not quantified in muscles, fatand lungs following the first collection time, with the exception of one lungs samplein one rabbit sacrificed at the 24h time-point (77 mg.kg-1). FLU titrable concentrationswere not observed in any of the tissues collected at the 48h and 72h time-points,with the exception of a sample of liver containing 139 mg.kg-1 at the 48h time-point.
Many individual variations were also observed in this experimental phase, but, as mentioned above, these could not be attributed to the different individual drugassumptions that, as reported in Table 3, were quite homogeneous. Additionally,individual variability and digestive tract repletion differences in this trial are probablythe main reasons for the substantial differences observed within the groups.
In the tissue distribution study, the livers, kidneys and fat showed significant differences in the concentrations assayed only between 1h and 12h (P<0.05), musclesand fat between 1h and 9h (P<0.05), and in the kidneys significant differences werealso observed between 3h and 12h (P<0.05).
In the residue study, the statistical analysis of the livers and kidneys showed a significant difference between the concentration assayed at 0h and those assayed at RESIDUE STUDY OF FLUMEQUINE IN RABBITS Table 2: FLU concentrations (mg.kg-1) after repeated administration (5 days) of medicated water (150 ppm).
LOQ muscles, fat and lungs = 25 mg.kg-1; LOQ liver and kidney = 50 mg.kg-1; *ND = not detected, considered as 0 in the statistical analyses; ** <LOQ = residues below the LOQ were considered as LOQ in statistical analyses; (§) = significantly different from concentrations at 48h (P<0.01); (#) = significantly different from concentrations at 72h (P<0.001); (£) = significantly different fromconcentrations at 24h (P<0.001); (&) = significantly different from concentrations at 48h (P<0.001);($) = different from concentrations at 72h (P<0.05); s.d. = standard deviation.
VILLA et al.
48h (P<0.01) and at 72h (P<0.001). With respect to the lungs, the concentrations at0h were always significantly different from all the other time points (P<0.001). Asfor the fat, a significant difference in the residues was only observed between 0hand 72h (P<0.05), while in the muscles no statistical differences were observed.
FLU concentrations recorded for kidneys and livers in the distribution study were found to be in accordance with our expectations. These concentrations weregreater than the MIC values available in the literature on the most susceptible microorganisms for the entire 12h experimental period in kidney samples and for 6hours in liver samples (Table 4). The FLU levels recorded in the other tissuesinvestigated was lower. In particular, for muscles, fat and lungs the drugconcentrations were never higher than MICs . For tissue collected at the 6h time- point FLU was detectable at measured levels in two out of four muscles samplesand in three out of four lungs samples, while the drug was not quantified in fat Table 3: FLU assumption (mg.kg-1) during the 5-days
period of administration of medicated water (150 ppm ).
SD = standard deviation.
RESIDUE STUDY OF FLUMEQUINE IN RABBITS samples. These results reveal a very short duration of FLU efficacy in the control ofsystemic infections sustained by susceptible bacteria.
However, the experimental data recorded in the distribution study shows that the antibiotic diffuses rapidly (1 hour post-administration), although in differentconcentrations, in all the tissues studied.
The FLU levels observed at the first time of sacrifice (1 hour) following the gavage administration of the single dose of 15 mg.kg-1 were higher than thoserecorded at the first time-point (0h) following treatment with medicated water. Theseresults are probably attributable to the different types of treatment: administrationby gavage in the distribution study and by continuous assumption (medicated water)during the 5-day period in the residue depletion study. In fact, in the first type oftreatment the drug dose was administered as a bolus in a unique administration,whereas in the second type of treatment the same dose was assumed during a 24hperiod. The drug was, therefore, available for a longer period but at smallerconcentrations.
Table 4: Minimal inhibitory concentrations MIC (mg.mL-1) of
flumequine for bacterial strains isolated from several species (from CHEVALEIER et al., 1982; ZIV et al., 1986; ATEV et al., 1987; HANNAN et al., 1989; PIJPERS et al., 1989).
Escherichia coli VILLA et al.
The analytical method used for the detection of FLU in the different matrixes was specific, linear, reproducible and accurate. The extraction procedure is easilyapplicable, but not very rapid as only a few samples can be processed in a day.
In conclusion, the results of the experiment enable us to suggest that in the rabbit an oral dose of 15 mg.kg-1 administered as a bolus by gastric gavage is adequateto ensure effective concentrations for at least 6-8 hours only in livers and kidneys,whereas this dose seems to be insufficient to control respiratory, muscoloskeletaland adipose infections sustained by susceptible bacteria.
The same conclusions can also be drawn a considering the results of the residue study in which FLU was administered via drinking water. In fact, at the cessation oftreatment (5 consecutive days), potentially effective drug concentrations wererecorded only in the livers and kidneys.
Finally, the results deriving from the residue depletion study allow us to conclude that a 2-day withdrawal time for FLU preparations administered via medicated waterfor about 5 days at the dosing regimen, which is normally adopted in the commercialbreeding sector, can be considered sufficient to guarantee the respect of establishedMRLs.
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