Pharmacological assessment of netobimin as a potential anthelmintic for use in horses: Plasma disposition, faecal excretion and efficacy

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Pharmacological assessment of netobimin as a potential anthelmintic for use in horses: Plasma disposition, faecal excretion and efficacy
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Pharmacological assessment of netobimin as a potential anthelminticfor use in horses: Plasma disposition, faecal excretion and efficacy C. Gokbulut a,b, * , V.Y. Cirak c , B. Senlik c , F. Yildirim d , Q.A. McKellar e a Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Adnan Menderes, Aydin, Turkey b Research and Development Laboratory, University of Adnan Menderes, Aydin, Turkey c Department of Parasitology, Faculty of Veterinary Medicine, University of Uludag, Bursa, Turkey d General Directory of Agricultural Farms, Karacabey Agricultural Farm, Bursa, Turkey e The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK  a r t i c l e i n f o  Article history: Accepted 1 October 2008 Keywords: HorseNetobiminAlbendazoleAlbendazole sulphoxideEnantiomersPharmacokineticsEfficacyStrongyles a b s t r a c t This study aimed to determine the plasma disposition and faecal excretion of netobimin (NTB) and itsrespective metabolites as well as the efficacy against strongyles in horses following oral administration.Netobimin(10mg/kg)wasadministeredorallyto8horses. Bloodandfaecal sampleswerecollectedfrom1to120hpost-treatment andanalysedbyhighperformance liquidchromatography (HPLC). Using achi-ral phase-based HPLC, plasma disposition of ABZSO enantiomers produced was also determined. Faecalstrongyle egg counts (EPG) were performed by a modified McMaster’s technique before and after thetreatment. Neither NTB nor ABZ were present and only albendazole sulphoxide (ABZSO) and sulphonemetabolites (ABZSO 2 ) were detected in the plasma samples. Maximum plasma concentration of ABZSO(0.53±0.14 l g/ml) and ABZSO 2  (0.36±0.09 l g/ml) were observed at ( t  max ) 10.50 and 19.50h, respec-tively following administration of NTB. The area under the curve (AUC) of the two metabolites was sim-ilar to each other. Netobimin was not detected, and ABZ was predominant in faecal samples. Themaximumplasmaconcentration( C  max ) of (  )ABZSOwas significantly higherthan (+)ABZSO, but theareaunder thecurves(AUCs) oftheenantiomerwere notsignificantlydifferent eachotherinplasmasamples.TheenantiomersofABZSOwereclosetoracemateinthefaecalsamplesanalyzed.NetobiminreducedtheEPG by 100%, 100%, 77%, 80% and 75% 2, 4, 6, 8 and 10 weeks post-treatment, respectively. The specificbehaviourof thetwoenantiomers probablyreflectsdifferentenantioselectivityoftheenzymatic systemsof the liver which are responsible for sulphoxidation and sulphonation of ABZ. Considering the pharma-cokinetic and efficacy parameters NTB could be used as an anthelmintic in horses.   2008 Elsevier Ltd. All rights reserved. 1. Introduction Netobimin (NTB) is a pro-drug of the benzimidazole sulphide,albendazole. It is used against liver flukes, tapeworms and gastro-intestinal and lung nematodes in ruminants. Netobimin is con-verted into albendazole (ABZ) by gastrointestinal microflorafollowing oral or intra-ruminal administration to sheep and cattle(Delatour et al., 1986; Lanusse and Prichard, 1990 ) . Almost all of theABZabsorbedfromtheintestineinruminantsisrapidlymetab-olized into its anthelm i ntically active albendazole sulphoxide(ABZSO) and inactive sulphone (ABZSO 2 ) metabolites by liver en-zymes (Delatour et al., 1986, Fig. 1). Sulphoxide benzimidazoles (ABZSO and oxfendazole-OFZ),which have a chiral centre about the sulphur atom, are formed asmetabolites of sulphides and are metabolised into sulphones(Fig. 1). Thesulphonesareanthelminticallyinactive,whereassulp-hidesandsulphoxidesarebothactive(Laceyetal.,1987).Theplas-ma dispositions of the two enantiomers of ABZSO andfenbendazole sulphoxide (FBZSO) have been investigated in manyspecies after oral administration of pro-chiral ABZ and fenbendaz-ole(FBZ)(Delatouretal.,1990,1991a,b;McKellaretal.,2002;Gou-dah, 2003). Moreover the  in vitro  enantioselectivity of ruminal andintestinal flora in sulphoxidationof ABZ, sulphonationand sulpho-reduction of ABZSO has been also shown by using the ruminal andintestinal fluids of sheep and cattle (Virkel et al., 2002).Previous studies have reported that the gastrointestinal envi-ronment of equine species probably has a great reductive capacityfor benzimidazole drugs (McKellar et al., 2002; Gokbulut et al.,2002, 2006a). It has been shown that the benzimidazoles licensedfor use in horses in some countries (e.g. FBZ, OFZ and oxibendaz-ole-OXB) have relatively poor bioavailability, since they are exten-sivelymetabolised totheir inactivemetabolitesby gastrointestinalmicroflora and/or first-pass metabolism compared to ruminant 0034-5288/$ - see front matter   2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.rvsc.2008.10.001 *  Corresponding author. Address: Department of Pharmacology and Toxicology,Faculty of Veterinary Medicine, University of Adnan Menderes, Aydin, Turkey. Tel.:+90 256 247 03 40; fax: +90 256 247 07 00. E-mail address: (C. Gokbulut).Research in Veterinary Science 86 (2009) 514–520 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage:  Author's personal copy species. The detailed characterization of the kinetic behaviour andmetabolic pathways of NTB, has markedly contributed to its opti-mized use in ruminants. However, there is no information avail-able on the pharmacological behaviour or efficacy of NTB inequine species. Therefore, the aims of this study were to determinethe plasma disposition and faecal excretion of NTB, and its metab-olites (ABZ, ABZSO and ABZSO 2 ) including the enantiospecific pro-duction of ABZSO and to evaluate the efficacy against strongylesfollowing oral NTB administration to horses. 2. Materials and methods  2.1. Experimental animals Eight thoroughbred horses with a mean weight of 520(516–550 kg) were used in the study. The animals had a historyof grazing pasture contaminated with equine nematode parasitesbut were kept indoors and fed with hay and concentrated horsefeed during the study. They had not been treated with anthelmin-tics during the previous three months. Water was provided  adlibitum  throughout the course of the study.  2.2. Treatments and sampling  A commercially available formulation of NTB (Hapadex, 15%oral suspension, Schering–Plough) was orally administered to ani-mals at a dose rate of 10 mg/kg bodyweight in the study. Heparin-ized blood samples were collected by jugular venipuncture prior todrug administration and 1, 2, 4, 8, 12, 16, 20, 24, 32, 48, 56, 72, 96and 120 h thereafter. Faecal samples (>5 g) were collected per rec-tum throughout the blood-sampling period, before drug adminis-tration and then at 4, 8, 12, 20, 24, 32, 48, 56, 72 and 96 h inorder to determine faecal excretion of the NTB and its respectivemetabolites during the study. Blood samples were centrifuged at3000  g   for 20 min and plasma was transferred to plastic tubes. Allthe plasma and faecal samples were stored at  20   C until estima-tion of drug concentration.  2.3. Analytical procedures Plasma concentrations of NTB, ABZ, ABZSO and ABZSO 2  wereestimated by high performance liquid chromatography (HPLC)with a liquid–liquid phase extraction procedure adapted from thatdescribed by Marriner and Bogan (1980).  2.4. Extraction from plasma Pure standard compounds of NTB (Schering Plough Ltd., Wel-wyn Garden City, UK), ABZ, rac-ABZSO and ABZSO 2  (SmithKlineBeecham, West Sussex, UK) and internal standard OFZ were ob-tained from Sigma Chemical Co. (St Louis, USA). These were dilutedwith acetonitrile to give 0.5, 1, 2.5, 5, 10 and 10, 50, 500 l g/mlstandard solutions for plasma and faecal samples, respectively forcalibration as standard curves and to add to drug-free plasmaand faecal samples to determine the recovery.Drug-free plasma samples (1 ml) were spiked with standards of NTB, ABZ, ABZSO and ABZSO 2  to reach the following final concen-trations: 0.05, 0.1, 0.5, 1 and 5 l g/ml. Ammonium hydroxide(100 l l, 0.1 N, pH 10) was added to 10 ml-ground glass tubes con-taining 1 ml spiked or experimental plasma samples. Oxfendazole(0.5 l g/ml) was used as an internal standard. After mixingfor 15 s, 6 ml ethyl acetate was added. The sample tubes were CH 3 CH 2 CH 2 -S N=CNO 2 NH-CH 2 -CH 2 SO 3 HNH-C-O-CH3ONNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SNNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO  .. NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO ..   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SOO NTB(-)ABZSO (+)ABZSOABZABZSO 2 CH 3 CH 2 CH 2 -S N=CNO 2 NH-CH 2 -CH 2 SO 3 HNH-C-O-CH3OCH 3 CH 2 CH 2 -S N=CNO 2 NH-CH 2 -CH 2 SO 3 HNH-C-O-CH3O   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -S    NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SNNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO  ..   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO  .. NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO ..   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SO ..   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SOO   NNHNH-CO 2 CH 3 CH 3 CH 2 CH 2 -SOO NTB(-)ABZSO (+)ABZSOABZABZSO 2 Fig. 1.  Metabolic pathways of netobimin (NTB), albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO 2 ). C. Gokbulut et al./Research in Veterinary Science 86 (2009) 514–520  515  Author's personal copy stoppered and shaken for 10 min on a slow rotary mixer. After cen-trifugation at 3000  g   for 10 min, the upper organic phase (4 ml) wastransferred to a thin-walled 10 ml-conical glass tube and evapo-ratedtodrynessat40   Cinarotavapour(Maxi-Dryplus,Heto,Den-mark).Thedryresiduewasreconstitutedwith400 l lmobilephase.Then the tubes were placed in an ultrasonic bath and finally, 50 l lof this solution was injected into the chromatographic system.  2.5. Extraction from faeces Wet-faecal concentration of NTB, ABZ, ABZSO and ABZSO 2  wereestimated by HPLC with a liquid phase extraction procedureadapted from that described by Gokbulut et al. (2002). Briefly,wet-faecal samples were mixed finely with a spatula to obtainhomogeneous concentrations. Drug-free wet faecal samples(0.5 g) were spiked with benzimidazole standards to reach the fol-lowing final concentrations: 1, 5, 50, 100, 200 l g/g. Oxfendazole(10 l g/ml) was used as an internal standard. Sodium hydroxidebuffer (200 l l, 0.4 M, pH 10) and 1 ml acetonitrile were added to10 ml-ground glass tubes containing 0.5 g spiked or experimentalwet-faecal samples. After mixing, for 15 s, 8 ml ethyl acetate wasadded. The tubes were shaken on a slow rotary mixer for 15 min.After centrifugation at 3000  g   for 10 min, 4 ml upper organic phasewas transferred to a thin-walled 10 ml-conical glass tube andevaporated to dryness at 40   C in the sample concentrator. Thedry residue was resuspended with 500 l l dimethyl sulphoxide(DMSO). After ultrasonication for 1 min, the samples were filteredwith GF/C glass microfibre filter (Whatman International Ltd.,Maidstone, England). Finally, 10 l l of this solution was injectedinto the chromatographic system.  2.6. Chromatographic conditions The mobile phase was a mixture of acetonitrile-water to whichglacial acetic acid was added (0.5%, v/v). It was pumped throughthe column (Nemesis nukleosil C 18 , 4 l m, 250 mm  4.6 mm Phe-nomenex,Cheshire,UK)withnucleosilC 18  guardcolumn(Phenom-enex, Cheshire, UK) in a linear gradient fashion changing from10:90 (acetonitrile–water) to 85:15 for 8 min; 80:20 to 10:90 for1 min and the last ratio was maintained for 3 min. The flow ratewas 1 ml/min. Samples were processed on a computerized gradi-ent HPLC system (1100 series, Agilent Technologies, GmbH, Ger-many) comprising a degasser, a quaternary pump (G1354A), anauto sampler (G1313), a column oven (G1316A) and diode-arraydetector (G1315B) set at 360 nm for NTB and 292 nm for ABZ, ABZ-SO and ABZSO 2 . The retention times were 6.38 min (ABZSO)7.46 min (FBZSO), 7.93 (NTB), 8.33 (ABZSO 2 ) and 9.41 min (ABZ).The extracted samples were re-analysed by a chiral stationaryphase to determine the concentration of ABZSO enantiomers pro-duced. The enantiomers were estimated by using chiral chroma-tography adapted from that previously described by Delatouret al. (1990) with some modifications. Briefly, a mobile phase of acetonitrile:water (7:93) was pumped at a flow rate of 1 ml/minthrough a Chiral-AGP column (5 l , 150  40 mm, ChromTech,Cheshire, UK) with ultraviolet detection at 292 nm for 6 min andthen the mobile phase ratio was changed to 100% acetonitrileand maintained for 4 min to wash column for less polar moleculesand impurities and finally the ratio changed to initial proportionand (7:93) maintained for 3 min to prepare for next injection.Retention times were 3.27 min for (  )ABZSO and 4.57 min for(+)ABZSO.  2.7. Method of calibration Calibration graphs for the parent drug and the metabolites inthe range 0.05–5 l g/ml (plasma) and 1–200 l g/g (faeces) wereprepared using drug-free plasma and faeces from horses. Recoveryof the parent molecules and the metabolites under study was mea-suredbycomparison ofthepeak areasfrom spikedplasmasampleswith the areas resulting from direct injections of standards. The in-ter-assay precision of the extraction and chromatography proce-dures was evaluated by processing replicate aliquots of drug-freehorse plasma and faecal samples containing known amounts of the drugs on different days. The slope of the lines between peakareas and drug concentration was determined by least squares lin-ear regression and showed correlation coefficients between 0.996and 0.999.The limits of quantification (LOQ) were 0.05–0.08 l g/ml forplasma and 0.4–0.7 l g/g for faecal analysis. These values beingthe lowest concentrations detected with a coefficient of variations(CV) lower than 20%. The mean extraction recoveries for the plas-ma samples were 77.3% (SD = 8.82%) for NTB, 83.1% (SD = 5.34%)for ABZ, 94.1% (SD = 7.84%) for ABZSO and 95.1% (SD = 5.19%) ABZ-SO 2 ; and for faecal samples were 67.1% (SD = 8.41%) for NTB, 86.2%(SD = 10.37%) for ABZ, 94.5% (SD = 7.17%) for ABZSO and 74.2%(SD = 9.12%) ABZSO 2 . The overall accuracy (percent relative error)of the method was ranged from   7.8% to +3.1%.To determine the dry proportion of wet faecal samples, 2.0 g of wet faeces from each sample was weighed exactly into an evapo-rating bowl and heated in an oven at 75   C for 10 h. The weightof each was determined and the percentage of each dry samplewas calculated.  2.8. Parasitological analysis To determine the efficacy of NTB against strongyles, faecal sam-ples were collected at 2 week intervals during the 10 weeks aftertreatment. Strongyle egg counts per gram of faeces (EPG) weredetermined by a modified McMaster’s technique with a sensitivityof 50 EPG (MAFF, 1986). Pooled group faecal samples were incu-bated at 27   C for 7 days for larval identification (MAFF, 1986).Arithmetic group mean EPG’s were calculated before and afterthe treatment for each sampling date. The percentage faecal eggcount reduction (FECR) of NTB was calculated according to theformula:  2.9. Pharmacokinetic and statistical analysis of data The plasma or faecal concentration versus time curves obtainedafter each treatment in individual animals, were fitted with theWinNonlin software program (Version 4.1, Pharsight Corporation,Mountain View, CA, USA). Pharmacokinetic parameters for eachanimal were analysed using non-compartmental model analysiswith extravascular input. The maximum plasma concentration( C  max ) and time to reach maximum concentration ( t  max ) were ob-tained from the plotted concentration-time curve of each drug ineach animal. The linear trapezoidal rule was used to calculate thearea under the plasma concentration time curve (AUC): FECR  ð % Þ¼ mean EPG ð before treatment Þ mean EPG ð after treatment Þ mean EPG ð before treatment Þ  100 516  C. Gokbulut et al./Research in Veterinary Science 86 (2009) 514–520  Author's personal copy AUC 0  last  ¼ X ni ¼ 1 C  i þ C  i  1 2  ð t  i  t  i  1 Þ where C represents the plasma concentration,  i  1 and  i  are adjacentdata point times. The area under the first movement curve (AUMC)was calculated using the equation: AUMC 0  last  ¼ X ni ¼ 2 C  i t  i þ C  i  1 t  i  1 2  ð t  i  t  i  1 Þ Thus, the mean residence time (MRT) was calculated as: MRT 0  last  ¼ AUMC 0  last = AUC 0  last Terminal half life (t 1/2 k  z  ) was calculated as: t  1 = 2 k  z   ¼ ln ð 2 Þ = k  z  Where  k  z   represent the first order rate constant associated with theterminal (log-linear) portion of the curve. A minimum requirementis to have 3–4 observations in the terminal slope. The terminalelimination rate constant ( k  z  ) was estimated by means of log-linearregression on those observations. The pharmacokinetic parametersare reported as mean ± SD. Mean kinetic parameters of the enanti-omers were statistically compared by one-way analysis of variance(ANOVA, Minitab Release 12.0). Mean values were considered sig-nificantly different at  P   < 0.05. 3. Results No adverse reactions were observed in any of the horses treatedwith NTB. Neither NTB nor ABZ were detected in any plasma sam-ples analysed. Fig. 2 shows the mean (±SD) plasma concentrationversus time curves and Table 1 shows the mean (±SD) pharmaco-kinetic parameters of ABZSO and ABZSO 2 . The areas under thecurve (AUC) of ABZSO and ABZSO 2  were 8.63 ± 1.01 l g h/ml and8.21 ± 2.87 l g h/ml, respectively. 0 10 20 30 40 50 600.  ABZSO ABZSO 2    P   l  a  s  m  a  c  o  n  c  e  n   t  r  a   t   i  o  n   (  µ  g   /  m   l   ) Time (h) Fig. 2.  Mean (±SD) plasma concentrations ( l g/ml) of albendazole sulphoxide(ABZSO) and albendazole sulphone (ABZSO 2 ) following oral administration of netobimin (10 mg/kg) to horses ( n  = 8).  Table 1 Mean (±SD) pharmacokinetic parameters of total albendazole sulphoxide (ABZSO)(+)ABZSO, (  )ABZSO and albendazole sulphone (ABZSO 2 ) following oral administra-tion of netobimin (10 mg/kg) to horses ( n  = 8). Parameters ABZSO ABZSO 2 RacaemicABZSO(  )ABZSO (+)ABZSO t  max  (h) 10.50 ± 3.66 9.50 ± 2.98 12.50 ± 2.56 19.50 ± 3.96 C  max  ( l g/ml) 0.53 ± 0.14 0.35 ± 0.10 a 0.19 ± 0.05 0.36 ± 0.09AUC last  ( l g h/ml)8.63 ± 1.01 4.60 ± 0.85 3.78 ± 0.26 8.21 ± 2.87 t  1/2 k z  (h) 5.97 ± 1.59 5.86 ± 1.98 8.47 ± 3.25 7.44 ± 1.06AUMC last ( l g h 2 /ml)129.12 ± 20.10 55.38 ± 9.19 67.40 ± 15.00 177.55 ± 74.17MRT last  (h) 15.08 ± 2.50 12.17 ± 1.60 17.76 ± 3.41 21.33 ± 2.31 C  max , peak plasma concentration;  t  max , time to reach peak plasma concentration;AUC last , area under the (zero moment) curve from time 0 to the last detectableconcentration; AUMC last , area under the moment curve from time 0 to  t   lastdetectable concentration; MRT last , mean residence time;  t  1/2 k z , terminal half-life. a Mean parameters of (  )ABZSO are significantly different from those obtainedfor (+)ABZSO ( P   < 0.05).  (-)ABZSO (+)ABZSO 0102030405060708090100  % (-)ABZSO % (+)ABZSO    P  e  r  c  e  n   t  a  g  e  r  c   t 0.25  (-)ABZSO (+)ABZSO 0 10 20 30 40 50 60 Time (h)    P   l  a  s  m  a  c  o  n  c  e  n   t  r  a   t   i  o  n   (  µ  g   /  m   l   ) Time (h) 0 10 20 30 40 50 Fig. 3.  Mean (±SD) plasma concentrations ( l g/ml) of total, (  ) and (+) albendazolesulphoxide (ABZSO) following oral administration of netobimin (10 mg/kg) tohorses ( n  = 8). (Smaller graph: Mean percentage of (  ) and (+) enantiomers of albendazole sulphoxide (ABZSO)). 1101001000  ABZ ABZSO    D  r  y  -   f  a  e  c  a   l  c  o  n  c  e  n   t  r  a   t   i  o  n   (  µ  g   /  g   ) Time (h) 100150200250  ABZ ABZSO    D  r  y  -   f  a  e  c  a   l  c  o  n  c  e  n   t  r  a   t   i  o  n   (  µ  g   /  g   ) Time (h) 0 10 20 30 40 50 60 70 8050 0 10 20 30 40 50 60 70 80 Fig. 4.  Mean (±SD) dry-faecal concentrations ( l g/g) of albendazole (ABZ) andalbendazole sulphoxide (ABZSO) following oral administration of netobimin(10 mg/kg) to horses ( n  = 8). (Smaller graph: Semi log plot of mean±SD dry-faecalconcentration versus time curve).  Table 2 Mean (±SD) faecal kinetic parameters of albendazole (ABZ) and albendazole sulph-oxide (ABZSO) following oral administration of netobimin (10 mg/kg) to horses( n  = 8). Parameters ABZ ABZSO t  max  (h) 24.50 ± 3.34 26.00 ± 3.70 C  max  ( l g/g) 226 ± 144.18 10.16 ± 6.55 t  last  (h) 56.00 ± 7.41 52.00 ± 4.28AUC last  ( l g h 2 /g) 3178.88 ± 1153.85 199.78 ± 72.93MRT last  (h) 27.61 ± 2.25 29.43 ± 3.80 C  max , peak faecal concentration;  t  max , time to reach peak faecal concentration;  t  last ,time to the last detectable faecal concentration; AUC last , area under the (zeromoment) curve from time 0 to the last detectable concentration; MRT last , meanresidence time. C. Gokbulut et al./Research in Veterinary Science 86 (2009) 514–520  517
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