Pharmacokineticevaluation of co- administration of Triclabendazole and Tildipirosinin goats

*Azza M.M. Abdelmoteleb, **HallaM.Kallil, **Halla A., Elsaid, **Hala S. Abubaker

*Biochemistry, Toxicology and Feed Deficiency Dept., Animal Health Research Institute, Agricultural Research Center, Dokki, Giza, Egypt.

**Bacteriology Dept., Animal Health Research Institute, Agricultural Research Center, Dokki, Giza, Egypt.

Abstract

The existing researchaimed to explore triclabendazole effect (10 mg/kg b.wt., orally ) on disposition kinetic of tildipirosin (4 mg/kg b.wt) administered by intravenous or intramuscular routes in goats.Serum tildipirosin concentration was measured using microbiological technique by Bacillus subtilis. The concentration-time curve of tildipirosin was described via twocompartment open model. Concomitant administration with triclabendazole decreased tildipirosin concentrations in serum post IV and IM injection.

After IV administration, serum tildipirosin concentration at zero time (Co) showed lower in the triclabendazolepretreated goat group.The administration of triclabendazole significantly increased thetildipirosin distribution rate constant (alpha), the apparent peripheral compartment volume (V2); distribution volume at steady state (Vdss), total body clearance (CL) and inter-compartmental clearances (CL2) in the second group compared with first group. On the other hand, triclabendazole potentially reducedthe half-life of distribution (t1/2 Alpha), half- life of elimination (t1/2Beta), area under curve (AUC0-t), and mean residence time expressed as MRT in the triclabendazole co-administered group.

Following intramuscular injection, absorption half-life and corresponding t max exposedfasttildipirsonabsorption rate.Maximum serum concentration (Cmax)and area under the curve of tildipirosinshowed significantdeclineoftriclabendazole co-administered group compared with tildipirosin alone.Both t1/2 beta and MRT were decreasedpostI/M injection in the tildipirosin-triclabendazolegroup.

Simultaneous administration of triclabendazoleandtildipirosincaused significant variations in tildipirosin disposition kinetic.The interactions between the two drugs have clinical importance and so require tildipirosin dosage monitoring.

Keywords: Benzimidazole-Macrolides- Gram negative Bacteria-Microbiology -pharmacokinetic

INTRODUCTION

Macrolidesare bacteriostatic drugsfor vast majority of microorganismsbecause of their attachment to ribosomal ribonucleic acid at the 23S of the bacterial cell's ribosomalsubunits (50S) that restricting protein synthesis.Tildipirosin has 16–membered ring that has been authorized in macrolide veterinary medicine for cattle, pigs, sheep, rabbits and horserespiratory disease treatment(Bartram et al. 2016; Confer et al. 2016; Lei et al. 2018;Xiong et al. 2020; Abu Basha et al.2021).

Tildipirosin has been found to have a bacteriostatic impactonB. bronchiseptica and P. multocida, as well as a bactericidal action against H. somni, M. haemolytica and A. pleuropneumoniae and also H. parasuis in in vitro experiments.Cattle, mice, rabbits and dogs have all been studied for tildipirosin pharmacokinetics and proved that tildipirosin had extended half-life and high bioavailability following extravascular injection in these trials (Torres et al. 2016;Teixeira et al.2017;Arsic et al. 2018;Zenget al. 2018;EMA 2020).

Anthelmintic drugs are currently the major method for managing parasite infections.Nevertheless, the growth of drug-resistant portends treatment efforts.Triclabendazole (TCBZ) is a novel benzimidazole medication that works against Fasciola hepatica andFasciolagigantica(mature and immature stages) of cattle, sheep and goat (Sanyal1994;Mottier et al. 2004). The oxidized metabolites of triclabendazole include triclabendazolesulphoxide, triclabendazolesulphone, and ketotriclabendazoleshowed efficient effects against parasite (Halferty et al. 2009;Belal et al. 2014; Robles-Pérez et al. 2015; Wang et al. 2019).

Medication combinations are widely used in veterinary medicine. These tested combinations in goats exhibited either pharmacological potentiation or decreased effect(Sidhu et al. 2006;Gould 2016). In veterinary medicine, minimum attention has been paid to the pharmacokinetic interactions of anthelmintic with antibacterial drugs. To date, there is no data on the pharmacokinetics of compiled tildipirosin with triclabendazoletreatment in goats.Thus, the objective of this research was to appraisethe influence of oral triclabendazole administration on intravenous (IV) and also intramuscular (IM) administration of tildipirosin pharmacokinetic in goats.

Materials and Methods

Drugs and Chemicals:

-      Tildipirosin (Zuprevo® ,solution containing  180 mg/ml) was obtained fromIntervet, Netherlands.

-      Triclabendazole(tricazanamed®,suspension containing 100 mg/ml ) was obtained from Sigma Pharmaceutical Industries , Egypt .

Animals:

Twelve healthy male adult Baladi goats weighed 45 ± 5 kg body and confirmed to be healthy by physical examination. Goatswere fed a diet of barely grain and green feedin hygienic place for two months prior to the start of the experiment without any medications. The Ethics Committee was approved during the animal study.

Sampling,isolationand identification of bacterial strain:

Milk and intestinal swabs from goat farms suffering from diarrhoea, weight loss, and fever were grown on MacConkey agar and incubated for 24-48 hour at 37^oC.Escherichia coli colony wasLactose fermenting (pink colonies) were performed on Gram's negative response testing as submitted to TSI (Triple Sugar Iron), citrate, urease, and oxidase testing.

Salmonella typhimurium colonies were identified as non-lactose fermenting colonies and tested for Gram’s reaction. Colonies of Gram negative bacilli were subjected for xld agar,TSI,citrate, urease, and oxidase test.Bacillus subtilis(medium, whitish, convex, rod coloneis) were tested for Gram positive reaction and subjected for gelatin, aesculin, casein, H₂S production, nitrate tests.

Identificationby vitek 2 compact system

Identification wereperformed to the manufacture’s instruction (Biomeriux VITEK-2 Compact ref Manual – Ref-414532,BioMe'rieux 2006) at Bacteriological Department, Animal Health Research Institute, Egypt. A sterile swab was used fortransmission of supposed colonies of culture to 3.0 ml of sterile saline (aqueous 0.45% till 0.50% NaCI, PH 4.5-7.0). The turbidity was monitored to the equivalent of 0.5-0.63 McFarland turbidity with a VITEK2 Densi-Check (biomerieux, France).Gram negative cards were inoculated with microorganism suspension for each isolate. Cardwas recognised by forty seven biochemical tests.  Fullcassette comprising microorganism suspension located in a vacuum chamber station.Airis re-introduced keen on the station. Theorganism suspension was transferred into micro-channels in wells.Inoculated cards are passed by cutting off the transfer tube and seal card prior to filling into cartridge incubator. Cardswere incubated for six hours at 35.5 + 1.0 °C.  Reading was recorded every 15 min automatically during incubation cards. Finalresults were printed within 6-8 hours. Cardswere allotted into waste container automatically.

Experimental design:

All goats were fastedfor twelve hours, weighed and equally classified into four groups of three goats each:

-The first group was received a single dose of tildipirosin at 4 mg/kg body weightI/V through the left jugular vein.

-The second group was administered a single dose of 10 mg/kg body weight of triclabendazoleorally one hour before I/V injection of tildipirosin in a dose of 4 mg/kg body weight according to Abo El-Sooud (2003).

-The third group was injected with a single dose of tildipirosin at 4 mg/kg body weightI/Minto the left gluteal muscle.

- The fourth group was administered a single dose of 10 mg/kg body weight of triclabendazoleorally one hour before I/M injection of tildipirosin in a dose of  4 mg/kg body weight.

Sampling:

Blood samples from a jugular vein of all groups were collected at 5, 10, 15, 30 minutes and 1, 2, 4, 6, 8, 12 and 24 hours after drug administration then left 30 minutes to clot. Samples centrifuged at 3000 rpm for 15 minutes to get serum and retained at -20^oC till assayed.

Antimicrobial activity of antibiotics:

 Antimicrobial activities of tildipirosin against Bacillus subtilis, E.coli (O78) andSalmonella typhimuriumwere assessedvia agar well diffusion method. 0.1 ml of diluted inoculum was swabbed on Mueller Hinton plates. Wells (6mm diameter) were cut into the agar and 100 µL of different concentrations of antibacterial agents ranging from 50 to 0.09μg/ml are used. Activity was measured as inhibition zone in millimeters around the well (Balouiri et al. 2016) as recorded in Table 1.

Drug bioassay:

Serum tildipirosin concentrations detected by microbiological assay usingBacillus subtilisas a choice organism. Standard curve remained linear above the range of 0.39 -100 µg /ml, minimal detectable limit was0.39 µg / ml (Arret et al. 1971).

Pharmacokinetic and statistical analysis:

The serum tildipirosin concentration time profile of each animal following intravenous and intramuscular administration was used to determine the pharmacokinetic variables using PK Solver (An add-in program for Micro- soft Excel, version 2). The data generated were exposed to statistical investigationusing the Student′s t-test with P. P(Snedecor andCochran 1967 and Zhang et al. 2010).

Results

In vitro, tildipirosin was effective in the range of 50 to 0.09μg/ml against Bacillus subtilis with zone of inhibition ranged between 30 to 10 mm while MIC of tildipirosin was 3.125μg/ml against Salmonella typhimurium and 25μg/ml against E.coli. From that Bacillussubtilis appeared to be the more sensitive organism so used in drug bioassay as shown in table (1).

Table (1): Antibacterial activity (zone of inhibition) of tildipirosin against Bacillussubtilis, E. coli and Salmonella typhimurium by agar well diffusion method

ND: Non Detected

Semi-logarithmic plot of serum tildipirosin concentration-time data after intravenous injection alone or in combination triclabendazoleindicated a two-compartment system of pharmacokinetics in goats.Tildipirosin was identified up to 24 h after I/V dosing in both groups. Serum tildipirosin concentration was declined in second group than first group (0.14 and 0.13μg/ml) as shown in Figure 1.

Fig.1. Semilogarithmic graph ofthe mean (±SE) serum tildipirosinof first (tildipirosin) and second (tildipirosin+ triclabendazole) goat groups

After IV administration, the serum tildipirosin concentration at zero time (Co) showed lower in the second group (2.73 ± 0.06μg/ml) than those of the first group (2.82 ± 0.04μg/ml).The administration of triclabendazole had the ability to significant increase of distribution rate constant (alpha) and supported by high value of first order elimination rate constant obtained from central compartment (k10), first-order rate constants for tildipirosin distribution between the central and peripheral compartments (K12 and K21).Apparentvolume of peripheral compartment (V2),total body clearance (CL),volume of distribution at steady state (Vdss)and inter-compartmental clearances (CL2)were elevated inthe second group compared withfirst group.On the other hand, triclabendazole has the potential to reduce half-life of distribution and elimination ((t_1/2 Alpha and t_1/2Beta), the area under the curve (AUC0-t), the area under the curve from zero to infinity and mean residence time in the second group as illustrated in table 2.

Table (2): Pharmacokinetic parameters of tildipirosin following a single intravenous dose (4 mg/kg b wt.) alone or with triclabendazole (10 mg/kg b.wt. orally) in goats (Mean ± SD)

C0: ''serum drug concentration at t=0, A:''zero time intercept of the distribution phase'', Alpha: ''distribution rate constant'', B : ''zero time intercept of the elimination phase'', Beta: ''Elimination rate constant'', k10: ''first–order elimination rate constant from central compartment'', K12 and K21:''first-order rate constants for drug distribution between the central and peripheral compartments'', t_1/2Alpha: ''distribution half-life'', t_1/2Beta: ''elimination half- life''; V: ''The apparent volume of central compartment'', V2 : ''The apparent volume of peripheral compartment'',Vdss: ''volume of distribution at steady state''; CL: total body clearance; CL2: ''inter-compartmental clearances'', AUC0-t : ''area under the curve'', AUC 0-inf: ''area under the curve from zero to infinity'', AUMC: '' area under the first moment curve", MRT: ''mean residence time''.

Data presented as Mean ± SD (P<0.05)

Following intramuscular injection,tildipirosin was detectedpost dose up to 24 h (0.15 and 0.12ug/ml) in third and fourth groups respectivelyas illustrated in figure 2.

Fig.2. Semilogarithmic graph of the mean (±SE) serum tildipirosin in third (tildipirosin) and fourth (tildipirosin+ triclabendazole) goat groups

There was a substantial increase in both distribution and elimination rate constants (alpha & beta) in third group contrary in fourthgroup, indicating a slower drop in serum tildipirosin concentrations.The values of time to peak concentration were1.14 ± 0.02 and 1.23 ± 0.02h in the third and fourth groups, respectively.Mean residence time (MRT) was significantly higher in third group(22.46± 3.83h) than fourth group(18.64 ± 1.69h) and that was supported by longer elimination half-life (17.71 ± 3.12h)

The shorter absorption half-life (t1/2ab) in fourth group than thirdgroup indicated rapid absorption from the injection site.

The values of AUC0-t, AUC 0-inf and AUMC and MRT in the fourth group were declined supported by shorter elimination half-lifecompared to the values ofthird group as illustrated in table 3.

Table (3): Pharmacokinetic parameters of tildipirosin following a single intramuscular dose (4 mg/kg b.w.) alone or with triclabendazole (10 mg/kg b.w. orally) in goats (Mean ± SD)

A:''zero time intercept of the distribution phase'', Alpha: ''distribution rate constant'', B : ''zero time intercept of the elimination phase'', Beta: ''Elimination rate constant'', kab: "absorption rate constant", k10: ''first–order elimination rate constant from central compartment'', K12 and K21:''first-order rate constants for drug distribution between the central and peripheral compartments'', t_1/2Alpha: ''distribution half-life'', t_1/2Beta: ''elimination half- life''; t_1/2ab: "absorption half-life"; Cmax:" peak drug concentration; Tmax:" time to peak concentration"; AUC 0-t : ''area under the curve'', AUC 0-inf: ''area under the curve from zero to infinity'', AUMC: ''the area under the first moment curve, MRT: ''mean residence time'' .

Data presented as Mean ± SD (P<0.05)

Discussion

It is recognized that co-administration of binary drugs could affect the pharmacokinetics of both agents (BenetandSheiner 1985). Pharmacokinetic communications between anthelmintic and antimicrobial drugs in veterinary medicine in case of Fasciolaparasite and respiratory disease have deficiency researches given their frequent clinical use in combination.

Evaluation semi-logarithmic plot of serum tildipirson concentration-time data following its administration intravenously alone or with triclabendazole is used a two compartment open model. This behaviourassociated with previously recorded byLeietal. (2018) and Galecio et al. (2020).

Serum tildipirson concentrations following intravenous and intramuscular administration   in goats post oral dose of triclabendazolewere declined than dataof goats given only tildipirson.Oral triclabendazole was rapidly converted to its sulphoxide and sulphone derivatives in normal goats. The maximal plasma concentrations of sulphoxide and sulphone were comparable, ranging from 9 to 19 Hg/ml, and were reached an average of 12.8 and 25.6 hours after delivery, respectively. Both metabolites were removed slowly from plasma, with sulphoxide elimination half-lives of 22.4 h and sulphone elimination half-lives of 19.4 h. Triclabendazolesulphone and sulphoxide were identified in plasma 2 hours after triclabendazole injection (Kinab and Bogan 1988).

Thesefindingswere supported by Abo El-Sooud(2003) and Atef et al.(2010)who found that albendazole could lowerserum concentration of enrofloxacin and tylosin, respectively.

Co-administration of triclabendazole significantly shortened tildipirsondistribution and elimination half-life (t_1/2α and t_1/2β) after intravenous dosing. A previous study of Abo El-Sooud(2003)directed that serum enrofloxacinelimination half-life is lowered by administration of albendazole to lactating goats.

Following intravenous administration,the volume of tildipirsondistribution at steady state (Vdss) waspointedlygreateringoats given tildipirson with triclabendazole(5.23 ±0.07 mg/μg/ml)indicated large extravascular distribution of the drug.AUC0-t and AUMC oftildipirson were significantly decreased in goats pretreated with triclabendazole(7.56± 0.30μg.h/ml and 101.28 ± 10.6 μg.h2/ml, respectively).The drug was highly cleared in goats pretreated with triclabendazoleas the value ofCLwas0.45± 0.02mg /(μg/ml)/h.The rapid clearance and higher volume of distribution could clarify the lower tildipirsonconcentration and AUC0-tpretreated with triclabendazole in goats. This was in parallel with the results of Atef et al.2010 who reported lower AUC0-t and AUMC of   tylosinwithalbendazole group than tylosin group in lactating goats.

Following intramuscular dosing,tildipirsonabsorbed from the injection site asabsorption half-life value (tab)recorded0.23 ± 0.01h whilethis value lowered by triclabendazole(0.20 ± 0.02h).The distribution of tildipirson was significantly decreased in triclabendazole pretreated group as the value of t1/2Alpha was 1.63± 0.41h.The results revealed that intramuscular tildipirson injection with triclabendazole resulted in quicker elimination, with a t1/2beta value of 13.22± 1.25 h and a lower MRT (18.64 ±1.69 h). This discrepancy might be attributable to the action of triclabendazole on liver microsomal enzymes. This is consistent with the findings of Escobar-Garcia et al.(2001) and Bapiro et al.(2002) who identifiedactivation of cytochrome P(450) CYP1A1 and CYP1A2 enzyme by albendazol at the transcriptional level that may be clinically significant in the metabolism of other drugs e encourages.According to Abo ElSooud (2003), the declineof florfenicollevel pretreated with albendazole than other treatments in goats might be clarified by its influence of liver microsomal enzymes. A research on deer, cattle, sheep, and pigs found substantial increase in liver microsomal biotransformation of albendazole (Velik et al. 2005). Furthermore, Albendazole and benzimidazole were reported to strongly activate cytochrome P-450 enzymes in rats and pigs(Asteinza et al. 2000;Baliharova et al.2004).

Tildipirosin had MIC50 values of 0.5,1, 2 and 2 g/mL against P multocida, H parasuis, A pleuropneumoniae, and Bordetellabronchiseptica, respectively as recorded by EMA (2015).  In our study, tildipirosin serum concentration at 24 h after intramuscular treatment was greater than MIC against theaforementioned bacterial pathogens in both groups (Tildipirosin and Tildipirosin plus triclabendazole), with values of 0.15 and 0.12 g / ml, respectively. That indicated triclabendazole did not effect on the duration of tildipirosin administration in goats by I/M route.

Our researchconcluded that triclabendazole causes majorvariations in disposition kinetic of tildipirosin that enhances the distribution and elimination rate of tildipirosin in goats.

There are no adjustments to the dose regimen for tildipirosin in concurrent therapy with triclabendazole being investigated in goats because of investigations shortageof disposition kinetics effects.