There is a need to introduce more effective veterinary antibiotics, particularly as many infectious diseases of animals are endemic and resistant to classical antibiotics. In this project, two compounds not used in humans and not currently used to treat infectious diseases in sheep were evaluated for this purpose. Trovafloxacin (TVX) is a highly effective fluoroquinolone antibacterial agent withdrawn from human markets. Marbofloxacin (MBX) is a veterinary fluoroquinolone antibiotic used mainly in companion animals. However, their pharmacological profiles in sheep have not been carefully evaluated previously. The overall aim of this thesis is, therefore, to characterize the pharmacokinetics (PK), pharmacodynamics (PD) and safety profiles of TVX and MBX in sheep to examine the possibility of using these drugs for the treatment of resistant infectious diseases in that species.
The plasma concentration-time profiles of TVX and MBX were obtained after a single bolus intravenous (IV) or intramuscular (IM) injection. The PK properties of these drugs in sheep were also investigated after multiple IM injections at two (low and high) dose levels. The drug levels in tissues, urine, faeces and bile were also quantified using a validated high performance chromatography (HPLC) assay. PD profiles were defined by the prediction of antibacterial activity of both agents by integration of plasma drug concentrations versus minimum inhibitory concentrations (MICs) and microbiological analysis of faeces samples after multi-dosing administration of both antibiotics. The drug safety was evaluated by the adverse clinical signs, haematological and biochemical parameters, tissue histopathology and cytokines levels in sheep. The penetration of both agents into sheep neutrophils was directly imaged by multiphoton microscopy.
The PK profiles of both drugs were examined using compartmental, non-compartmental and numerical deconvolution pharmacokinetic methods. Both MBX and TVX exhibited apparent linear bi-exponential PK profile after IV dosing. MBX appeared to display first order absorption kinetics after IM injection, whereas TVX exhibited variable plasma concentration time profiles between individual animals. In some sheep, a secondary peak in the profile was observed at approximately 6-10 h after IM injection. The PK profiles for TVX were shown to be consistent with precipitation of TVX at the injection site, noting also that TVX has low water solubility. The high probability of precipitation was confirmed by Reflectance Confocal Microscopy (RCM), where TVX crystal formation was detected after a simulated IM injection in an in vitro experiment using sheep meat.
MBX was found to have a lower clearance (0.28 ± 0.05 vs 0.58 ± 0.17 L/h/kg) and a longer elimination half-life (2.42 ± 0.40 h) compared to TVX (1.82 ± 0.21 h), with smaller volumes of distribution (10.15 ± 3.17 vs 14.20 ± 8.63 L/kg respectively for Vdβ and 3.83 ± 0.87 vs 4.94 ± 3.22 2 L/kg respectively for Vss). IM and IV injection of MBX at doses of 2 mg/kg or IV administration of TVX at 3 mg/kg resulted in adequate plasma concentrations for effective treatment of certain bacterial diseases in sheep.
Following IM dosing, there was no apparent accumulation of MBX in the body after 5 days of treatment, while TVX showed accumulation after 3 days of treatment. About 30% of the total MBX dose was recovered in urine. In contrast, only about 3% of the total dose of TVX was recovered in the urine, indicating TVX is mainly cleared by non-renal mechanisms.
No obvious adverse effects and no haematological or histological changes were observed after multiple doses of MBX. However, ALT, AST and LDH enzyme levels were elevated after multiple TVX doses. In addition, some significant adverse effect signs, including neutrophilia and liver tissue damage were observed in some TVX treated sheep. No significant immunological changes were found after MBX multiple treatments, while IL10 and TNFα plasma levels in sheep were decreased after 3 days injection of the high dose of TVX (12 mg/kg).
The numbers of isolated bacterial colonies from sheep faeces were reduced gradually within a few days of antibiotic treatment. Furthermore, TVX and MBX were highly effective against Gram negative organisms in the sheep faecal flora and in selected Gram positive strains which predominated in the samples collected at the completion of the study. Penetration of TVX and MBX into sheep neutrophils was determined by MPM. Results were evidenced by the higher intracellular concentration of both drugs compared to extracellular drug concentration after incubation of the drug with neutrophils.
In summary, the PK, PD and safety profiles of two fluoroquinolones were investigated to evaluate the possibility of using these drugs as alternative antibiotics for the treatment of incurable bacterial infections in sheep. It was found that MBX given either IV or IM exhibited predictable and desirable PK profiles and antibacterial activity, suggesting that MBX should be evaluated further, to include a full safety profile, for treatment of such infections in sheep. This work has shown that TVX has variable absorption profiles after IM dosing. In addition, there is evidence of hepatotoxicity in the sheep consistent with previous human data, making it unlikely to be approved for veterinary use.