Feasibility of establishing a veterinary marker to total residue in edible tissues with non-radiolabeled study using high-resolution mass spectrometry.

Traditionally, in vivo metabolism and total residue studies in veterinary drug research were conducted using radiolabeled drug where information on metabolite profiles and marker residue to total residue ratio is obtained. The Veterinary International Conference on Harmonisation (VICH) guideline GL46 indicates that the metabolism and residue kinetics in food-producing animals may be documented by an alternative approach, one other than the traditional radiolabeled study. High-resolution mass spectrometry (HRMS) has been widely used in human pharmaceutical R&D from metabolite profiling and identification in early drug discovery to first-in-human (FIH) studies in development. Recent advances in data mining tools have greatly improved the metabolite profiling capability with HRMS. It is now routine to study metabolism using non-radiolabeled samples without missing any major metabolites. In the current paper, we explored the feasibility of conducting non-radiolabeled marker residue studies to obtain metabolism information using HRMS. Metabolite profiles of gamithromycin in edible tissues of sheep treated with 6 mg/kg body weight subcutaneous injections were obtained with HRMS. The semi-quantitative relationship between the level of gamithromycin and the total treatment-related residues was established by determining the percentages of extracted ion chromatograms for metabolites and parent compound residues in each tissue. Major components (gamithromycin and its metabolite, declad) were measured quantitatively using a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Metabolite profiles in excreta were also obtained and the major components measured quantitatively with a LC-MS/MS method to ensure no major metabolite was missing. Combining previous knowledge of marker residue studies in cattle and swine, as well as an in vitro comparative metabolism study with metabolite data across various species, gamithromycin was designated as the marker residue in sheep edible tissues. The marker to total residue ratios were established using a combination of the semi-quantitative HRMS results and quantitative results with the major components: the marker residue and declad. The pros and cons of the HRMS method as well as the appropriate use of the method for marker residue studies are discussed.

Pharmacokinetics of a novel endectoparasiticide topical formulation for cats, combining esafoxolaner, eprinomectin and praziquantel.

Esafoxolaner, a purified enantiomer of afoxolaner with insecticidal and acaricidal properties, is combined with eprinomectin and praziquantel in NexGard® Combo, a novel topical endectoparasiticide formulation for cats. The parasiticide potencies of topical esafoxolaner, eprinomectin and praziquantel, are based on transcutaneous absorption, systemic distribution, and exposure of respective target parasites. For each compound, the pharmacokinetic profile, non-interference, dose linearity/proportionality after one administration, and the accumulation and time to reach a steady state after repeated monthly administrations of the novel formulation, were investigated. After one topical application of NexGard® Combo at the minimum recommended dose, the mean plasma concentration of esafoxolaner immediately reached (and remained at) a level supporting rapid onset and sustained efficacy against ectoparasites for at least 1 month. The mean Cmax, Tmax, T1/2, and the topical bioavailability of esafoxolaner were 130 ng/mL, 7.1 days, 21.7 days and 47.2%, respectively, and the plasma profiles of eprinomectin and praziquantel supported their known endoparasiticide properties. No relevant interference between the three compounds was observed. Dose proportionality was demonstrated for the three compounds over a range of 0.5× to 2× the minimum recommended dose. Steady state after repeated monthly administrations was reached by the second dose for praziquantel and by the fifth dose for esafoxolaner and eprinomectin. Accumulation was limited and drug plasma concentrations were maintained within a safe level.

TITLE: Pharmacocinétique d'une nouvelle formulation topique d'endectoparasiticide pour chats, combinant esafoxolaner, éprinomectine et praziquantel. ABSTRACT: L'esafoxolaner, un énantiomère purifié d'afoxolaner aux propriétés insecticides et acaricides, est combiné à l'éprinomectine et au praziquantel dans NexGard® Combo, une nouvelle formulation endectoparasiticide topique pour chats. Les pouvoirs parasiticides de l'esafoxolaner topique, de l'éprinomectine et du praziquantel sont basés sur l'absorption transcutanée, la distribution systémique et l'exposition des parasites cibles respectifs. Pour chaque composé, le profil pharmacocinétique, la non-interférence, la linéarité/proportionnalité de dose après une administration, ainsi que l'accumulation et le temps nécessaire pour atteindre un état d'équilibre après des administrations mensuelles répétées de la nouvelle formulation, ont été étudiés. Après une application topique de NexGard® Combo à la dose minimale recommandée, la concentration plasmatique moyenne d'esafoxolaner a immédiatement atteint et est restée à un niveau soutenant une apparition rapide et soutenue de l'efficacité contre les ectoparasites pendant au moins un mois. La Cmax moyenne, la Tmax, la T1/2, et la biodisponibilité topique de l'esafoxolaner était respectivement de 130 ng/mL, 7,1 jours, 21,7 jours et 47,2 %, et les profils plasmatiques de l'éprinomectine et du praziquantel ont confirmé leurs propriétés endoparasiticides connues. Aucune interférence significative entre les trois composés n'a été observée. La proportionnalité de la dose a été démontrée pour les trois composés sur une plage de 0,5 × à 2 × la dose minimale recommandée. L'état d'équilibre après des administrations mensuelles répétées a été atteint par la deuxième dose de praziquantel et par la cinquième dose d'esafoxolaner et d'éprinomectine. L'accumulation était limitée et les concentrations plasmatiques du médicament étaient maintenues à un niveau sûr.

Gamithromycin in swine: Pharmacokinetics and clinical evaluation against swine respiratory disease.

The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half-life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3-12 mg/kg BWT. No gender-related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for 'treatment success' or 'treatment failure' (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non-inferiority hypothesis test (non-inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%).

UV Photolysis of Pyrazine and the Production of Hydrogen Isocyanide.

Photolysis of the diazine heterocycle, pyrazine, following irradiation at 308, 248, and 193 nm was examined using nanosecond time-resolved Fourier transform infrared emission spectroscopy. The resulting time-resolved IR emission spectra reveal that for 308 and 248 nm vibrationally highly excited pyrazine is produced, but no photolysis products were detected. However, at 193 nm excitation, the measured IR emission spectra consist solely of resonances originating from rovibrationally excited photofragments, including acetylene (HCCH), hydrogen cyanide (HCN), and hydrogen isocyanide (HNC), indicating that photofragmentation proceeds from vibrationally highly excited pyrazine on the ground electronic state. Spectral fit analysis of the time-resolved HCN and HNC IR emission band shapes and intensities allowed an estimate of the nascent product population distributions, from which a lower bound estimate of the HNC/HCN branching ratio was deduced as Φ ≥ 0.07. Additionally, ab initio calculations were performed in order to examine the propensity of photoinduced reactions on the ground- and lowest-energy excited-state surfaces. The calculations provide a basis for understanding the wavelength dependence of the UV photolysis of pyrazine, the photolytic production of HNC, and also explain previous experimental observations in the literature.

The intravenous and oral pharmacokinetics of afoxolaner used as a monthly chewable antiparasitic for dogs.

The pharmacokinetics of afoxolaner in dogs was evaluated following either intravenous or after oral administration of NEXGARD(®), a soft chewable formulation. Afoxolaner is a member of one of the newest classes of antiparasitic agents, known as antiparasitic isoxazolines. The soft chewable formulation underwent rapid dissolution, and afoxolaner was absorbed quickly following oral administration of the minimum effective dose of 2.5mg/kg, with maximum plasma concentrations (Cmax) of 1,655 ± 332 ng/mL observed 2-6h (Tmax) after treatment. The terminal plasma half-life was 15.5 ± 7.8 days, and oral bioavailability was 73.9%. Plasma concentration-versus-time curves fit a 2-compartment model and increased proportionally with dose over the oral dose range of 1.0-4.0mg/kg, and over the oral dose range from 1.0 to 40 mg/kg. Following an intravenous dose of 1mg/kg, the volume of distribution (Vd) was 2.68 ± 0.55 L/kg, and the systemic clearance was 4.95 ± 1.20 mL/h/kg. Afoxolaner plasma protein binding was >99.9% in dogs. One major metabolite, formed following hydroxylation of afoxolaner, was identified in dog plasma, urine and bile. When afoxolaner is administered orally, there is a strong correlation between afoxolaner plasma concentration and efficacy with EC90 values of 23 ng/mL for Ctenocephalides felis and ≥ 100 ng/mL for Rhipicephalus sanguineus sensu lato and Dermacentor variabilis. The pharmacokinetic properties of afoxolaner are suited for a monthly administration product because the fast absorption and long terminal half-life support a rapid onset of action while ensuring month-long efficacy.

Reevaluation of efficacy against nematode parasites and pharmacokinetics of topical eprinomectin in cattle.

A study was conducted to confirm the efficacy of topical eprinomectin against nematodes and to evaluate the pharmacokinetics in cattle prevented from having physical contact with other cattle and from self-grooming. Sixteen male Brown Swiss calves were infected with larvae of recently isolated nematode parasites. Inoculation was scheduled so that the nematodes were expected to be adults at the time of treatment. Animals were blocked based on pretreatment body weight and randomly allocated to the untreated control group or the group treated with EPRINEX® Pour-On (Merial; 0.5 mg eprinomectin per kilogram body weight). Plasma samples were collected prior to and between 1 and 21 days following treatment and analysed for eprinomectin (B1a component) concentrations. For parasite recovery, identification and counting, animals were humanely euthanized 21 days after treatment. Calves treated with eprinomectin had significantly (p < 0.05) fewer (>99 % reduction) adult Dictyocaulus viviparus, Bunostomum phlebotomum, Cooperia oncophora, Cooperia surnabada, Cooperia punctata, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Ostertagia lyrata, and Trichostrongylus axei and inhibited fourth-stage Nematodirus and Ostertagia larvae than the controls. The main pharmacokinetic parameters of eprinomectin B1a were: AUC(inf), 124 ± 24 day ng/mL; T (1/2), 5.2 ± 0.9 days; and C (max), 9.7 ± 2.2 ng/mL. Individual maximal concentrations were observed 3-7 days after treatment. This study confirmed the continued high level of efficacy of topically administered eprinomectin against a wide range of recently isolated nematodes. In addition, this study demonstrates that oral ingestion is not required to achieve adequate exposure for efficacy following topical administration of eprinomectin.

Photodissociation of vinyl cyanide at 193 nm: Nascent product distributions of the molecular elimination channels.

The photodissociation dynamics of vinyl cyanide (H(2)CCHCN, acrylonitrile) and deuterated vinyl cyanide (D(2)CCDCN) at 193 nm are examined using time-resolved Fourier transform infrared emission spectroscopy. Prior photofragment translational spectroscopy studies [D. A. Blank et al., J. Chem. Phys. 108, 5784 (1998)] of the dissociation have observed the presence of four main dissociation channels; two molecular and two radical in nature. However, with the exception of a<0.01 quantum yield determined for the CN radical loss channel, the branching ratios of the remaining three elimination channels were not measured. The time-resolved emission spectra, including those from the deuterated samples, revealed the presence of acetylene, hydrogen cyanide (HCN), as well as the energetically less stable isomer hydrogen isocyanide (HNC). Acetylene is found in two distinct energetic distributions, suggesting that both three- and four-centered elimination reactions are occurring significantly in the dissociation. In contrast to prior ab initio studies that have suggested the dominant nature of the three-center elimination of molecular hydrogen (H(2)) and cyanovinylidene (:C=CHCN), we find this reaction channel to be of little importance as there is no evidence to support any significant presence of rovibrationally excited cyanoacetylene. Spectral modeling of the product distributions allows for the first experimental determination of the relative occurrence of the three-centered (resulting in HCN+vinylidene) versus four-centered (HNC+acetylene) elimination channels as 3.34 to 1.00, in contrast to the previously calculated value of 126:1. Rice-Ramsperger-Kassel-Marcus analysis depicts that the transition state energy of the four-centered reaction should be about 10 kcal mole(-1) lower than the three-centered reaction.

Pharmacokinetics of firocoxib after administration of multiple consecutive daily doses to horses.

OBJECTIVE: To determine pharmacokinetic parameters and variables, firocoxib concentrations in urine and plasma, urine-to-plasma ratios, and the urine depletion profile of firocoxib and to evaluate whether the pharmacokinetic behavior of firocoxib was governed by linear processes after multiple doses of firocoxib were administered IV and orally. ANIMALS: 6 healthy female horses (5 Paint horses and 1 Quarter Horse) in experiment 1 and 12 healthy male and female horses in experiment 2. PROCEDURES: In experiment 1, 6 horses were orally administered firocoxib paste once daily for 12 consecutive days, and plasma and urine samples were obtained and analyzed. In a second experiment, 12 horses received IV injections of firocoxib solution once daily for 9 consecutive days, and plasma was obtained and analyzed. RESULTS: Mean +/- SD clearance and steady-state volume of distribution of firocoxib were 40.5 +/- 14.7 mL/h/kg and 2.3 +/- 0.7 L/kg, respectively. Mean half-life was 44.2 +/- 21.6 hours and 36.5 +/- 9.5 hours for IV and oral administration, respectively. The urine concentration- time curve decreased in parallel with the plasma concentration-verus-time curve. Renal clearance (0.26 +/- 0.09 mL/kg/h) was low, compared with total body clearance, which indicated that the main route of elimination was hepatic clearance. CONCLUSIONS AND CLINICAL RELEVANCE: The pharmacokinetics of firocoxib during prolonged use were determined. Use of plasma or urine to ascertain drug concentrations in horses is scientifically valid because the plasma-to-urine ratio was consistent over time and among horses.

Automated liquid chromatography-tandem mass spectrometry method for the analysis of firocoxib in urine and plasma from horse and dog.

A rugged, sensitive and efficient liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 microL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column. The mobile phase was 45% acetonitrile and 55% of a 2 mM ammonium formate buffer. The method was accurate (88-107%) and precise (CV<12.2%) within and between sets. Extraction efficiencies (recovery)>93% were achieved and ionization efficiencies (due to matrix effects) were >72%. Extensive stability and ruggedness testing was also performed; therefore, the method can be used for pharmacokinetic studies as well as drug monitoring and screening. The data presented here is the first LC-MS/MS method for the quantitation of firocoxib in plasma (LLOQ of 1 ng/mL), a 25-fold improvement in sensitivity over the HPLC-UV method and the first quantitative method for firocoxib in urine (LLOQ of 5 ng/mL). Additionally the sample preparation process has been automated to improve efficiency.

Evaluating barriers to bioavailability in vivo: validation of a technique for separately assessing gastrointestinal absorption and hepatic extraction.

PURPOSE: The purpose of this study was to develop and validate a method for separately evaluating the roles of gastrointestinal absorption and hepatic extraction as barriers to oral bioavailability (BA). The method was validated using five reference compounds known to have different absorption and hepatic extraction properties. Dose-dependence was also investigated for one reference compound. METHODS: Five reference compounds, amoxicillin, antipyrine, atenolol, propranolol, and testosterone, were administered as a cassette intravenouly (IV), via the hepatoportal vein (IPV), intraduodenally (ID), and intracolonically (IC) to male Sprague-Dawley rats. Blood samples were taken at nine time points, and the compounds were extracted from plasma using solid phase extraction. Plasma concentrations of each compound were determined using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Pharmacokinetic parameters including bioavailability were calculated for each compound for each route of administration. RESULTS: Testosterone BA was less than 10% by ID, IC, and IPV routes, due to high hepatic extraction, consistent with its high systemic clearance (63 ml x min(-1) x kg(-1)) and short terminal plasma half-life (23 min). The IPV BA of amoxicillin was 95%+/-6% indicating the absence of hepatic extraction in the rat, but with an ID BA of approximately 39% suggesting incomplete GI absorption to be the main barrier to bioavailability. Absorption was poor from the colon, demonstrating site-dependence consistent with literature reports of site-dependent absorption. Low oral BA of propranolol was due in part to first-pass hepatic extraction (IPV BA of 36%). The IPV BA of propranolol was dose-dependent, most likely due to saturation of the P450 enzymes. Atenolol was incompletely bioavailable due to incomplete intestinal absorption, with no contribution of hepatic first-pass metabolism. Antipyrine was highly bioavailable by all routes. CONCLUSIONS: This in vivo rat model is demonstrated to be useful for identifying and quantifying the causes of incomplete bioavailabilty. It separately evaluates intestinal absorption, hepatic extraction, and site-dependent absorption. Concentration-dependence of saturable processes can also be examined.