The bioequivalence of a single intravenous administration of the anesthetic alfaxalone in cyclodextrin versus alfaxalone in cyclodextrin plus preservatives in cats.

To demonstrate the bioequivalence of alfaxalone in cyclodextrin (Reference Product) to a formulation of alfaxalone in cyclodextrin also containing the preservatives ethanol, chlorocresol, and benzethonium chloride (Test Product) when administered for the purpose of inducing anesthesia in the cat. Blinded, single-dose, randomized, two-period, two-sequence, cross-over bioequivalence study with a 7-day washout period between treatments. Twenty-four (12 neutered males and 12 intact females), healthy, adult cats weighing 4.1±0.9 kg. Cats were administered 5 mg/kg IV of alfaxalone in the Reference or Test Product using a randomized cross-over design. One-milliliter venous blood samples were collected at predetermined time points to 12 hr after drug administration to determine alfaxalone plasma concentration over time. Alfaxalone concentrations were determined by a validated analytical testing method using HPLC-MS/MS. Plasma profiles of alfaxalone concentration against time were analyzed by noncompartmental analysis. The pivotal variables for bioequivalence were AUClast and Cmax . Equivalence was achieved if the 90% confidence interval for AUClast and Cmax fell into the asymmetric ±20% interval (0.80-1.25). Physiological variables, quality of anesthesia visual analog scale (VAS) scoring and anesthetic event times were recorded. ANOVA or ANCOVA (single time point), RMANOVA or RMANCOVA (multiple time point) was used for normally distributed data. GLIMMIX was used for nonnormally distributed data. VAS scores were analyzed as for blood bioequivalence data. Variables were evaluated for safety and assessed at alpha = 0.10. Cmax and AUClast for Reference and Test Products were statistically bioequivalent. No physiological variables except for a drug by time interaction for respiratory rate differed between treatment groups, and this difference was not clinically relevant. No anesthetic event times or VAS scores for quality of anesthesia were different between treatment groups. Neither formulation caused pain upon injection. The Reference and Test Products are pharmaceutically bioequivalent formulations when administered as a single intravenous administration for the purpose of induction of anesthesia in cats.

Plasma pharmacokinetics of alfaxalone after a single intraperitoneal or intravenous injection of Alfaxan(®) in rats.

Alfaxalone (3α-hydroxy-5α-pregnane-11, 20-dione) is a neuroactive steroid with anaesthetic properties and a wide margin of safety. The pharmacokinetic properties of alfaxalone administered intravenously and intraperitoneally in rats (n = 28) were investigated. Mean t(1/2elim) for 2 and 5 mg/kg i.v. was 16.2 and 17.6 min, respectively, but could not be estimated for IP dosing, due to sustained plasma levels for up to 60 min after injection. Clp for i.v. injection was calculated at 57.8 ± 23.6 and 54.3 ± 6.8 mL/min/kg, which were 24.5% and 23% of cardiac output, respectively. The observed C(max) was 3.0 mg/L for IP administration, and 2.2 ± 0.9 and 5.2 ± 1.3 mg/L for 2 and 5 mg/kg i.v. administration, respectively. AUC(0-60) was 96.2 min.mg/L for IP dosing. The relative bioavailability for IP dosing was 26% and 28% compared to i.v. dosing. Differences in t(1/2elim) and Cl(p) from previous pharmacokinetic studies in rats are likely due to variations in alfaxalone formulation rather than sex differences. Alfaxan® given IP caused sustained levels of alfaxalone, no apnoea and longer sleep times than i.v. dosing, although immobilization was not induced in 30% of rats given Alfaxan® IP. A pharmacodynamic study of the effects of combining IP injection of Alfaxan® with other premedication agents is worthwhile, to determine whether improved anaesthesia induction could ultimately provide an alternative anaesthetic regimen for rats.

Demonstrating bioequivalence using clinical endpoint studies.

For drug products not amenable to blood level studies, clinical endpoint studies have been used as an indirect measure of formulation difference in bioavailability between test and reference products. However, clinical endpoint studies are not as sensitive in detecting formulation differences as blood level studies and offer numerous challenges to both regulatory authorities and sponsors. The objective of this article is not to suggest new regulatory policies, but to explore new methodologies and alternative solutions to clinical endpoint bioequivalence (BE) studies, which are used when a blood level study is not considered to be appropriate. To achieve this objective, this article identifies situations where a clinical endpoint study might be appropriate, lists the advantages and disadvantages of this type of study design, and discusses possible alternative solutions. It is concluded that future evidence-based research is needed to explore new methodologies such as clinical trial simulations of various study designs, new statistical methods, and new in vitro methods to demonstrate BE.

Challenges associated with the demonstration of bioequivalence of intramammary products in ruminants.

This article explores the numerous challenges encountered when the goal is to demonstrate bioequivalence (BE) between test and reference intramammary (IMM) products in ruminants. Numerous pathophysiological factors of mastitis and physicochemical properties of IMM formulations are implicated in the difficulties in confirming BE for this dosage form. Advantages and disadvantages of current BE study designs are discussed, and alternative perspectives are outlined. Ongoing and future research increasing our knowledge of the pharmacokinetics and pharmacodynamics of antimicrobial drugs delivered through this route is crucial to better understanding the implications of clinically significant formulation differences in the demonstration of BE and may also help in developing more effective IMM formulations for ruminants.

The pharmacokinetics and pharmacodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses.

This study aimed to determine the pharmacokinetic parameters and pharmacodynamics of alfaxalone in a 2-hydroxypropyl-beta-cyclodextrin alfaxalone formulation (Alfaxan), Jurox Pty Ltd, Rutherford, NSW, Australia) in cats after single administration at clinical and supraclinical dose rates and as multiple maintenance doses. First, a prospective two-period cross-over study was conducted at single clinical and supraclinical doses. Second, a single group multiple dose study evaluated the effect of maintenance doses. Eight (five female and three male) domestic cats completed the cross-over experiment and six female cats completed the multiple dose study. In the first experiment, alfaxalone was administered intravenously (IV) at 5 or 25 mg/kg with a washout period of 14 days. In the second experiment, alfaxalone was administered IV at 5 mg/kg followed by four doses each of 2 mg/kg, administered at onset of responsiveness to a noxious stimulus. Blood was collected at prescribed intervals and analysed by LCMS for plasma alfaxalone concentration. Noncompartmental pharmacokinetics were used to analyse the plasma alfaxalone data. The plasma clearance of alfaxalone at 5 and 25 mg/kg differed statistically at 25.1 and 14.8 mL/kg/min respectively. The elimination half lives were 45.2 and 76.6 min respectively. Alfaxalone has nonlinear pharmacokinetics in the cat. Nevertheless, for cats dosed with sequential maintenance doses, a regression line through their peak plasma concentrations indicated that there was no clinically relevant pharmacokinetic accumulation. The duration of nonresponsiveness after each maintenance dose was similar at approximately 6 min, indicating a lack of accumulation of pharmacodynamic effect. The cardiovascular and respiratory parameters measured in cats after administration of the labelled doses of Alfaxan were stable. In conclusion, the pharmacokinetics of alfaxalone in cats are nonlinear. At clinical dose rates, however, neither alfaxalone nor its effects accumulated to a clinically relevant extent. Further, in the un-premedicated cat the induction and maintenance of surgical anaesthesia was free of untoward events after a dose of 5 mg alfaxalone/kg body weight followed by four sequential doses of 2 mg/kg as needed (i.e., approximately 7 to 8 mg/kg/h).

Plasma prostaglandin E2 concentrations after single dose administration of ketorolac tromethamine (Toradol) in dogs.

Ketorolac tromethamine (Toradol) is a relatively new, potent, non-narcotic analgesic with cyclooxygenase (COX) inhibitory activity and has been associated with gastric and renal toxicity in people and dogs. The objectives of this study were to establish whether endogenous PGE2 exists in the plasma of healthy dogs and to determine if, and to what magnitude, ketorolac alters PGE2 plasma concentrations after administration. Enzyme immunoassay measurement of a stable PGE2 derivative, bicyclo PGE2, showed that after i.v. administration of 0.5 mg/kg ketorolac tromethamine, 1 and 24 h plasma samples contained significantly (P < or = 0.01) less PGE2 than did plasma samples collected from dogs before the drug treatment. After p.o. administration, 1 h plasma samples contained significantly (P < or = 0.01) less PGE2 than did pretreatment samples, and the 24 h post-drug administration samples contained significantly (P < or = 0.01) less plasma PGE2 than the 96 h plasma samples. The results of this study suggest that a clinically effective single i.v. or p.o. dose of ketorolac tromethamine to healthy dogs causes a significant but reversible decrease in endogenous PGE2 production which may partially explain the drug's low therapeutic index.

Multicentre, randomised clinical trial evaluating the efficacy and safety of alfaxalone administered to bitches for induction of anaesthesia prior to caesarean section.

OBJECTIVE: To determine the clinical safety and efficacy of alfaxalone in bitches undergoing caesarean section (CS) and their puppies when it is administered for induction of anaesthesia followed by maintenance with isoflurane and oxygen and in conjunction with perioperative pharmaceuticals. DESIGN: A multicentre, randomised, positive-controlled clinical study. METHODS: A total of 74 bitches were enrolled in the study with 48/74 (65%) and 26/74 (35%) receiving alfaxalone and propofol, respectively, for induction of anaesthesia. Bitches were examined prior to induction and monitored during induction, surgery and recovery. Assessments were made for quality of induction, maintenance and recovery from anaesthesia. Assessments were made on pup viability for suction, dorsal flexion, withdrawal and anogenital reflexes. RESULTS: Of the 48 bitches receiving alfaxalone, 47 (98%) and 39 (81%) scored a top score of excellent for induction and anaesthesia effectiveness, respectively. For the same parameters with propofol in 26 bitches, 23 (88%) and 17 (65%) scored excellent. Average scores for recovery were not different between the two treatment groups with alfaxalone 46/48 (96%) and 25/26 (96%) of propofol induced bitches scoring a good or excellent rating. Bitches tolerated a number of concurrent medications throughout the peri-operative period. No bitch fatalities were observed in this study. There were no statistically significant differences between treatment groups for the puppy variables. Live puppies born by CS to bitches having been administered alfaxalone or propofol had similar survival rates 24 h after birth (i.e. 205/213 (96%) and 124/131 (95%), respectively). CONCLUSION: This study confirms the safety and efficacy of alfaxalone for the purpose of anaesthetic induction for CS in the bitch. In addition, alfaxalone had a negligible effect on the neonate with >95% of puppies alive 24 h after the bitch had recovered from anaesthesia with alfaxalone induction.

Alfaxalone and medetomidine intravenous infusion to maintain anaesthesia in colts undergoing field castration.

REASONS FOR PERFORMING THE STUDY: The use of alfaxalone and medetomidine administered as an i.v. infusion to maintain anaesthesia has not previously been reported in the horse. OBJECTIVES: To investigate the use of alfaxalone in hydroxpropyl-beta-cyclodextrin (Alfaxan) and medetomidine infusion as a field anaesthetic for short-term surgical procedures in the horse. HYPOTHESIS: Alfaxalone-medetomidine anaesthesia is suitable for short-term field anaesthesia in horses. METHODS: Eleven healthy colts underwent 45 min of anaesthesia with an i.v. infusion of alfaxalone (2 mg/kg bwt/h) and medetomidine (5 µg/kg bwt/h) for routine field castration. Horses were premedicated with i.v. acepromazine (0.03 mg/kg bwt), medetomidine (7 µg/kg bwt) and guaiphenesin (35 mg/kg bwt) before i.v. induction with alfaxalone (1 mg/kg bwt). Colts were intubated with an endotracheal tube and 100% oxygen insufflated at 10 l/min. The physiological variables monitored included pulse rate, respiratory rate, direct arterial blood pressure, arterial blood gases and the quality of the inductions and recoveries were scored. RESULTS: Overall, the anaesthetic period and surgical conditions were acceptable and the quality of the anaesthetic inductions and recoveries was good to excellent. All colts stood on their first attempt (mean ± s.d.) 37 ± 13.5 min after the infusion was stopped. During anaesthesia, cardiopulmonary data, presented as range of mean values at each time point were: heart rate: 45-47 beats/min; mean blood pressure: 104-112 mmHg; respiratory rate: 8 breaths/min; PaO2 : 117-172 mmHg; PaCO2 : 50-56 mmHg and pH 7.34-7.37. CONCLUSIONS AND POTENTIAL RELEVANCE: The co-administration of alfaxalone and medetomidine as an i.v. infusion after anaesthetic induction with alfaxalone was suitable for short-term field anaesthesia in the horse.

Clinical evaluation of alfaxalone as an anaesthetic induction agent in cats less than 12 weeks of age.

OBJECTIVE: To assess the clinical suitability of alfaxalone as an anaesthetic induction and maintenance agent for kittens aged less than 12 weeks. MATERIALS AND METHODS: The study group comprised 34 kittens aged less than 12 weeks that were presented for surgical desexing. They were aged by dentition, examined and weighed prior to premedication with acepromazine, atropine and morphine. At 20-30 min after premedication, animals were anaesthetised with intravenous alfaxalone administered to effect, using a target maximum expected dose of 5 mg/kg. All cats were intubated: 25 were maintained with isoflurane in oxygen administered with a non-rebreathing circuit and 8 were maintained by supplemental intravenous administration of alfaxalone. Subjective measures of anaesthetic quality and vital signs were recorded from enrolment to recovery. Cats receiving supplemental alfaxalone for maintenance were evaluated for time to first supplemental dose and the total dose of supplemental alfaxalone (mg/kg/h). Descriptive and comparative statistics were used to analyse and present collected data. RESULTS: The mean (± SD) dose of alfaxalone for induction was 4.7 ± 0.5 mg/kg body weight. Subjective measures of anaesthetic quality indicated acceptable induction, maintenance and recovery standards. Measured cardiovascular and respiratory parameters were well maintained. CONCLUSION: Alfaxalone in 2-hydroxypropyl-beta-cyclodextrin (Alfaxan®) is a suitable injectable anaesthetic induction agent for juvenile cats aged less than 12 weeks requiring anaesthesia. Maintenance of anaesthesia with supplemental doses of alfaxalone may be a suitable alternative in kittens when the use of inhalant anaesthetic maintenance is not feasible.

Clinical evaluation of alfaxalone as an anaesthetic induction agent in dogs less than 12 weeks of age.

OBJECTIVE: To assess the efficacy of alfaxalone as an anaesthetic induction agent for dogs aged less than 12 weeks. MATERIALS AND METHODS: The study group comprised 25 juvenile dogs aged less than 12 weeks that were presented for surgical desexing. Dogs were aged by dentition, examined and weighed prior to premedication with acepromazine, atropine and morphine. At 20-30 min after premedication, animals were anaesthetised with intravenous alfaxalone administered to effect, using a target maximum expected dose of 2 mg/kg. Dogs were intubated and anaesthesia was maintained with isoflurane in oxygen administered with a non-rebreathing system. Subjective measures of anaesthetic quality and vital signs were recorded from enrollment to recovery. Descriptive and comparative statistics were used to analyse and present collected data. RESULTS: The mean (± SD) dose of alfaxalone for induction was 1.7 (± 0.3) mg/kg body weight. Subjective measures of anaesthetic quality indicated acceptable induction, maintenance and recovery standards. Measured cardiovascular and respiratory parameters were well maintained. CONCLUSION: Alfaxalone in 2-hydroxypropyl-beta-cyclodextrin (Alfaxan®) is a suitable injectable anaesthetic induction agent for dogs aged less than 12 weeks requiring anaesthesia.

Pharmacokinetics of ketorolac after intravenous and oral single dose administration in dogs.

The pharmacokinetics of ketorolac (Toradol), a human non-narcotic, nonsteroidal anti-inflammatory drug (NSAID) of the pyrrolo-pyrrole group, was studied in six mixed breed dogs of varying ages (1-5 years). The study was performed using a randomized crossover design, with each dog initially assigned to one of two groups (intravenous (i.v.) or oral (p.o.)). Each group of three dogs received either the injectable or oral formulation of ketorolac tromethamine at 0.5 mg/kg. Serial blood samples were collected before and over 96 h following treatment. Samples were analysed by reverse phase HPLC. Individual ketorolac plasma concentration-time curves were initially evaluated by computerized curve stripping techniques followed by nonlinear least squares regression. Following i.v. administration mean (+/- SD) pharmacokinetic parameters were: elimination half-life (t1/2 beta) = 4.55 h, plasma clearance (Clp) = 1.25 (1.13) mL/kg/min, and volume of distribution at steady state (Vss) = 0.33 (0.10) L/kg. Mean (+/- SD) p.o. pharmacokinetic values were: t1/2 beta = 4.07 h, time to reach maximum concentration (tmax) = 51.2 (40.6) min, and p.o. bioavailability (F) = 100.9 (46.7)%. These results suggest that the pharmacodisposition characteristics of a clinically effective 0.5 mg/kg i.v. or p.o. single dose of ketorolac tromethamine administered to dogs is fairly similar to that observed in humans.

Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus faecalis.

The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1-phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.