Some pharmacokinetic indices of oral fluconazole administration to koalas (Phascolarctos cinereus) infected with cryptococcosis.

Three asymptomatic koalas serologically positive for cryptococcosis and two symptomatic koalas were treated with 10 mg/kg fluconazole orally, twice daily for at least 2 weeks. The median plasma Cmax and AUC0-8 h for asymptomatic animals were 0.9 µg/mL and 4.9 µg/mL·h, respectively; and for symptomatic animals 3.2 µg/mL and 17.3 µg/mL·h, respectively. An additional symptomatic koala was treated with fluconazole (10 mg/kg twice daily) and a subcutaneous amphotericin B infusion twice weekly. After 2 weeks the fluconazole Cmax was 3.7 µg/mL and the AUC0-8 h was 25.8 µg/mL*h. An additional three koalas were treated with fluconazole 15 mg/kg twice daily for at least 2 weeks, with the same subcutaneous amphotericin protocol co-administered to two of these koalas (Cmax : 5.0 µg/mL; mean AUC0-8 h : 18.1 µg/mL*h). For all koalas, the fluconazole plasma Cmax failed to reach the MIC90 (16 µg/mL) to inhibit C. gattii. Fluconazole administered orally at either 10 or 15 mg/kg twice daily in conjunction with amphotericin is unlikely to attain therapeutic plasma concentrations. Suggestions to improve treatment of systemic cryptococcosis include testing pathogen susceptibility to fluconazole, monitoring plasma fluconazole concentrations, and administration of 20-25 mg/kg fluconazole orally, twice daily, with an amphotericin subcutaneous infusion twice weekly.

In vitro activity of chloramphenicol, florfenicol and enrofloxacin against Chlamydia pecorum isolated from koalas (Phascolarctos cinereus).

OBJECTIVE: To determine the in vitro susceptibilities of koala isolates of Chlamydia pecorum to enrofloxacin and chloramphenicol, which are frequently used to treat koalas with chlamydiosis, and florfenicol, a derivative of chloramphenicol. METHODS: The in vitro susceptibilities were determined by culturing three stored isolates and seven clinical swabs of C. pecorum. Susceptibility testing was undertaken using cycloheximide-treated buffalo green monkey kidney cells in 96 well microtitre plates. RESULTS: The minimum inhibitory concentrations (MICs) for all isolates were 0.25-0.50 µg/mL (enrofloxacin), 1-2 µg/mL (chloramphenicol), and 1-2 µg/mL (florfenicol). Minimum bactericidal concentration (MBC) values for five isolates were also determined and were within one two-fold dilution of MICs. The MICs and MBCs of these antimicrobials were within ranges previously reported for other chlamydial species. CONCLUSION: When combined with previously published pharmacokinetic data, the in vitro susceptibility results support chloramphenicol as a more appropriate treatment option than enrofloxacin for koalas with chlamydiosis. The susceptibility results also indicate florfenicol may be an appropriate treatment option for koalas with chlamydiosis, warranting further investigation.

Pharmacokinetics of fluconazole following intravenous and oral administration to koalas (Phascolarctos cinereus).

Clinically normal koalas (n = 12) received a single dose of 10 mg/kg fluconazole orally (p.o.; n = 6) or intravenously (i.v.; n = 6). Serial plasma samples were collected over 24 h, and fluconazole concentrations were determined using a validated HPLC assay. A noncompartmental pharmacokinetic analysis was performed. Following i.v. administration, median (range) plasma clearance (CL) and steady-state volume of distribution (Vss ) were 0.31 (0.11-0.55) L/h/kg and 0.92 (0.38-1.40) L/kg, respectively. The elimination half-life (t1/2 ) was much shorter than in many species (i.v.: median 2.25, range 0.98-6.51 h; p.o.: 4.69, range 2.47-8.01 h), and oral bioavailability was low and variable (median 0.53, range 0.20-0.97). Absorption rate-limited disposition was evident. Plasma protein binding was 39.5 ± 3.5%. Although fluconazole volume of distribution (Varea ) displayed an allometric relationship with other mammals, CL and t1/2 did not. Allometrically scaled values were approximately sevenfold lower (CL) and sixfold higher (t1/2 ) than observed values, highlighting flaws associated with this technique in physiologically distinct species. On the basis of fAUC/MIC pharmacodynamic targets, fluconazole is predicted to be ineffective against Cryptococcus gattii in the koala as a sole therapeutic agent administered at 10 mg/kg p.o. every 12 h.

Evaluation of enrofloxacin use in koalas (Phascolarctos cinereus) via population pharmacokinetics and Monte Carlo simulation.

Clinically normal koalas (n = 6) received a single dose of intravenous enrofloxacin (10 mg/kg). Serial plasma samples were collected over 24 h, and enrofloxacin concentrations were determined via high-performance liquid chromatography. Population pharmacokinetic modeling was performed in S-ADAPT. The probability of target attainment (PTA) was predicted via Monte Carlo simulations (MCS) using relevant target values (30-300) based on the unbound area under the curve over 24 h divided by the minimum inhibitory concentration (MIC) (fAUC0-24 /MIC), and published subcutaneous data were incorporated (Griffith et al., 2010). A two-compartment disposition model with allometrically scaled clearances (exponent: 0.75) and volumes of distribution (exponent: 1.0) adequately described the disposition of enrofloxacin. For 5.4 kg koalas (average weight), point estimates for total clearance (SE%) were 2.58 L/h (15%), central volume of distribution 0.249 L (14%), and peripheral volume 2.77 L (20%). MCS using a target fAUC0-24 /MIC of 40 predicted highest treatable MICs of 0.0625 mg/L for intravenous dosing and 0.0313 mg/L for subcutaneous dosing of 10 mg/kg enrofloxacin every 24 h. Thus, the frequently used dosage of 10 mg/kg enrofloxacin every 24 h subcutaneously may be appropriate against gram-positive bacteria with MICs ≤ 0.03 mg/L (PTA > 90%), but appears inadequate against gram-negative bacteria and Chlamydiae in koalas.

Pharmacokinetics of meloxicam in koalas (Phascolarctos cinereus) after intravenous, subcutaneous and oral administration.

The pharmacokinetic profile of meloxicam in clinically healthy koalas (n = 15) was investigated. Single doses of meloxicam were administered intravenously (i.v.) (0.4 mg/kg; n = 5), subcutaneously (s.c.) (0.2 mg/kg; n = 1) or orally (0.2 mg/kg; n = 3), and multiple doses were administered to two groups of koalas via the oral or s.c. routes (n = 3 for both routes) with a loading dose of 0.2 mg/kg for day 1 followed by 0.1 mg/kg s.i.d for a further 3 days. Plasma meloxicam concentrations were quantified by high-performance liquid chromatography. Following i.v. administration, meloxicam exhibited a rapid clearance (CL) of 0.44 ± 0.20 (SD) L/h/kg, a volume of distribution at terminal phase (Vz ) of 0.72 ± 0.22 L/kg and a volume of distribution at steady state (Vss ) of 0.22 ± 0.12 L/kg. Median plasma terminal half-life (t(1/2)) was 1.19 h (range 0.71-1.62 h). Following oral administration either from single or repeated doses, only maximum peak plasma concentration (C(max) 0.013 ± 0.001 and 0.014 ± 0.001 µg/mL, respectively) was measurable [limit of quantitation (LOQ) >0.01 µg/mL] between 4-8 h. Oral bioavailability was negligible in koalas. Plasma protein binding of meloxicam was ~98%. Three meloxicam metabolites were detected in plasma with one identified as the 5-hydroxy methyl derivative. This study demonstrated that koalas exhibited rapid CL and extremely poor oral bioavailability compared with other eutherian species. Accordingly, the currently recommended dose regimen of meloxicam for this species appears inadequate.

Pharmacokinetics of chloramphenicol following administration of intravenous and subcutaneous chloramphenicol sodium succinate, and subcutaneous chloramphenicol, to koalas (Phascolarctos cinereus).

Clinically normal koalas (n = 19) received a single dose of intravenous (i.v.) chloramphenicol sodium succinate (SS) (25 mg/kg; n = 6), subcutaneous (s.c.) chloramphenicol SS (60 mg/kg; n = 7) or s.c. chloramphenicol base (60 mg/kg; n = 6). Serial plasma samples were collected over 24-48 h, and chloramphenicol concentrations were determined using a validated high-performance liquid chromatography assay. The median (range) apparent clearance (CL/F) and elimination half-life (t(1/2)) of chloramphenicol after i.v. chloramphenicol SS administration were 0.52 (0.35-0.99) L/h/kg and 1.13 (0.76-1.40) h, respectively. Although the area under the concentration-time curve was comparable for the two s.c. formulations, the absorption rate-limited disposition of chloramphenicol base resulted in a lower median C(max) (2.52; range 0.75-6.80 µg/mL) and longer median tmax (8.00; range 4.00-12.00 h) than chloramphenicol SS (C(max) 20.37, range 13.88-25.15 µg/mL; t(max) 1.25, range 1.00-2.00 h). When these results were compared with susceptibility data for human Chlamydia isolates, the expected efficacy of the current chloramphenicol dosing regimen used in koalas to treat chlamydiosis remains uncertain and at odds with clinical observations.

Plasma concentrations of chloramphenicol after subcutaneous administration to koalas (Phascolarctos cinereus) with chlamydiosis.

Nine mature koalas with chlamydiosis, typically keratoconjunctivitis and/or urogenital tract infection, were treated with daily subcutaneous injections of chloramphenicol at 60 mg/kg for 45 days (five koalas), or for a shorter duration (four koalas). All koalas were initially positive for Chlamydia pecorum as determined by real-time polymerase chain reaction (qPCR). Plasma chloramphenicol concentrations were determined at t = 0, 1, 2, 4, 8, and 24 h after the day 1 injection (nine koalas) and after the day 15 injection (seven koalas). Chloramphenicol reached a median (and range) maximum plasma concentration of 3.03 (1.32-5.03 µg/mL) at 4 (1-8 h) after the day 1 injection and 4.82 (1.97-27.55 µg/mL) at 1 (1-2 h) after day 15. The median (and range) of AUC(0-24) on day 1 and day 15 were 48.14 (22.37-81.14 µg·h/mL) and 50.83 (28.43-123.99 µg·h/mL), respectively. The area under the moment curve (AUMC)(0-24) median (and range) for day 1 and day 15 were 530.03 (233.05-798.97 h) and 458.15 (291.72-1093.58 h), respectively. Swabs were positive for chlamydial DNA pretreatment, and all koalas except one, produced swabs negative for chlamydial DNA during treatment and which remained so, for 2-63 days after treatment, however whether chloramphenicol treatment prevented long-term recrudescence of infection was not established. At this dose and dosing frequency, chloramphenicol appeared to control mild chlamydial infection and prevent shedding, but severe urogenital disease did not appear to respond to chloramphenicol at this dosage regime. For koalas affected by severe chlamydiosis, antibiotics alone are not sufficient to effect a cure, possibly because of structural or metabolic changes associated with chronic disease and inflammation.

DI@L-log by design: an integrated tele-care communication infrastructure.

A spoken dialogue system for the acquisition of type 2 diabetes mellitus (T2DM) home monitored patient data known as DI@L-log is presented. The purpose of the system is to collect weight, blood sugar and blood pressure readings from a cohort of hypertensive T2DM patients on a weekly basis using their home telephone. The recent voice-data convergence affords an arguably improved means for doctors to track patient health states at a distance in order to provide health institutions with more frequent and accurate patient profiles. Our system architecture integrates VoiceXML and the standard PSTN, with a Pan-European open source for hosting Internet telephony applications. This paper reports on recent developments in the design of DI@L-log, which aims to serve as a communication intervention that disparages the traditional paper logbook used to document readings by the patient.

Hazardous drugs.

Engineering controls and personal protective equipment are crucial for health care professionals working in situations where cytotoxic agents, pharmaceuticals such as ribavirin and pentamidine, and other potentially hazardous drugs are present. Plans for exposure control, spill clean-up, and medical surveillance must be instituted to protect the employee.

Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.

Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Mono- and bis(aminomethyl)phenylacetic acid esters as short-acting antiarrhythmic agents. 2.

The synthesis, antiarrhythmic activity, and blood hydrolysis properties of a series of mono- and bis(aminomethyl)phenylacetic acid esters related to a previously reported class Ic antiarrhythmic agent (ACC-9358) are described. Of the various oxa-, aza-, thia-, and carbacyclic esters initially prepared in the bis(pyrrolidinomethyl)-4-hydroxyphenylacetic acid series, the 1,4-benzodioxanyl-2-methyl(3q) and the thienyl-2-methyl(31) esters were evaluated in vivo for antiarrhythmic efficacy. In addition, a number of monoappended phenylacetic esters of 3q with or without the 4-hydroxy group were also prepared for evaluation of antiarrhythmic, lipophilic, and metabolic properties. Of these compounds, 3q possessed the most desirable pharmacological and pharmacokinetic profile.

Ester derivatives of 2,6-bis(1-pyrrolidinylmethyl)-4-benzamidophenol as short-acting antiarrhythmic agents. 1.

In an effort to find a replacement for the iv antiarrhythmic drug lidocaine having reduced systemic and central nervous system effects, activity against supraventricular as well as ventricular arrhythmias, and a biological half-life of less than 15 min, derivatives of the orally active class Ic clinical agent 2,6-bis(1-pyrrolidinylmethyl)-4-benzamidophenol, 1 (ACC-9358), were synthesized and tested. Compounds with ester groups attached to the phenyl ring were either weakly active or toxic. Replacement of the formanilide function with alkyl esters afforded compounds with antiarrhythmic activity in the range of 1. When the ester carboxyl was separated from the bis(aminomethyl)phenol by methylene units, very short half-lives were observed in human blood. In general, these compounds also had low lipophilic character.

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--II. Evidence that the epitope recognized is involved in a late step in the cytolytic mechanism.

A monoclonal antibody (RH1-38) which blocks multiple systems of cell-mediated cytotoxicity was functionally characterized. RH1-38 specifically blocks, in the absence of complement, natural killer (NK) activity (K562 targets) without any effect on NK-K562 conjugate formation. Kinetic studies suggested that the antibody blocks a step that occurs 30-120 min after effector populations are mixed with target cells. Single-cell cytotoxicity assays in agarose, combined with standard 51Cr release assays and Michaelis-Menten analysis revealed that RH1-38 markedly decreases Vmax and the number of active NK cells, again without any effect on the number of target-binding cells. The maximum recycling capacity was usually decreased, but in some experiments unchanged, in the presence of the monoclonal antibody. RH1-38 inhibited equally well whole peripheral blood mononuclear leukocytes (PBML), Percoll-fractionated lymphocytes enriched for NK activity, and interferon (IFN)-boosted NK activity. PBML exposed to RH1-38 and then washed mediated depressed NK activity which was partially reversed by subsequent treatment with IFN. These studies are most consistent with the hypothesis that RH1-38 inhibits a step late in the NK cytolytic mechanism rather than through an effect on conjugate formation. The primary effect is probably not on the IFN-generating or boosting mechanism, but a secondary effect on IFN-related mechanisms cannot be ruled out. Inhibition through an effect on a small lymphocyte modulator of NK activity is also unlikely but not rigorously excluded. Thus, RH1-38 appears to inhibit NK activity through a direct effect on NK effector cells, probably by interfering with a cell-surface molecule which is important in the expression of NK activity. The companion paper demonstrates that this monoclonal antibody immunoprecipitates a molecule which is very similar or identical to the LFA-1 antigen. Thus, RH1-38 recognizes either a novel epitope on the LFA-1 molecule or alternatively a distinct, functional killer cell surface molecule. The epitope appears to be involved in a late step in the cytolytic mechanism, possibly part of the effector cell lytic machinery.

Analysis of procainamide hydrochloride and acecainide hydrochloride in rat feed.

An extraction and GLC assay procedure was developed for quantitation of procainamide hydrochloride and acecainide hydrochloride in rat feed. 4-Amino-N-[2-(dipropylamino)ethyl]benzamide hydrochloride was synthesized and utilized as an internal standard. The assay has good precision and accuracy and was used to establish the stability of acecainide hydrochloride and procainamide hydrochloride in rat feed.

Production of enterotoxin A in milk.

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.

Serological identification of enterotoxigenic staphylococci from cheese.

Single and double gel-diffusion techniques were employed to examine serologically coagulase-positive staphylococci from cheese for enterotoxigenicity. Supernatant fluid from sac cultures was examined for enterotoxins A and B. The results indicated that 9 of 155 cultures from market cheese and 7 of 77 cultures from food-poisoning cheese produced enterotoxin A, and that none of the cultures produced detectable levels of enterotoxin B. Results of serological tests were confirmed by intravenous injection of cats.