Pharmacokinetics of posaconazole in koalas (Phascolarctos cinereus) after intravenous and oral administration.

The pharmacokinetic profile of posaconazole in clinically normal koalas (n = 8) was investigated. Single doses of posaconazole were administered intravenously (i.v.; 3 mg/kg; n = 2) or orally (p.o.; 6 mg/kg; n = 6) with serial plasma samples collected over 24 and 36 hr, respectively. Plasma concentrations of posaconazole were quantified by validated high-performance liquid chromatography. A noncompartmental pharmacokinetic analysis of data was performed. Following i.v. administration, estimates of the median (range) of plasma clearance (CL) and steady-state volume of distribution (Vss ) were 0.15 (0.13-0.18) L hr-1  kg-1 and 1.23 (0.93-1.53) L/kg, respectively. The median (range) elimination half-life (t1/2 ) after i.v. and p.o. administration was 7.90 (7.62-8.18) and 12.79 (11.22-16.24) hr, respectively. Oral bioavailability varied from 0.43 to 0.99 (median: 0.66). Following oral administration, maximum plasma concentration (Cmax ; median: 0.72, range: 0.55-0.93 µg/ml) was achieved in 8 (range 6-12) hr. The in vitro plasma protein binding of posaconazole incubated at 37°C was 99.25 ± 0.29%. Consideration of posaconazole pharmacokinetic/pharmacodynamic (PK/PD) targets for some yeasts such as disseminated candidiasis suggests that posaconazole could be an efficacious treatment for cryptococcosis in koalas.

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.

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.

In vitro hepatic microsomal metabolism of meloxicam in koalas (Phascolarctos cinereus), brushtail possums (Trichosurus vulpecula), ringtail possums (Pseudocheirus peregrinus), rats (Rattus norvegicus) and dogs (Canis lupus familiaris).

Quantitative and qualitative aspects of in vitro metabolism of the non-steroidal anti-inflammatory drug meloxicam, mediated via hepatic microsomes of specialized foliage (Eucalyptus) eating marsupials (koalas and ringtail possums), a generalized foliage eating marsupial (brushtail possum), rats, and dogs, are described. Using a substrate depletion method, intrinsic hepatic clearance (in vitro Clint) was determined. Significantly, rates of oxidative transformation of meloxicam, likely mediated via cytochromes P450 (CYP), were higher in marsupials compared to rats or dogs. The rank order of apparent in vitro Clint was brushtail possums (n=3) (mean: 394µL/min/mg protein), >koalas (n=6) (50), >ringtail possums (n=2) (36) (with no significant difference between koalas and ringtail possums), >pooled rats (3.2)>pooled dogs (in which the rate of depletion, as calculated by the ratio of the substrate remaining was <20% and too slow to determine). During the depletion of meloxicam, at a first-order rate constant, 5-hydroxymethyl metabolite (M1) was identified in the brushtail possums and the rat as the major metabolite. However, multiple hydroxyl metabolites were observed in the koala (M1, M2, and M3) and the ringtail possum (M1 and M3) indicating that these specialized foliage-eating marsupials have diverse oxidation capacity to metabolize meloxicam. Using a well-stirred model, the apparent in vitro Clint of meloxicam for koalas and the rat was further scaled to compare with published in vivo Cl. The closest in vivo Cl prediction from in vitro data of koalas was demonstrated with scaled hepatic Cl(total) (average fold error=1.9) excluding unbound fractions in the blood and microsome values; whereas for rats, the in-vitro scaled hepatic Cl fu(blood, mic), corrected with unbound fractions in the blood and microsome values, provided the best prediction (fold error=1.86). This study indicates that eutherians such as rats or dogs serve as inadequate models for dosage extrapolation of this drug to marsupials due to differences in hepatic turnover rate. Furthermore, as in vivo Cl is one of the pharmacokinetic indexes for determining therapeutic drug dosages, this study demonstrates the utility of in vitro to in vivo scaling as an alternative prediction method of drug Cl in koalas.

Acupuncture for treatment of arthralgia secondary to aromatase inhibitor therapy in women with early breast cancer: pilot study.

BACKGROUND: Aromatase inhibitors (AIs) are recommended as adjuvant hormone treatment for postmenopausal women with early breast cancer. A substantial proportion of women taking AIs experience joint pain and stiffness. Studies have suggested that acupuncture may be effective in treating joint pain. OBJECTIVE: A pilot study was conducted to evaluate the feasibility, safety and efficacy of using acupuncture to treat AI-induced arthralgia. METHODS: A total of 32 patients were randomised to receive either sham or real electroacupuncture (EA) twice weekly for 6 weeks. Outcomes of joint pain, stiffness and physical function were measured with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), overall pain severity and interference with the BPI-SF and quality of life (QOL) with the Functional Assessment of Cancer Therapy-General (FACT-G) instrument. Hand strength was assessed by a grip test, and a serum marker of inflammation (C reactive protein (CRP)) was also measured. All assessments were performed at baseline, 6 weeks and 12 weeks, except for blood samples at baseline and 6 weeks only. RESULTS: No serious adverse events were reported during or after acupuncture treatments. There were no significant differences in outcome measures. However, positive trends were observed in stiffness and physical function at week 12 in favour of real EA. CONCLUSIONS: Findings suggest that acupuncture is feasible and safe in patients with breast cancer with joint pain caused by AI. A larger study with adequately powered to confirm these results and detect clinically relevant effects is needed.

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.

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.

Twist contributes to hormone resistance in breast cancer by downregulating estrogen receptor-α.

The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.

Susceptibility of rapidly growing mycobacteria isolated from cats and dogs, to ciprofloxacin, enrofloxacin and moxifloxacin.

Rapidly growing mycobacteria (RGM) cause infections in cats and dogs which require prolonged antibacterial medication for resolution. In Australia, pathogens from the Mycobacterium fortuitum and Mycobacterium smegmatis clusters are responsible for most of the RGM infections in cats and dogs. As fluoroquinolones are often recommended for treating such infections, 14 M. fortuitum isolates, 51 isolates from the M. smegmatis cluster and 2 M. mageritense isolates, collected from feline and canine patients, underwent susceptibility testing to the second generation fluoroquinolones ciprofloxacin and enrofloxacin and the newer generation fluoroquinolone moxifloxacin. Using microbroth dilution, the MIC(90) of ciprofloxacin, enrofloxacin, and moxifloxacin that inhibited growth of M. fortuitum isolates were 0.500, 0.250 and 0.063 µg/mL respectively. For the M. smegmatis cluster isolates the corresponding MIC(90) was 0.500, 0.250 and 0.125 µg/mL respectively. E-test results showed similar trends but MICs were lower than those determined by microbroth dilution. Additionally, moxifloxacin was administered to 10 clinically normal cats (50mg per cat, once daily for 4 days). The plasma moxifloxacin concentration 2h after the last dose was determined by liquid chromatography as 2.2 ± 0.6 µg/mL. The plasma concentration at 2h:MIC(90) ratios for moxifloxacin for M. fortuitum and M. smegmatis cluster was 34.9 and 17.6 respectively which exceeded the recommended threshold of 10, indicating that moxifloxacin has good theoretical efficacy for treatment of those M. fortuitum and M. smegmatis infections in cats and dogs that have become refractory to other antibacterial drug classes.

Isolation and characterization of new mutagens from fried ground beef.

Frying of ground beef at 250 degrees C results in the formation of a series of mutagenic heterocyclic amines, possibly as many as 10 distinct compounds. In this study, the mutagens are separated by aqueous/acid extraction from the beef, XAD adsorption, acid/neutral/base-liquid/liquid extraction, preparative reverse phase h.p.l.c., normal phase h.p.l.c., and analytical reverse phase h.p.l.c. Identification is by low and high resolution mass spectrometry, u.v. absorption spectroscopy and nitrite sensitivity assays. More than 30% of the mutagenic material is identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). The previously described beef mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) contributes less than 12% of the total mutagenicity at high temperatures (300 degrees C) and less than 4% at lower temperatures. In addition to MeIQx major mutagens are seen with molecular ions at m/z 227 (C12H13N5), 209 (C13H11N3), and 176 (C9H12N4). Two very polar and two non-polar peaks making a relatively minor contribution to the total mutagenicity (less than 16% total) have not yet been identified. Estimates of the mass of these mutagens in the original cooked beef were made from the mass spectral analysis and the mutagenic recoveries. From one kilogram of cooked ground beef, 1.0 micrograms MeIQx, 0.02 micrograms of IQ, and up to 1.5 micrograms of additional mutagens are formed.

Tetrachlorodibenzo-p-dioxin quantitation in stack-collected coal fly ash.

Gas chromatography-high resolution mass spectrometry has been used to quantitate tetrachlorodibenzo-p-dioxin (TCDD) in fly ash collected from the stack of a typical commercial coal-fired power plant. No TCDD was detected in this fly ash, but minute traces may be present below the detection limit of 1.2 parts per trillion (by weight). This finding indicates that this type of fossil-fueled power plant is not a large source of this compound in environmental samples, in contrast to the conclusions presented in a recent industrial report.