Andrew G. Ellis

10 Perfusion Methods for the Study of Gill Physiology

Publisher Summary This chapter discusses perfusion methods for the study of gill physiology. A large number of perfused gill preparations have been developed to study gill physiology. In general, isolated or in situ perfused gills allow to control or measure many variables concerning gill function. The control of perfusate composition, pressure, and flow and the exclusion of postbranchial circulation, together with an increased accuracy of measurements, are the principal advantages of perfusion methods. Perfused gill preparations fall into four main groups: isolated branchial arches, branchial baskets including all arches, heads, and whole bodies. Isolated branchial arches exclude tissues, such as the central nervous system, buccal and opercular epithelium, and pseudobranch, and represent truly isolated gills. The cost of isolation is the impairment of recurrent circulation, abnormal ventilation, and mechanical stresses. The lamellar blood channels of fish gills are perfused with blood under arterial pressure and in that sense bear a close resemblance to the capillaries of a glomerular kidney. Experiments designed to study branchial ion transport have also been conducted, primarily on intact animals.

Is Fosfomycin a Potential Treatment Alternative for Multidrug-Resistant Gram-Negative Prostatitis?

BACKGROUND Multidrug-resistant gram-negative bacterial (MDR-GNB) infections of the prostate are an increasing problem worldwide, particularly complicating transrectal ultrasound (TRUS)-guided prostate biopsy. Fluoroquinolone-based regimens, once the mainstay of many protocols, are increasingly ineffective. Fosfomycin has reasonable in vitro and urinary activity (minimum inhibitory concentration breakpoint ≤64 µg/mL) against MDR-GNB, but its prostatic penetration has been uncertain, so it has not been widely recommended for the prophylaxis or treatment of MDR-GNB prostatitis. METHODS In a prospective study of healthy men undergoing a transurethral resection of the prostate for benign prostatic hyperplasia, we assessed serum, urine, and prostatic tissue (transition zone [TZ] and peripheral zone [PZ]) fosfomycin concentrations using liquid chromatography-tandem mass spectrometry, following a single 3-g oral fosfomycin dose within 17 hours of surgery. RESULTS Among the 26 participants, mean plasma and urinary fosfomycin levels were 11.4 ± 7.6 µg/mL and 571 ± 418 µg/mL, 565 ± 149 minutes and 581 ± 150 minutes postdose, respectively. Mean overall prostate fosfomycin levels were 6.5 ± 4.9 µg/g (range, 0.7-22.1 µg/g), with therapeutic concentrations detectable up to 17 hours following the dose. The mean prostate to plasma ratio was 0.67 ± 0.57. Mean concentrations within the TZ vs PZ prostate regions varied significantly (TZ, 8.3 ± 6.6 vs PZ, 4.4 ± 4.1 µg/g; P = .001). Only 1 patient had a mean prostatic fosfomycin concentration of <1 µg/g, whereas the majority (70%) had concentrations ≥4 µg/g. CONCLUSIONS Fosfomycin appears to achieve reasonable intraprostatic concentrations in uninflamed prostate following a single 3-g oral dose, such that it may be a potential option for prophylaxis pre-TRUS prostate biopsy and possibly for the treatment of MDR-GNB prostatitis. Formal clinical studies are now required.

Inhibition of etoposide elimination in the isolated perfused rat liver by Cremophor EL and Tween 80

Abstract Cremophor EL, a surfactant used in the clinical formulation of cyclosporine and paclitaxel, will reverse the multidrug resistance (MDR) phenotype in vitro. As other MDR modulators can alter the pharmacokinetics of cytotoxic drugs, the aim of this study was to examine the effect of Cremophor and another MDR-reversing surfactant, Tween 80, on the hepatic elimination and biliary excretion of etoposide. Using the isolated perfused rat-liver model with 80 ml recirculating perfusate containing 20% red blood cells and 4% bovine serum albumin, etoposide (1.6 mg) with and without Cremophor (800 or 80 mg) or Tween 80 (80 mg) was given into the perfusate reservoir, and perfusate and bile samples were collected for 3 h. Etoposide was measured by high-performance liquid chromatography (HPLC) and Cremophor was measured using a bioassay. Both surfactants changed the etoposide elimination profile from biphasic to monophasic. High-dose Cremophor increased the AUC (from 334±23 to 1540±490 μg min ml-1, P<0.05) and decreased the total clearance (from 4.8±0.3 to 1.1±0.3 ml/min, P<0.05) and biliary clearance (from 2.6±1.1 to 0.5±0.2 ml/min, P<0.05) but decreased the elimination half-life (from 62±17 to 40±5 min, P<0.05) and volume of distribution (from 424±85 to 65±19 ml, P<0.05). Low-dose Cremophor and Tween 80 caused intermediate effects on these parameters that were statistically significant for total clearance, half-life, and volume of distribution. Cremophor had no adverse effect on liver function, whereas Tween 80 caused haemolysis and cholestasis. The initial high-dose Cremophor perfusate concentration was 0.8 mg/ml, which previous studies have shown to be clinically relevant and close to the optimal level for MDR reversal in vitro (1.0 mg/ml). Cremophor may be a clinically useful MDR modulator, but it may alter the pharmacokinetics of the cytotoxic drug.

Study Protocol--accurate assessment of kidney function in Indigenous Australians: aims and methods of the eGFR study.

BackgroundThere is an overwhelming burden of cardiovascular disease, type 2 diabetes and chronic kidney disease among Indigenous Australians. In this high risk population, it is vital that we are able to measure accurately kidney function. Glomerular filtration rate is the best overall marker of kidney function. However, differences in body build and body composition between Indigenous and non-Indigenous Australians suggest that creatinine-based estimates of glomerular filtration rate derived for European populations may not be appropriate for Indigenous Australians. The burden of kidney disease is borne disproportionately by Indigenous Australians in central and northern Australia, and there is significant heterogeneity in body build and composition within and amongst these groups. This heterogeneity might differentially affect the accuracy of estimation of glomerular filtration rate between different Indigenous groups. By assessing kidney function in Indigenous Australians from Northern Queensland, Northern Territory and Western Australia, we aim to determine a validated and practical measure of glomerular filtration rate suitable for use in all Indigenous Australians.Methods/DesignA cross-sectional study of Indigenous Australian adults (target n = 600, 50% male) across 4 sites: Top End, Northern Territory; Central Australia; Far North Queensland and Western Australia. The reference measure of glomerular filtration rate was the plasma disappearance rate of iohexol over 4 hours. We will compare the accuracy of the following glomerular filtration rate measures with the reference measure: Modification of Diet in Renal Disease 4-variable formula, Chronic Kidney Disease Epidemiology Collaboration equation, Cockcroft-Gault formula and cystatin C- derived estimates. Detailed assessment of body build and composition was performed using anthropometric measurements, skinfold thicknesses, bioelectrical impedance and a sub-study used dual-energy X-ray absorptiometry. A questionnaire was performed for socio-economic status and medical history.DiscussionWe have successfully managed several operational challenges within this multi-centre complex clinical research project performed across remote North, Western and Central Australia. It seems unlikely that a single correction factor (similar to that for African-Americans) to the equation for estimated glomerular filtration rate will prove appropriate or practical for Indigenous Australians. However, it may be that a modification of the equation in Indigenous Australians would be to include a measure of fat-free mass.

Accurate assessment of kidney function in indigenous Australians: the estimated GFR study.

Study equation (8.9 [95% CI, 7.9-11.1] mL/min/1.73 m 2 less than mGFR) than with the CKD-EPI equation (3.8 [95% CI, 2.5-5.6] mL/min/1.73 m 2 less than mGFR). Stratified by mGFR group (Table S1), bias was greatest in indigenous participants with mGFR 90 mL/min/1.73 m 2 when the MDRD Study equation was used, but improved with use of the CKD-EPI equation (without the correction factor). The accuracy of eGFR was not significantly different between the 2 equations. Thus, the magnitude of bias using the MDRD Study equation in indigenous participants was similar with or without use of theAfrican American correction factor; however, the direction of bias differed (GFR was overestimated with the correction factor and underestimated without it). For the CKD-EPI equation, bias and accuracy were improved significantly by omitting the correction factor. WithomissionoftheAfricanAmericancorrectionfactor,eGFRCKDEPI provided a reasonably unbiased and accurate estimate of GFR, whereas the MDRD Study equation significantly underestimated GFRinIndigenousAustralians(comparedwithnonindigenousAustralians). This may relate to inherent limitations of the MDRD Study equation rather than body build or other differences in Indigenous Australians.The CKD-EPI equation has been shown to perform better at higher mGFRs (approximately 60 mL/min/1.73 m 2 ), and the MDRD Study performs better at lower GFRs. 9 The mean mGFR of the Indigenous Australian cohort (93 mL/min/1.73 m 2 ) was closer to that of the CKD-EPI than the MDRD development cohort, and this

Inhibition of paclitaxel elimination in the isolated perfused rat liver by Cremophor EL

Purpose: Cremophor can alter the pharmacokinetics of cytotoxic drugs, including doxorubicin and etoposide. In view of its presence in the formulation of paclitaxel, the aim of this study was to investigate the influence of Cremophor on the hepatobiliary elimination of paclitaxel. Methods: In a recirculating isolated perfused rat-liver system the elimination of 1.7 mg paclitaxel given as a bolus into the perfusate reservoir was monitored in perfusate and bile in controls and after the administration of either 80 or 800 μl Cremophor. The higher dose of Cremophor yields clinically relevant perfusate concentrations. Paclitaxel was measured in perfusate, bile, and liver tissue by high-performance liquid chromatography. Results: Cremophor caused a dose-dependent inhibition of the elimination of paclitaxel, with a statistically significant mean value ± SD, n = 3; (P < 0.05 versus controls Bonferroni t-test) 9-fold increase in AUC (2227±106 versus 245 ± 40 g ml−1min), 9-fold decrease in total clearance (0.8±0.1 versus 7.0±1.1 ml/min), and 5-fold increase in elimination half-life (92±14 versus 18±4 min) being observed after a dose of 800 μl Cremophor. With the addition of Cremophor the amount of paclitaxel remaining after 3 h increased in perfusate from none to 20, increased in liver tissue from 4 to 18, and remained constant in bile at 11–13%. In the control group, 86 of the paclitaxel dose was recovered in bile as five putative metabolites, which were measured in paclitaxel equivalents, with the major metabolite. M3 co-eluting with 3′-p-hydroxypaclitaxel. This decreased to 45 of the dose on the addition of Cremophor, and the ratio of M3 to paclitaxel in bile decreased. Conclusions: Cremophor inhibits the hepatic elimination of paclitaxel in the isolated perfused rat liver, primarily by preventing the drug from reaching sites of metabolism and excretion. The presence of Cremophor in the paclitaxel formulation may therefore contribute to the nonlinear pharmacokinetics and pharmacodynamics of paclitaxel.

Relationship of Hepatic Functional Imaging to Irinotecan Pharmacokinetics and Genetic Parameters of Drug Elimination

PURPOSE The marked variability of irinotecan (Ir) clearance warrants individualized dosing based on hepatic drug handling. The aims of this trial were to identify parameters from functional hepatic nuclear imaging (HNI) that correlate with (1) Ir pharmacology, and (2) single-nucleotide polymorphisms (SNPs) for the ABCB1 (P-glycoprotein) and UGT-1A1 genes, known to influence Ir handling. METHODS Patients underwent genotyping for ABCB1 SNPs and UTUGT-1A1*28 carriage and HNI with 99mTc-DIDA (acetanilidoiminodiacetic acid)/99mTc-DISIDA (disofenin) and MIBI (99mTc-sestamibi) scans, probes for biliary transport proteins ABCC1 and -2, and ABCB1 function. HNI data were analyzed by noncompartmental and deconvolutional analysis to provide hepatic extraction and biliary excretion parameters. Patients received Ir, fluorouracil, and folinic acid using a weekly x2, every-3-weeks schedule. Plasma was taken for Ir and SN-38 analysis on day 1, cycle 1. RESULTS Of the 21 patients accrued, Ir pharmacokinetics data were obtained from 16 patients. 99mTc-DIDA/DISIDA percent retention at 1 hour (1-hour RET) correlated to baseline serum bilirubin (P = .008). Both 99mTc-DIDA/DISIDA and MIBI 1-hour RET correlated with SN-38 area under the curve (AUC; P < .01). On multiple regression analysis, SN-38 AUC = -215 + 18.68 x bilirubin + 4.27 x MIBI 1-hour RET (P = .009, R2 = 44.2%). HNI parameters did not correlate with Ir toxicity or UGT1A1*28 carriage. MIBI excretion was prolonged in patients with the ABCB1 exon 26 TT variant allele relative to wild-type (P = .015). CONCLUSION Functional imaging of hepatic uptake and excretory pathways may have potential to predict Ir pharmacokinetics. Evaluation of a larger cohort as well as polymorphisms in other biliary transporters and UGT1A1 alleles is warranted.

Phase I and pharmacokinetic evaluation of intravenous hyaluronic acid in combination with doxorubicin or 5-fluorouracil

Background: Pre-clinically, hyaluronan (HA) has been demonstrated to systemically target chemotherapeutic drugs to tumours while ameliorating treatment toxicities. This study is a preliminary clinical investigation to determine if HA could be safely used in combination with 5-fluorouracil (5-FU) and doxorubicin (DOX). Methods: Thirty patients with metastatic cancer were intravenously administered 500 mg/m2 HA in combination with escalating doses of DOX (30–60 mg/m2) or 5-FU (cumulative dose of 1,350–2,250 mg/m2 per cycle). The effect of pre-administration of 20 mg/m2 of folinic acid on HA/5-FU chemotherapy was also investigated. Patients were randomized to receive either HA/chemotherapy or chemotherapy alone in their first treatment cycle and vice versa for the second cycle. Patients received HA and chemotherapy in all subsequent cycles. Results:Treatment was well tolerated, tumour responses were observed and the co-administration of HA did not alter the pharmacokinetics of clinically relevant doses of 5-FU or DOX. Conclusion:High doses of intravenous high-molecular-weight HA can be safely co-administered with clinical doses of chemotherapy without significantly altering the toxicity or pharmacokinetics of the drugs or HA.

Optimal timing of oral fosfomycin administration for pre-prostate biopsy prophylaxis

OBJECTIVES As the optimal administration time for fosfomycin peri-procedural prophylaxis is unclear, we sought to determine optimal administration times for fosfomycin peri-procedural prophylaxis. METHODS Plasma, peripheral zone and transition zone fosfomycin concentrations were obtained from 26 subjects undergoing transurethral resection of the prostate (TURP), following a single oral dose of 3 g of fosfomycin. Population pharmacokinetic modelling was completed with the Nonparametric Adaptive Grid (NPAG) algorithm (Pmetrics package for R), with a four-compartment model. Plasma and tissue concentrations were simulated during the first 24 h post-dose, comparing these with EUCAST susceptibility breakpoints for Escherichia coli, a common uropathogen. RESULTS Non-compartmental-determined pharmacokinetic values in our population were similar to those reported in the package insert. Predicted plasma concentrations rapidly increased after the first hour, giving more than 90% population coverage for organisms with an MIC ≤4 mg/L over the first 12 h post-dose. Organisms with higher MICs fared much worse, with organisms at the EUCAST breakpoint being covered for <10% of the population at any time. Transitional zone prostate concentrations exceeded 4 mg/L for 90% of the population between hours 1 and 9. Peripheral zone prostate concentrations were much lower and only exceeded 4 mg/L for 70% of the population between hours 1 and 4. CONCLUSIONS Until more precise plasma and tissue data are available, we recommend that fosfomycin prophylaxis be given 1-4 h prior to prostate biopsy. We do not recommend fosfomycin prophylaxis for subjects with known organisms with MICs >4 mg/L.

Fosfomycin for treatment of prostatitis: new tricks for old dogs

Treatment options for prostatitis caused by multidrug-resistant gram-negative bacilli are limited. We report two cases cured with oral fosfomycin and provide a pharmacokinetic analysis of fosfomycin predose concentrations during treatment.