John C. Lukas

Detection of Mycobacterium avium subsp. paratuberculosis in Retail Cheeses from Greece and the Czech Republic

ABSTRACT We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis, albeit at a low level for viable cells.

Effect of P-glycoprotein inhibition on methadone analgesia and brain distribution in the rat.

Methadone is an opiate drug that has been identified as an in‐vitro substrate of the efflux pump P‐glycoprotein (P‐gp), active in the intestinal epithelium and in the blood–brain barrier (BBB), among other sites. The objective of this study was to test in vivo, in the rat model, the role of P‐gp modulation on the analgesic effect and brain uptake of methadone, as well as identify the most relevant site via dual oral and intravenous (i.v.) experiments. The P‐gp specific inhibitor (valspodar or PSC833) was preadministered (10 mg kg−1 i.v.) to test groups. Analgesia was measured using the tailflick test. The ED50 for oral methadone (2, 3, 6 and 8 mg kg−1) decreased three‐fold in valspodar groups compared with controls (2.23 + 0.002 mg kg−1 and 6.07 + 0.07 mg kg−1; P < 0.0001). The overall analgesic effect (% antinociception) was elevated 3.1 times in pretreated compared with control rats (90.65% + 0.22 vs 29.23% + 14.0; P < 0.01) after 6 mg kg−1 oral methadone and 2.8 times after i.v. (0.35 mg kg−1) administration (91.75% + 4.27 vs 32.45% + 9.0; P < 0.01). The brain:plasma distribution ratio was higher in pretreated animals and AUCbrain (overall brain concentration) was 6 times higher after oral methadone and 4 times higher after i.v. compared with controls, disproportionally increased relative to plasma, implying an active process at the BBB. P‐gp, and hence substrate comedication, plays a critical role in the evolution of the methadone analgesic effect and in its brain uptake, independent of the administration route.

Mycobacterium avium subsp. paratuberculosis, Genetic Susceptibility to Crohn's Disease, and Sardinians: the Way Ahead

ABSTRACT The present study was performed to determine what proportion of people in Sardinia with or without Crohns disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod2, an intracellular protein involved in Crohns disease susceptibility. Genetic analysis of the alleles of the NOD2/CARD15 gene (insC3020, G908R, and R702W alleles), linked to susceptibility or genetic predisposition to Crohns disease in humans, was carried out on specimens from 37 Crohns disease patients and 34 patients without Crohns disease. Our results show that more than 70 percent of people in Sardinia with Crohns disease carry at least one of the susceptibility-associated NOD2/CARD15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis.

Population pharmacokinetics of recombinant factor VIII:C (ReFacto®) in adult HIV-negative and HIV-positive haemophilia patients

PurposeThe aim of this study was to explore possible differences in the pharmacokinetics (PK) of recombinant factor VIII:C (ReFacto® - ReFacto ) in HIV+ vs. HIV– patients and also differences in the chromogenic substrate bioassay (CHS) and one-stage clotting (OSC) methods.MethodsTwenty-eight haemophilia A adults (20 HIV– and eight HIV+) were assayed with both the CHS and OSC methods. An average of two and six samples were collected per patient for HIV–/+, respectively, after one, and occasionally two more, prophylactic doses (mean 2,003 IU; range 1,000–4,300 IU). The observations were analysed with the mixed-effects (population) compartmental PK modelling package NONMEM (nonlinear mixed-effects modelling) and the FOCE (first-order conditional estimation) method. Base modelling was performed independently for the CHS and OSC bioassays for comparison, and covariate models and simulation tests were done only for the commonly used OSC bioassay. The final covariate model was validated using the bootstrap method. Monte Carlo simulations were used to estimate the expected probability of exceeding 20%, 40% or 60% of normal factor VIII:C in plasma after a single dose, corresponding to required levels for preventing mild, moderate and life-threatening haemorrhages.ResultsOne-compartment base-model population PK parameters were [mean parameter (interpatient variability %)] for CHS: clearance (CL) = 2.56 dl h−1 (33.2%); volume of distribution (V) = 34.8 dl (12.8%); and for OSC: CL = 3.83 dl h−1 (47.8%), V = 53.7 dl (22.4%). The volumes differed significantly between the CHS and OSC methods (p < 0.0001), and variabilities were higher for OSC. Nevertheless, the empirical half-lives (t1/2 = l n (2) × V/CL) were similar for CHS and OSC, [(mean ± standard deviation (SD)], 9.5 ± 3 h and 10.2 ± 4 h, respectively. In covariate modelling with the OSC-derived model, HIV status (VIR) was a significant categorical predictor (p < 0.005) for V. The final covariate models with OSC were for CL = 3.93 + 0.09 × (WT-75) and for V = 48.6 × (1 + 0.36 × VIR) + 0.55 × (WT-75); therefore, V for the typical HIV+ patient was 36% higher than for the HIV– patient.ConclusionsBoth HIV– and HIV+ patients showed 100% success with the 20% threshold at doses >20 IU/kg. HIV– patients receiving >50 IU/kg had a 100% expected chance of success for all thresholds. HIV+ patients for moderate or life-threatening haemorrhage treatment need 10 IU/kg more than the HIV– patient equivalent to have the same probability of success.

The role of unbound drug in pharmacokinetics/pharmacodynamics and in therapy.

The evolution of research on drug protein binding is discussed with the unbound concentration (Cu) and the unbound fraction (fu) as protagonists. Particular attention is paid to the mechanisms via which alterations in binding affect the pharmacokinetics (PK) and the effect, or independently the pharmacodynamics (PD). Apart from albumin, the important alpha-acid glycoprotein (AGP), as well as specific drug classes and applications in the clinic and development (routine monitoring, cancer and HIV therapy, allometry) are addressed. The flaws with the classical method of indirectly calculating the Cu or the unbound PK/PD parameters, based on the fu in vitro, are related to the intrinsic complexity and variability in the outcomes. Increased focus is urged on directly estimating the unbound PK/PD and also on using population statistical methods.

Sex specificity in methadone analgesia in the rat: a population pharmacokinetic and pharmacodynamic approach.

AbstractPurpose. To quantify the extent to which a sex-specific dichotomy in the temporal evolution of the analgesic effect, after intravenous (i.v.) methadone injection in the rat, relates to the pharmacokinetics (PK) and pharmacodynamics (PD) that mediate the dose-to-effect pathway. Methods. Tail-flick analgesia was measured after i.v. methadone injection (0.35 mg/kg) in female (n = 16) and male (n = 16) Sprague-Dawley rats. The PK were evaluated in separate female (n = 56) and male (n = 56) rats after they had received the same dose of methadone i.v. (0.35 mg/kg). A bicompartmental model described the kinetics and a sigmoid Emax model-related drug effect vs. simulated concentrations (pharmacodynamics) at the times of effect measurement. All model parameters as well as interanimal and assay variabilities were estimated with a mixed-effects population method using the program NONMEM. Results. The area under the effect-time curve (AUCE0-120) was (mean ± interanimal SD) 1859 ± 346 min in the females, which was significantly lower than the 4871 ± 393 min in the males (P < 0.0001). On the contrary, the profiles of concentration vs. time were higher in females and, therefore, corresponded inversely to the effect vs. time-relative magnitudes. The central volume of distribution, V1, was 1.94 ± 0.37 l/kg for female rats and 3.01 ± 0.33 l/kg for male rats. Also, the central clearance was 0.077 ± 0.006 l/min/kg and 0.102 ± 0.005 l/min/kg, respectively, for female and male rats. Both parameters differed significantly between sexes (P < 0.0001). The pharmacodynamic maximum observed effect parameter (Emax) was 37% ± 29% in female rats and 85% ± 16% in male rats, and these values were significantly different (P < 0.0001). The parameter for the concentration eliciting half of Emax (EC50) was 24.1 ± 7.5 μg/l in female rats and 20.3 ± 2.9 μg/l in male rats, and the Hill-related exponent, γ, was 6.3 ± 3.9 in female rats and 5.5 ± 4.1 in male rats. These parameters did not differ significantly (at the P < 0.05 level). Conclusions. A sex-specific dichotomy in the methadone antinociceptive effect, in the rat, was not proportionally related to plasma concentrations. Each sex corresponded to a distinct subpopulation of the PK parameters and one of the pharmacodynamic parameters (Emax). When the course of a drug involves PK or PD subpopulations, PK/PD modeling can afford the safest prediction of the effect-time evolution for a particular dose.

Altered dose-to-effect of propofol due to pharmacokinetics in rats with experimental diabetes mellitus.

Pathology related alterations in the pharmacokinetics or the pharmacodynamics of propofol could contribute to the observed large variability in the hypnotic dose. We have tested the influence of diabetes mellitus on the induction dose and the pharmacokinetics and pharmacodynamics of propofol in rats. Diabetes was induced in rats by administration of streptozotocin (60 mg kg−1, i.p.) while control rats received vehicle intraperitoneally. All animals had glucose, cholesterol, triglycerides and albumin levels measured. In‐vitro protein binding was determined by ultrafiltration. Rats were randomly split into set 1 (dose‐concentration‐effect study) with control and streptozotocin rats, and set 2 (pharmacokinetic study), with control and streptozotocin rats. Rats in the effect set received either a variable infusion of 6 mg kg−1 min−1 propofol until onset (induction dose) of the hypnotic effect (loss of the righting reflex), or a 15 mg kg−1 bolus to assess offset time (recovery of the righting reflex). Blood (Cblood) and brain (Cbrain) propofol concentrations at onset and offset were assayed by HPLC. In the pharmacokinetic study, propofol was administered intravenously at 6 mg kg−1 min−1 for 2 min. Arterial blood samples were collected between 0.5 and 540 min and assayed for propofol. A mixed effects compartmental pharmacokinetic modelling method (NONMEM) was used to analyse the observations and variabilities. The dose necessary for onset differed between streptozotocin and controls, and so did the pharmacokinetics with two‐ and three‐compartment descriptions, respectively. Cblood and Cbrain at onset and offset were similar, possibly rejecting changes in pharmacodynamics. The total and unbound volume of distribution was significantly lower in the streptozotocin group with no differences in clearance (CL) between streptozotocin and controls, (mean (inter‐animal CV%)) CL = 0.026 (17%) and 0.025 (62%) L min−1, respectively. Individual Bayes Vdss (volume of distribution at steady state) were different, (mean (s.d.)) Vdss = 7.7 (2.67) and 1.11 (0.09) L, respectively. The pharmacokinetic model was validated by comparison with the data from set 1. Simulations of total and unbound Cblood, for both groups, at the hypnotic dose for the controls, revealed differences throughout the time course of the pharmacokinetics. The difference observed in the induction dose of propofol to streptozotocin and control rats was due to alterations in the pharmacokinetics, secondary to the pathology.

Prediction of unbound Propofol concentrations in a diabetic population

Propofol is a short-acting general intravenous anesthetic characterized by a wide interindividual variability in the response after the same dose. Its binding to serum proteins exceeds 98%, so small changes in protein concentrations can be amplified in the unbound fraction of the drug and hence possibly in the effect. It is then likely that part of the variability in the response could be attributed to differences in protein levels among individuals and particularly among those with pathologies such as diabetes. The aim of this study was to establish predictive regression models in a diabetes mellitus (DM) population between unbound:bound propofol ratios and demographic and biochemical indices. Unbound:bound propofol ratios can be routinely obtained in the clinic as opposed to the free fraction of the drug. In DM patients (30 women and 37 men aged between 17 and 78 y) with mellitus type 1 (n = 37) and type 2 (n = 30) diabetes, the authors measured the lipoproteins (HDL, LDL, and VLDL), cholesterol, triglycerides, albumin, &agr;1-acid glycoprotein (AAG), free fatty acids (FFA), glycosylated hemoglobin, and the unbound fraction (Fu) and the bound/free ratio (B/F) of propofol. A linearized regression model between the above variables—as well as age, sex, and type of diabetes—and Fu was then developed. Patients had blood drawn and sera separated by centrifugation and spiked with propofol to a concentration of 10 &mgr;g/mL. The Fu was determined via ultrafiltration. Multiple linear regression analysis was used to identify significant predictor variables of Fu in this population and two models were originated: one with lipoprotein serum concentrations as explanatory variables (Model A) and another that depended on cholesterol and triglycerides (Model B). Both models presented high correlation coefficients (r2 = 0.71 and 0.68, respectively;P < 0.0001), and each was used to predict Fu in an independent group of 15 DM patients of similar characteristics and biochemical indices as the model development group. Bias and precision were for Model A, 0.9% and 7.8%, and for Model B, 3.0% and 8.7%, respectively. Both models were compared with each other and to a naive predictor (the mean) and each was better than the naive model in predicting the unbound fraction of propofol. Model A and model B could be used in estimating Fu of propofol in DM patients based on the more routine clinical measures of lipoprotein serum concentrations or cholesterol and triglyceride levels.

Pharmacokinetics of Teicoplanin in An ICU Population of Children and Infants

AbstractPurpose. Better dosing is needed for antibiotics, including teicoplanin (TEI), to prevent emergence of resistant bacterial strains. Here, we assess the TEI pharmacokinetics (PK) related to a 10 mg/l minimum inhibitory concentration (MIC) target in ICU children (4 to 120 months; n = 20) with gram+ infections. Methods. Standard administration of TEI was with three 10 mg/kg Q12h, loading infusions, and maintainance with 10 mg/kg or 15 mg/kg Q24h. During maintenance, 9 samples (3/day) were collected per patient and the PK analyzed with Nonlinear Mixed Effects Model (NONMEM). Results. Thirty-five percent of concentrations in older children (≥2 months) vs. 8% in younger infants (<12 months) were below the target MIC. The global bicompartmental population PK parameters were [mean (interindividual CV%)] CL = 0.23 l/h [72%], V = 3.16 l [58%], k12 = 0.23 h−1, and k21 = 0.04 h−1. Two PK subpopulations were identified. The older children had CL = 0.29 [23%] l/h, V = 3.9 l and the younger infants, CL = 0.09 [37%] l/h, V = 1.05 l. Residual error was reduced from 52% to around 30% in the final models. Conclusions. Older children in the ICU may require relatively higher doses of teicoplanin. However, a study in a larger population is needed.

Evaluation of Fentanyl Disposition and Effects in Newborn Piglets as an Experimental Model for Human Neonates

Background Fentanyl is widely used off-label in NICU. Our aim was to investigate its cerebral, cardiovascular and pulmonary effects as well as pharmacokinetics in an experimental model for neonates. Methods Fentanyl (5 µg/kg bolus immediately followed by a 90 minute infusion of 3 µg/kg/h) was administered to six mechanically ventilated newborn piglets. Cardiovascular, ventilation, pulmonary and oxygenation indexes as well as brain activity were monitored from T = 0 up to the end of experiments (T = 225–300 min). Also plasma samples for quantification of fentanyl were drawn. Results A “reliable degree of sedation” was observed up to T = 210–240 min, consistent with the selected dosing regimen and the observed fentanyl plasma levels. Unlike cardiovascular parameters, which were unmodified except for an increasing trend in heart rate, some of the ventilation and oxygenation indexes as well as brain activity were significantly altered. The pulmonary and brain effects of fentanyl were mostly recovered from T = 210 min to the end of experiment. Conclusion The newborn piglet was shown to be a suitable experimental model for studying fentanyl disposition as well as respiratory and cardiovascular effects in human neonates. Therefore, it could be extremely useful for further investigating the drug behaviour under pathophysiological conditions.