Determination of Tobramycin in M9 Medium by LC-MS/MS: Signal Enhancement by Trichloroacetic Acid.

It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods. Here, we report that trichloroacetic acid increases the MS signal of tobramycin. To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed. Aliquots of 25 µL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid. The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode. ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification. The calibration curve concentration range was 50-25000 ng/mL. Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold). The method is simple, fast, and reliable. Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.

Determination of Free 25(OH)D Concentrations and Their Relationships to Total 25(OH)D in Multiple Clinical Populations.

Context: The optimal measure of vitamin D status is unknown. Objective: To directly measure circulating free 25-hydroxyvitamin D [25(OH)D] concentrations and relationships to total 25(OH)D in a clinically diverse sample of humans. Design: Cross-sectional analysis. Setting: Seven academic sites. Patients: A total of 1661 adults: healthy (n = 279), prediabetic (n = 479), outpatients (n = 714), cirrhotic (n = 90), pregnant (n = 20), nursing home resident (n = 79). Interventions: Merge research data on circulating free 25(OH)D (directly-measured immunoassay), total 25(OH)D (liquid chromatography/tandem mass spectrometry), D-binding protein [DBP; by radial (polyclonal) immunodiffusion assay], albumin, creatinine, intact parathyroid hormone, and DBP haplotype. Main outcome measures: Distribution of free 25(OH)D (ANOVA with Bonferroni correction for post hoc comparisons) and relationships between free and total 25(OH)D (mixed-effects modeling incorporating clinical condition, DBP haplotype with sex, race, estimated glomerular filtration rate (eGFR), body mass index (BMI), and other covariates). Results: Free 25(OH)D was 4.7 ± 1.8 pg/mL (mean ± SD) in healthy persons and 4.3 ± 1.9 pg/mL in outpatients, with levels of 0.5 to 8.1 pg/mL and 0.9 to 8.1 pg/mL encompassing 95% of healthy persons and outpatients, respectively. Free 25(OH)D was higher in patients with cirrhosis (7.1 ± 3.0 pg/mL; P < 0.0033) and nursing home residents (7.9 ± 2.1 pg/mL; P < 0.0033) than in other groups and differed between whites and blacks (P < 0.0033) and between DBP haplotypes (P < 0.0001). Mixed-effects modeling of relationships between free and total 25(OH)D identified clinical conditions (patients with cirrhosis > nursing home residents > patients with prediabetes > outpatients > pregnant women) and BMI (lesser effect) as covariates affecting relationships but not eGFR, sex, race, or DBP haplotype. Conclusions: Total 25(OH)D, health condition, race, and DBP haplotype affected free 25(OH)D, but only health conditions and BMI affected relationships between total and free 25(OH)D. Clinical importance of free 25(OH)D needs to be established in studies assessing outcomes.

Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms.

OBJECTIVES: The selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model. METHODS: Pharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7. RESULTS: Meropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone. CONCLUSIONS: Simulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

Mathematical Modeling of Biofilm Structures Using COMSTAT Data.

Mathematical modeling holds great potential for quantitatively describing biofilm growth in presence or absence of chemical agents used to limit or promote biofilm growth. In this paper, we describe a general mathematical/statistical framework that allows for the characterization of complex data in terms of few parameters and the capability to (i) compare different experiments and exposures to different agents, (ii) test different hypotheses regarding biofilm growth and interaction with different agents, and (iii) simulate arbitrary administrations of agents. The mathematical framework is divided to submodels characterizing biofilm, including new models characterizing live biofilm growth and dead cell accumulation; the interaction with agents inhibiting or stimulating growth; the kinetics of the agents. The statistical framework can take into account measurement and interexperiment variation. We demonstrate the application of (some of) the models using confocal microscopy data obtained using the computer program COMSTAT.

New in vitro model to study the effect of human simulated antibiotic concentrations on bacterial biofilms.

A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h(-1)) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

Determination of meropenem in bacterial media by LC-MS/MS.

To support the development of a dynamic in vitro human pharmacokinetic/pharmacodynamic simulation model for biofilm-mediated infections and study stability of meropenem, an LC-MS/MS method for the determination of meropenem in Luria Bertani (LB) media was developed and validated in an API2000 LC-MS/MS system. A partial validation was also performed in M9 media. Sample aliquots of 100µL (or 25µL for M9 media) were mixed with the internal standard (IS) ceftazidime and filtered. The filtrate was directly injected onto a C8 column eluted with ammonium formate (10mM, pH 4) and acetonitrile (0.1% formic acid) in a gradient mode. ESI(+) and MRM with ion pair m/z 384→68 for meropenem and m/z 547→468 for the IS were used for quantification. The calibration curve concentration range was 50 to 25,000ng/mL. The recovery was over 98%. In LB media, significant signal suppression was observed throughout the time period of detection when compared with mobile phase solvents, but the matrix effect was compensated well with the IS. In M9 media, much less signal suppression was observed. The method is simple, fast, and reliable. Using the method, stability of meropenem in LB and M9 media were tested. No significant degradation was observed for at least 8h in both LB media (37°C) and M9 media (30°C), but more than 15% degradation was observed overnight (∼20h). The method was transferred to an API5000 LC-MS/MS system using meropenem-d6 as the IS.

Gentamicin pharmacokinetics and dosing in neonates with hypoxic ischemic encephalopathy receiving hypothermia.

STUDY OBJECTIVE: To evaluate the pharmacokinetics of gentamicin in neonates with hypoxic ischemic encephalopathy (HIE) receiving hypothermia and to identify an empiric gentamicin dosing strategy in this population that optimizes achievement of target peak and trough concentrations. DESIGN: Population pharmacokinetic study using retrospective medical record data. SETTING: Tertiary neonatal intensive care unit. PATIENTS: A total of 29 full-term neonates diagnosed with HIE treated with hypothermia who received gentamicin and underwent therapeutic drug monitoring MEASUREMENT AND MAIN RESULTS: Patient demographics and gentamicin concentration data were retrospectively collected over a 2-year period. A population-based pharmacokinetic model was developed using nonlinear mixed-effects modeling (NONMEM). Using the developed model, Monte Carlo simulations were performed to evaluate the probability of achieving target peak (> 6 mg/L) and trough (< 2 mg/L) gentamicin concentrations for various potential dosing regimens. A one-compartment model best described the available gentamicin concentration data. Birthweight and serum creatinine significantly influenced gentamicin clearance. For the typical study neonate (birthweight 3.3 kg, serum creatinine 0.9 mg/dl), clearance was 0.034 L/hour/kg and volume was 0.52 L/kg. At a 24-hour dosing interval, Monte Carlo simulations predicted target gentamicin peak and trough concentrations could not be reliably achieved at any dose. At a 36-hour dosing interval, a dose of 4-5 mg/kg is predicted to achieve target gentamicin peak and trough concentrations in more than 90% of neonates. CONCLUSIONS: Gentamicin clearance is decreased in neonates with HIE treated with hypothermia compared with previous reports in nonasphyxiated normothermic full-term neonates. A prolonged 36-hour dosing interval will be needed to achieve target gentamicin trough concentrations in this population. Further prospective evaluation of this dosing recommendation is needed.

Population pharmacokinetics of unbound mycophenolic acid in adult allogeneic haematopoietic cell transplantation: effect of pharmacogenetic factors.

AIM: To evaluate pharmacogenetic factors as contributors to the variability of unbound mycophenolic acid (MPA) exposure in adult allogeneic haematopoietic cell transplantation (alloHCT) recipients. METHODS: A population-based pharmacokinetic (PK) model of unbound MPA was developed using non-linear mixed-effects modelling (nonmem). Previously collected intensive unbound MPA PK data from 132 adult alloHCT recipients after oral and intravenous dosing of the prodrug mycophenolate mofetil (MMF) were used. In addition to clinical covariates, genetic polymorphisms in UGT1A8, UGT1A9, UGT2B7 and MRP2 were evaluated for their impact on unbound MPA PK. RESULTS: Unbound MPA concentration-time data were well described by a two compartment model with first order absorption and linear elimination. For the typical patient (52 years of age, creatinine clearance 86 ml min(-1)), the median estimated values [coefficient of variation, %, (CV)] of systemic clearance, intercompartmental clearance, central and peripheral volumes of MPA were 1610 l h(-1) (37.4%), 541 l h(-1) (75.6%), 1230 l (37.5%), and 6140 l (120%), respectively. After oral dosing, bioavailability was low (0.56) and highly variable (CV 46%). No genetic polymorphisms tested significantly explained the variability among individuals. Creatinine clearance was a small but significant predictor of unbound MPA CL. No other clinical covariates impacted unbound MPA PK. CONCLUSIONS: In adult alloHCT recipients, variability in unbound MPA AUC was large and remained largely unexplained even with the inclusion of pharmacogenetic information. Targeting unbound MPA AUC in a patient will require therapeutic drug monitoring.

Nevirapine pharmacokinetics and risk of rash and hepatitis among HIV-infected sub-Saharan African women.

OBJECTIVES: To estimate nevirapine (NVP) pharmacokinetics and examine its association with rash and/or hepatotoxicity in women starting antiretroviral treatment in the AIDS Clinical Trials Group A5208/OCTANE study in Africa. DESIGN: In HIV-infected, nonpregnant women with screening CD4 cell count less than 200 cells/µl randomized to NVP (twice daily, after 14-day once-daily lead-in period) and tenofovir/emtricitabine, single NVP blood samples were collected 14 and 28 days following randomization. Rash and hepatotoxicity that occurred during therapy, or within 7 days after the last dose of NVP, were defined as toxicity. METHODS: NVP pharmacokinetics were modeled by population pharmacokinetic analysis. Individual Bayesian pharmacokinetic estimates were used to calculate clearance, 24-h area under the curve, and predicted plasma concentrations. RESULTS: Median week 4 NVP clearance was 2 l/h. Among the 359 women, 194 (54%) developed a rash of any grade; 82 (23%) had grade 2+ and nine (3%) had grade 3+ rash. Median clearance was 1.7 l/h for participants exhibiting 3+ rash versus 2 l/h in women without 3+ rash (P = 0.046). The odds of developing 3+ rash was 50% higher for every 20% decrease in clearance (P = 0.046). NVP discontinuation due to rash/liver toxicity was significantly more common among women with pretreatment CD4 cell count more than 250 cells/µl (P = 0.003). CONCLUSION: In this study, HIV-infected African women starting a NVP-based antiretroviral regimen had a lower NVP clearance compared to previous reports. Severe rash, but not hepatotoxicity, was associated with higher NVP exposure. Albeit observed in a small number of women, baseline CD4 cell count at least 250 cells/µl was significantly associated with NVP toxicity.

A method to quantify illicit intake of drugs from urine: methamphetamine.

Qualitative urinalysis can verify abstinence of drug misuse but cannot detect changes in drug intake. For drugs with slow elimination, such as methamphetamine (MA), a single episode of abuse can result in up to 5 days of positive urine drug screens. Thus, interventions that produce substantial decreases in drug use but do not achieve almost complete abstinence are classified as ineffective. Using nonpharmacologic doses of deuterium-labeled l-methamphetamine (l-MA-d(3)) we have developed a simple, robust method that reliably estimates changes in MA intake. Twelve subjects were dosed with 5 mg of l-MA-d(3) daily and challenged with 15, 30, and 45 mg of nonlabeled d-MA (d-MA-d(0)) after reaching plasma steady status of l-MA-d(3). Urinary concentration ratios of d-MA-d(0) to l-MA-d(3) provided clear separation of the administered doses with as little as 15-mg dose increments. Administered doses could not be resolved using d-MA-d(0) concentrations alone. In conclusion, the urinary [d-MA-d(0)]:[l-MA-d(3)] provides a quantitative, continuous measure of illicit MA exposure. The method reliably detects small, clinically relevant changes in illicit MA intake from random urine specimens, is amenable to deployment in clinical trials, and can be used to quantify patterns of MA abuse.

Fractional dynamics pharmacokinetics-pharmacodynamic models.

While an increasing number of fractional order integrals and differential equations applications have been reported in the physics, signal processing, engineering and bioengineering literatures, little attention has been paid to this class of models in the pharmacokinetics-pharmacodynamic (PKPD) literature. One of the reasons is computational: while the analytical solution of fractional differential equations is available in special cases, it this turns out that even the simplest PKPD models that can be constructed using fractional calculus do not allow an analytical solution. In this paper, we first introduce new families of PKPD models incorporating fractional order integrals and differential equations, and, second, exemplify and investigate their qualitative behavior. The families represent extensions of frequently used PK link and PD direct and indirect action models, using the tools of fractional calculus. In addition the PD models can be a function of a variable, the active drug, which can smoothly transition from concentration to exposure, to hyper-exposure, according to a fractional integral transformation. To investigate the behavior of the models we propose, we implement numerical algorithms for fractional integration and for the numerical solution of a system of fractional differential equations. For simplicity, in our investigation we concentrate on the pharmacodynamic side of the models, assuming standard (integer order) pharmacokinetics.

Fractional compartmental models and multi-term Mittag-Leffler response functions.

Systems of fractional differential equations (SFDE) have been increasingly used to represent physical and control system, and have been recently proposed for use in pharmacokinetics (PK) by (J Pharmacokinet Pharmacodyn 36:165-178, 2009) and (J Phamacokinet Pharmacodyn, 2010). We contribute to the development of a theory for the use of SFDE in PK by, first, further clarifying the nature of systems of FDE, and in particular point out the distinction and properties of commensurate versus non-commensurate ones. The second purpose is to show that for both types of systems, relatively simple response functions can be derived which satisfy the requirements to represent single-input/single-output PK experiments. The response functions are composed of sums of single- (for commensurate) or two-parameters (for non-commensurate) Mittag-Leffler functions, and establish a direct correspondence with the familiar sums of exponentials used in PK.

Oral heroin in opioid-dependent patients: pharmacokinetic comparison of immediate and extended release tablets.

In diacetylmorphine prescription programs for heavily dependent addicts, diacetylmorphine is usually administered intravenously, but this may not be possible due to venosclerosis or when heroin abuse had occurred via non-intravenous routes. Since up to 25% of patients administer diacetylmorphine orally, we characterised morphine absorption after single oral doses of immediate and extended release diacetylmorphine in 8 opioid addicts. Plasma concentrations were determined by liquid chromatography-mass spectrometry. Non-compartmental methods and deconvolution were applied for data analysis. Mean (+/-S.D.) immediate and extended release doses were 719+/-297 and 956+/-404 mg, with high absolute morphine bioavailabilities of 56-61%, respectively. Immediate release diacetylmorphine caused rapid morphine absorption, peaking at 10-15 min. Morphine absorption was considerably slower and more sustained for extended release diacetylmorphine, with only approximately 30% of maximal immediate release absorption being reached after 10 min and maintained for 3-4h, with no relevant food interaction. The relative extended to immediate release bioavailability was calculated to be 86% by non-compartmental analysis and 93% by deconvolution analysis. Thus, immediate and extended release diacetylmorphine produce the intended morphine exposures. Both are suitable for substitution treatments. Similar doses can be applied if used in combination or sequentially.

A mechanistic model for the sex-specific response to nalbuphine and naloxone in postoperative pain.

We develop a mechanistic model for post-operative pain and apply it to describe the pharmacodynamic effects of the kappa-opioids nalbuphine and naloxone administered either alone or in combination in patients after surgical removal of one or more madibular third molar teeth. Data were obtained from 6 clinical studies in which a total of 304 patients were intravenously administered single doses of 2.5, 5, 10 or 20 mg of nalbuphine. Some groups also received 0.2 or 0.4 mg of naloxone. A total of 3,040 Visual analog scale (VAS) pain ratings were recorded at 20 min intervals for 3 h after drug administration. We used a two-site indirect action model to describe early and late pain and to incorporate the effect of nalbuphine and naloxone on pain over time. A mixed effects statistical model was used to account for inter- and intra-individual variability. Our model estimated the population average baseline pain score in men to be lower than that in women (68 vs. 76 mm on the 100 mm VAS scale). The model confirmed a late increase in pain (anti-analgesia) and estimated the lag time for the start of anti-analgesia to be 73 min after study drug administration. The maximum early phase pain score is 81.6 mm while the maximum anti-analgesia is 16.1 mm. The nalbuphine dose required to reduce early pain by 50% (ED(50)) was estimated to be 5.85 mg and the naloxone dose required to reduce late phase pain by 50% was estimated to be 0.5 mg. The model confirmed the results from conventional statistical analyses performed previously on individual studies.

Reversible jump Markov chain Monte Carlo for deconvolution.

To solve the problem of estimating an unknown input function to a linear time invariant system we propose an adaptive non-parametric method based on reversible jump Markov chain Monte Carlo (RJMCMC). We use piecewise polynomial functions (splines) to represent the input function. The RJMCMC algorithm allows the exploration of a large space of competing models, in our case the collection of splines corresponding to alternative positions of breakpoints, and it is based on the specification of transition probabilities between the models. RJMCMC determines: the number and the position of the breakpoints, and the coefficients determining the shape of the spline, as well as the corresponding posterior distribution of breakpoints, number of breakpoints, coefficients and arbitrary statistics of interest associated with the estimation problem. Simulation studies show that the RJMCMC method can obtain accurate reconstructions of complex input functions, and obtains better results compared with standard non-parametric deconvolution methods. Applications to real data are also reported.

Improving data reliability using a non-compliance detection method versus using pharmacokinetic criteria.

Data from clinical trials present numerous problems for the data analyst. These include non-compliance with the prescribed dosing regimen and inaccurate recollection of dosing history by patients as well as mistakes in recording data. Several methods have been proposed to address these issues. One such technique by Lu et al. (Selecting reliable pharmacokinetic data for explanatory analyses of clinical trials in the presence of possible noncompliance. J. Pharmacokinet. Pharmacodyn. 28:343-362 (2001)) identifies occasions in pharmacokinetic (PK) data where the preceding dosing history is likely to be unreliable. We used this method, implemented in the software program NONMEM (beta) VI, to clean a dataset containing indinavir (IDV) plasma concentrations from HIV-1 infected patients. The data was also cleaned by inspection in Microsoft Excel using clinical PK criteria. A one-compartment model with first order absorption and elimination was fit to both sets of cleaned data. IDV population PK parameters obtained from these analyses were similar to those reported previously. It is established that IDV nephrotoxicity is related to high IDV exposure. However, no relationships were found between any PK parameters and nephrotoxicity in the "compliance cleaned" dataset. In the "PK cleaned" dataset, the oral clearance and apparent volume were lower by 9.1% and 6.6%, respectively in patients with any type of nephrotoxicity and the maximum IDV concentration (C(max)) was 12.1% higher. In patients suffering from nephrolithiasis in particular, C(max) was 15.5% higher. Accordingly, the use of the non-compliance detection method did not improve the reliability of our dataset over the usual method of applying clinical criteria. In fact, analyses on the compliance-cleaned dataset missed some exposure-toxicity relationships. Thus, automated methods must be tested rigorously with 'real life' datasets, used with caution, and always in conjunction with clinical reasoning to avoid overlooking a signal in noisy data.

A non-linear mixed effect dynamic model incorporating prior exposure and adherence to treatment to describe long-term therapy outcome in HIV-patients.

Long term therapy with antiretroviral agents in HIV-infected patients often result in failure to suppress the virus load. Imperfect adherence to prescribed antiviral drugs is an important factor explaining the resurgence of virus. A better understanding of the factors responsible for the virological failure is important for the development of new treatment strategies. Many complex non-linear models have been developed to describe and simulate the dynamics of HIV-1 virus. Those complicated viral dynamic models have not been used in clinical trials to estimate HIV dynamics parameters, due to their complexity, until the recent development of simplification and approximation techniques. The estimation of the parameters associated with the dynamics from real data has been mostly limited to linearized models that can only explain the decay (suppression) of the virus following antiviral treatment. Moreover, no complete characterization of typical clinical data in terms of inter-subject variability and identification of important covariates effecting HIV-1 dynamics has been attempted. The objective of our paper was to develop a hierarchical non-linear mixed effect model characterizing inter-subject variability in the long-term response to treatment of HIV-1 RNA, and show how the model can be used to quantify the effect of important covariates, such as physiological variables, adherence to treatment or previous exposure to treatment, on the dynamics of HIV-1 RNA. As an example we report the analysis of AIDS clinical trial data from AACTG 398, which shows that patients with previous exposure to treatment show faster death rates for HIV-1, and that higher adherence to treatment is associated with lower reproductive ratio.

Population analyses of amlodipine in patients living in the community and patients living in nursing homes.

OBJECTIVE: Our objective was to determine the effects of age, sex, and morbidity on the apparent oral clearance (CL/F) of amlodipine. METHODS: Population pharmacokinetic analyses were performed on data from 211 patients receiving oral racemic amlodipine (dose of 7.2 +/- 3.6 mg/d [mean +/- SD]) on a long-term basis. Of the patients, 105 were men, with a mean age of 72 +/- 13 years and lean body weight (LBW) of 60.7 +/- 7.6 kg, and 106 were women, with a mean age of 79 +/- 11 years and LBW of 44.2 +/- 6.0 kg; 119 lived in the community, 20 in assisted living facilities, and 72 in nursing homes. Amlodipine was measured by liquid chromatography-tandem mass spectrometry. Population analyses were performed by use of NONMEM with sex, age, race, living site, alcohol intake, and concomitant medications considered as covariates. The significance of covariates was determined by likelihood ratio tests. RESULTS: Female sex and living in a nursing home were associated with a faster CL/F compared with men and community-dwelling patients, respectively. The mean CL/F was 7.83 +/- 0.50 mL.min(-1).kg(-1) (LBW) in women compared with 6.31 +/- 1.01 in men and 8.68 +/- 1.00 mL.min(-1).kg(-1) in nursing home residents compared with 6.32 +/- 1.17 in community-dwelling patients. Increasing age was associated with decreasing CL/F only in community-dwelling patients and residents of assisted living facilities. CONCLUSIONS: In middle-aged and very old (>80 years) patients, amlodipine CL/F was faster in women compared with men and was faster in nursing home residents compared with community-dwelling patients, with increasing age decreasing CL/F only in community-dwelling patients and residents of assisted-living facilities.

Pharmacokinetic-pharmacodynamic modelling: history and perspectives.

A major goal in clinical pharmacology is the quantitative prediction of drug effects. The field of pharmacokinetic-pharmacodynamic (PK/PD) modelling has made many advances from the basic concept of the dose-response relationship to extended mechanism-based models. The purpose of this article is to review, from a historical perspective, the progression of the modelling of the concentration-response relationship from the first classic models developed in the mid-1960s to some of the more sophisticated current approaches. The emphasis is on general models describing key PD relationships, such as: simple models relating drug dose or concentration in plasma to effect, biophase distribution models and in particular effect compartment models, models for indirect mechanism of action that involve primarily the modulation of endogenous factors, models for cell trafficking and transduction systems. We show the evolution of tolerance and time-variant models, non- and semi-parametric models, and briefly discuss population PK/PD modelling, together with some example of more recent and complex pharmacodynamic models for control system and nonlinear HIV-1 dynamics. We also discuss some future possible directions for PK/PD modelling, report equations for general classes of novel semi-parametric models, as well as describing two new classes, additive or set-point, of regulatory, additive feedback models in their direct and indirect action variants.

Sample size computations for PK/PD population models.

We describe an accurate, yet simple and fast sample size computation method for hypothesis testing in population PK/PD studies. We use a first order approximation to the nonlinear mixed effects model and chi-square distributed Wald statistic to compute the minimum sample size to achieve given degree of power in rejecting a null hypothesis in population PK/PD studies. The method is an extension of Rochon's sample size computation method for repeated measurement experiments. We compute sample sizes for PK and PK/PD models with different conditions, and use Monte Carlo simulation to show that the computed sample size retrieves the required power. We also show the effect of different sampling strategies, such as minimal, i.e., as many observations per individual as parameters in the model, and intensive on sample size. The proposed sample size computation method can produce estimates of minimum sample size to achieve the desired power in hypothesis testing in a greatly reduced time than currently available simulation-based methods. The method is rapid and efficient for sample size computation in population PK/PD study using nonlinear mixed effect models. The method is general and can accommodate any type of hierarchical models. Simulation results suggest that intensive sampling allows the reduction of the number of patients enrolled in a clinical study.