Model-Informed Precision Dosing: Background, Requirements, Validation, Implementation, and Forward Trajectory of Individualizing Drug Therapy.

Model-informed precision dosing (MIPD) has become synonymous with modern approaches for individualizing drug therapy, in which the characteristics of each patient are considered as opposed to applying a one-size-fits-all alternative. This review provides a brief account of the current knowledge, practices, and opinions on MIPD while defining an achievable vision for MIPD in clinical care based on available evidence. We begin with a historical perspective on variability in dose requirements and then discuss technical aspects of MIPD, including the need for clinical decision support tools, practical validation, and implementation of MIPD in health care. We also discuss novel ways to characterize patient variability beyond the common perceptions of genetic control. Finally, we address current debates on MIPD from the perspectives of the new drug development, health economics, and drug regulations.

Simulation-Based Optimization of Sampling Schedules for Model-Informed Precision Dosing of Once-Daily and 4-Times-Daily Busulfan in Pediatric Patients.

BACKGROUND: Therapeutic drug monitoring (TDM) is crucial in optimizing the outcomes of hematopoietic stem cell transplantation by guiding busulfan (Bu) dosing. Limited sampling strategies show promise for efficiently adjusting drug doses. However, comprehensive assessments and optimization of sampling schedules for Bu TDM in pediatric patients are limited. We aimed to establish optimal sampling designs for model-informed precision dosing (MIPD) of once-daily (q24h) and 4-times-daily (q6h) Bu administration in pediatric patients. METHODS: Simulated data sets were used to evaluate the population pharmacokinetic model-based Bayesian estimation of the area under the concentration-time curve (AUC) for different limited sampling strategy designs. The evaluation was based on the mean prediction error for accuracy and root mean square error for precision. These findings were validated using patient-observed data. In addition, the MIPD protocol was implemented in the Tucuxi software, and its performance was assessed. RESULTS: Our Bayesian estimation approach allowed for flexible sampling times while maintaining mean prediction error within ±5% and root mean square error below 10%. Accurate and precise AUC0-24h and cumulative AUC estimations were obtained using 2-sample and single-sample schedules for q6h and q24h dosing, respectively. TDM on 2 separate days was necessary to accurately estimate cumulative exposure, especially in patients receiving q6h Bu. Validation with observed patient data confirmed the precision of the proposed limited sampling scenarios. Implementing the MIPD protocol in Tucuxi software yielded reliable AUC estimations. CONCLUSIONS: Our study successfully established precise limited sampling protocols for MIPD of Bu in pediatric patients. Our findings underscore the importance of TDM on at least 2 occasions to accurately achieve desired Bu exposures. The developed MIPD protocol and its implementation in Tucuxi software provide a valuable tool for routine TDM in pediatric hematopoietic stem cell transplantation.

Deep proteomics and phosphoproteomics reveal novel biological pathways perturbed by morphine, morphine-3-glucuronide and morphine-6-glucuronide in human astrocytes.

Tolerance and hyperalgesia associated with chronic exposure to morphine are major limitations in the clinical management of chronic pain. At a cellular level, neuronal signaling can in part account for these undesired side effects, but unknown mechanisms mediated by central nervous system glial cells are likely also involved. Here we applied data-independent acquisition mass spectrometry to perform a deep proteome and phosphoproteome analysis of how human astrocytes responds to opioid stimulation. We unveil time- and dose-dependent effects induced by morphine and its major active metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide that converging on activation of mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. We also find that especially longer exposure to M3G leads to significant dysregulation of biological pathways linked to extracellular matrix organization, antigen presentation, cell adhesion, and glutamate homeostasis, which are crucial for neuron- and leukocyte-astrocyte interactions.

Impact of enantiomer-specific changes in pharmacokinetics between infants and adults on the target concentration of racemic ketorolac: A pooled analysis.

AIMS: Ketorolac is a nonsteroidal anti-inflammatory racemic drug with analgesic effects only attributed to its S-enantiomer. The aim of this study is to quantify enantiomer-specific maturational pharmacokinetics (PK) of ketorolac and investigate if the contribution of both enantiomers to the total ketorolac concentration remains equal between infants and adults or if a change in target racemic concentration should be considered when applied to infants. METHODS: Data were pooled from 5 different studies in adults, children and infants, with 1020 plasma concentrations following single intravenous ketorolac administration. An allometry-based enantiomer-specific population PK model was developed with NONMEM 7.3. Simulations were performed in typical adults and infants to investigate differences in S- and R-ketorolac exposure. RESULTS: S- and R-ketorolac PK were best described with a 3- and a 2-compartment model, respectively. The allometry-based PK parameters accounted for changes between populations. No maturation function of ketorolac clearance could be identified. All model parameters were estimated with adequate precision (relative standard error <50%). Single dose simulations showed that a previously established analgesic concentration at half maximal effect in adults of 0.37 mg/L, had a mean S-ketorolac concentration of 0.057 mg/L, but a mean S-ketorolac concentration of 0.046 mg/L in infants. To match the effective adult S-ketorolac-concentration (0.057 mg/L) in typical infants, the EC50-racemic should be increased to 0.41 mg/L. CONCLUSION: Enantiomer-specific changes in ketorolac PK yield different concentrations and S- and R-ketorolac ratios between infants and adults at identical racemic concentrations. These PK findings should be considered when studies on maturational pharmacodynamics are considered.

Toward precision medicine in pediatric population using cytochrome P450 phenotyping approaches and physiologically based pharmacokinetic modeling.

The activity of drug-metabolizing enzymes (DME) shows high inter- and intra-individual variability. Genetic polymorphisms, exposure to drugs, and environmental toxins are known to significantly alter DME expression. In addition, the activity of these enzymes is highly age-dependent due to maturation processes that occur during development. Currently, there is a vast choice of phenotyping methods in adults using exogenous probes to characterize the activity of these enzymes. However, this can hardly be applied to children since it requires the intake of non-therapeutic xenobiotics. In addition, sampling may be challenging in the pediatric population for a variety of reasons: limited volume (e.g., blood), inappropriate sampling methods for age (e.g., urine), and metric requiring invasive or multiple blood samples. This review covers the main existing methods that can be used in the pediatric population to determine DME activity, with a particular focus on cytochrome P450 enzymes. Less invasive tools are described, including phenotyping using endogenous probes. Finally, the potential of pediatric model-informed precision dosing using physiologically based pharmacokinetic modeling is discussed.

Comparing Dried Blood Spots and Plasma Concentrations for Busulfan Therapeutic Drug Monitoring in Children.

BACKGROUND: Busulfan (Bu) is one of the conditioning regimen components for pediatric hematopoietic stem cell transplantation. Bu therapeutic drug monitoring (TDM) is essential for a successful treatment outcome and toxicity evasion. Dried blood spot (DBS) sampling is a rapid and simple method for Bu TDM, compared with conventional plasma sampling. This study evaluated the feasibility of using the DBS method for Bu TDM. The hematocrit (Hct) and conditioning day were also examined for their impact on the DBS method's performance. METHODS: Venous blood collected from 6 healthy volunteers was diluted, using their plasma into 4 samples of varying Hct values. Each sample was spiked with Bu calibrators (300, 600, and 1400 ng/mL), prepared using DBS and dried plasma spot (DPS) sampling and analyzed using a validated liquid-chromatography tandem-mass spectrometry method. Clinical blood samples (n = 153) from pediatric patients (n = 15) treated with Bu (mainly from doses 1, 2, 5, and 9) were used to prepare paired volumetric DBS and DPS samples. A Bland-Altman plot and Deming regression were used to define the agreement between the paired DBS and DPS measurements. Passing-Bablok regression analyses investigated the effects of Hct and conditioning day on the linearity between both methods. RESULTS: In vitro analyses showed good agreement between DBS and DPS measurements, with a mean difference of -5.4% and a 95% confidence interval on the limits of agreement of -15.3% to 4.6%. Clinical samples showed good correlation (Pearson correlation coefficient = 0.96; slope = 1.00) between the DBS and DPS methods. The DBS method met the clinical acceptance limits for clinical samples, with a bias <±20%. Bland-Altman plots showed good agreement, with only 5.8% of paired measurements exceeding the limits of agreement (±1.96 SD), although within its 95% confidence interval. Hct observations ranged from 21.7% to 34.7% and did not affect Bu concentrations measured from DBS in either the in vitro or in vivo studies. CONCLUSIONS: These results show that DBS is a useful method for Bu TDM, provided samples are analyzed on the collection day. DBS sampling offers advantages over traditional plasma sampling in infants and younger children because only small volumes of blood are required.

Variables of interest to predict glomerular filtration rate in preterm newborns in the first days of life.

BACKGROUND: Measurement of neonatal renal function is challenging, and accurate, easy-to-use markers to estimate glomerular filtration rate (eGFR) are lacking. This study aimed to evaluate principal determinants of GFR in neonates and develop a predictive equation. METHODS: GFR was measured, using single injection inulin clearance, at median day 3 of life in 48 newborns. Associations of clearance with height, gestational age, weight, creatinine, and cystatin C were explored and a multivariable model to estimate GFR developed. We also evaluated preexisting GFR equations (Schwartz, Zappitelli, combined Zappitelli). RESULTS: Forty-four clearances were measured, 36 very preterm neonates (28-32 weeks); 5 extremely preterm (< 28 weeks), and 3 term newborns. No patient presented acute renal insufficiency. Median inulin clearance in preterm infants was 18.83 ml/min/1.73 m2 (IQ 15.29; 24.99). Inulin clearance correlated with weight (ρ 0.74), gestational age (ρ 0.72), height (ρ 0.49), and creatinine (ρ - 0.42), but not cystatin C. In the multivariable model, predicted GFR equation was 2.32* (weight (g))0.64/(creatinine (mcmol/l))0.62. Mean error in predicting clearance was - 0.8 ml/min/1.73 m2 (- 3.0-1.4) ranging from - 14.9 to 13.3 ml/min/1.73 m2. Mean prediction error with Zappitelli and combined Zappitelli equations were 28.5 ml/min/1.73 m2 (95% CI 24.6-32.3) and 28.3 ml/min/1.73 m2 (95% CI 24.9-31.7), respectively, and 2 ml/min/1.73 m2 (95% CI - 0.6-4.6) for Schwartz equation. CONCLUSIONS: Weight and gestational age are crucial determinants of GFR in neonates. The Zappitelli models were not validated in our population. Our predictive model and Schwartz models performed better. Our model should be evaluated in another preterm population, particularly in those presenting renal insufficiency.

Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma.

Strategies that aim to limit the adaptive response to pathway inhibition in BRAF-mutated melanoma face the inherent limit of signaling redundancy and multiplicity of possible bypass mechanisms. Drug-induced expression of selected RNA-binding proteins, like the ubiquitously expressed HuR, has the potential to differentially stabilize the expression of many genes involved in the compensatory mechanisms of adaptive response. Here, we detect in BRAF-mutated melanoma cell lines having a higher propensity for adaptive response and in non-responding melanoma tumors, a larger proportion of HuRLow cells in the expression distribution of HuR. Using knockdown experiments, we demonstrate, through expression profiling and phenotypic assays, that increasing the proportion of HuRLow cells favors the adaptive response to BRAF inhibition, provided that the HuRLow state stays reversible. The MAPK dependency of melanoma cells appears to be diminished as the proportion of HuRLow cells increases. In single-cell assays, we demonstrate that the HuRLow cells display plasticity in their growth expression profile. Importantly, the adaptive over-proliferating cells emerge in the subpopulation containing the HuRLow cells. Therapeutic concentrations of lithium salts, although they moderately increase the global expression of HuR, are sufficient to suppress the HuRLow cells, induce an overall less resistant expression profile and attenuate in a HuR-dependent manner the adaptive response of melanoma cells in ex vivo assays. The therapeutic effectiveness of this approach is also demonstrated in vivo in mice xenografts. This study has immediate clinical relevance for melanoma therapy and opens a new avenue of strategies to prevent the adaptive response to targeted cancer therapy.

Therapeutic Drug Monitoring of Busulfan for the Management of Pediatric Patients: Cross-Validation of Methods and Long-Term Performance.

BACKGROUND: Busulfan (Bu) is an alkylating agent used as part of the conditioning regimen in pediatric patients before hematopoietic stem cell transplantation. Despite intravenous (IV) administration and dosing recommendations based on age and weight, reports have revealed interindividual variability in Bu pharmacokinetics and the outcomes of hematopoietic stem cell transplantation. In this context, adjusting doses to Bu's narrow therapeutic window is advised. We aimed to assess the utility of therapeutic drug monitoring (TDM) of Bu in children, the reliability of Bu quantification methods, and its stability in plasma when stored for up to 5 years. METHODS: Eighteen patients from our TDM center (252 samples) were included. All of them received a 2-hour Bu IV infusion 4 times daily for a total of 16 doses. The first dose of Bu was age/weight-based, and the subsequent doses were adjusted from third or fifth dose onward based on the estimated first dose pharmacokinetic parameters to target steady-state concentrations (Css) of 600-900 ng/mL. The performance of our unit's high-performance liquid chromatography with tandem mass spectrometry method was assessed using a quality control (QC, 35 series) chart. International, multicenter, cross-validation test (n = 21) was conducted to validate different analytical methods. To assess Bu stability, regression analyses and Bland-Altman plots were performed on measurements at repeated time points on samples stored at -80°C for up to 5 years. RESULTS: We observed a 4.2-fold interindividual variability in Bu Css after the first dose, with only 28% of children having a Css within the target range. During the 4 days of conditioning, 83% of children had their doses modified according to TDM recommendations. This achieved a Css within the target range in 75% of the children. Routine QC measurements were generally within the ±15% range around theoretical values, showing the optimal robustness of our center's analytical method. Two of the 21 Bu TDM centers returned inadequate results during cross-validation testing; both used a UV detection method. Storage at -80°C led to a fall in Bu content of 14.9% ± 13.4% at 2-4 years and of 20% ± 5% by 5 years (roverall = 0.92). CONCLUSIONS: We conclude that TDM is an effective method of achieving targeted Bu levels in children. QC programs are crucial to monitoring and maintaining the quality of an analytical method.

Prediction of drug-drug interactions using physiologically-based pharmacokinetic models of CYP450 modulators included in Simcyp software.

In recent years, physiologically based PharmacoKinetic (PBPK) modeling has received growing interest as a useful tool for the assessment of drug pharmacokinetics. It has been demonstrated to be informative and helpful to quantify the modification in drug exposure due to specific physio-pathological conditions, age, genetic polymorphisms, ethnicity and particularly drug-drug interactions (DDIs). In this paper, the prediction success of DDIs involving various cytochrome P450 isoenzyme (CYP) modulators namely ketoconazole (a competitive inhibitor of CYP3A), itraconazole (a competitive inhibitor of CYP3A), clarithromycin (a mechanism-based inhibitor of CYP3A), quinidine (a competitive inhibitor of CYP2D6), paroxetine (a mechanism-based inhibitor of CYP2D6), ciprofloxacin (a competitive inhibitor of CYP1A2), fluconazole (a competitive inhibitor of CYP2C9/2C19) and rifampicin (an inducer of CYP3A) were assessed using Simcyp® software. The aim of this report was to establish confidence in each CYP-specific modulator file so they can be used in the future for the prediction of DDIs involving new victim compounds. Our evaluation of these PBPK models suggested that they can be successfully used to evaluate DDIs in untested scenarios. The only noticeable exception concerned a quinidine inhibitor model that requires further improvement. Additionally, other important aspects such as model validation criteria were discussed.

Screening for genotypic and phenotypic variations in CYP450 activity in patients with therapeutic problems in a psychiatric setting, a retrospective study.

OBJECTIVES: This retrospective study aimed to assess to what extent an adverse drug reaction (ADR), an abnormal therapeutic drug monitoring (TDM) or a non-response, was attributable to an abnormal cytochrome P450 activity in a psychiatric setting. METHOD: We collected the results of investigations performed in these situations related to psychotropic drugs between January 2005 and November 2014. Activities of different cytochrome P450 were assessed by genotyping and/or phenotyping. Two experienced clinical pharmacologists assessed independently the possible association between the event and the results of the investigations. RESULTS: One hundred and thirty eight clinical or biological situations had a complete assessment of all major metabolic pathways of the target drug. A majority of clinical or biological situations were observed with antidepressants (n=93, 67.4%), followed by antipsychotics (n=28, 20.3%), benzodiazepines and hypnotics (n=13, 9.4%), and psychostimulants (n=4, 2.9%). Genotype and/or phenotype determination was mainly performed because of ADRs (n=68, 49.3%) or non-response (n=46, 33.3%). Inter-rate reliability of the scoring system between the pharmacologists was excellent (kappa=0.94). The probability of an association between ADR, TDM or non-response and metabolic status was rated as intermediate to high in 34.7% of all cases, with proportions of 30.4% and 36.7%, for non-response and ADR respectively. CONCLUSION: When indicated by clinical pharmacologists, ADR, TDM or non-response may be attributable to a variation of the metabolic status with an intermediate to high probability in 34.7% of patients, based on the congruent assessment made by two clinical pharmacologists. Further studies assessing the clinical relevance of prospective explorations and clarifying the appropriate method according to the clinical context are needed.

Plasma concentrations of transdermal fentanyl and buprenorphine in pigs (Sus scrofa domesticus).

OBJECTIVE: To determine the absorption characteristics of fentanyl and buprenorphine administered transdermally in swine. STUDY DESIGN: A randomized comparative experimental trial. ANIMALS: Twenty-four Yorkshire gilts weighing 27.8±2.2 kg (mean±standard deviation). METHODS: Animals were randomly assigned to different doses of transdermal patches (TPs) of fentanyl (50 µg hour-1, 75 µg hour-1 and 100 µg hour-1) or buprenorphine (35 µg hour-1 and 70 µg hour-1), once or twice. Thirteen blood samples were obtained for each TP applied. Plasma concentrations were determined, and the area under the curve, peak serum concentration (Cmax) and time to Cmax were calculated. RESULTS: Fentanyl: Cmax was observed at different time points: for the first TP application: 30 hours for 50 µg hour-1, 6 hours for 75 µg hour-1 and 100 µg hour-1 patches; and for the second TP application: 30 hours for 50 µg hour-1 and 36 hours for 75 µg hour-1 patches. Buprenorphine: serum concentrations were not detected for the 35 µg hour-1 patch; Cmax was observed at different times for the 70 µg hour-1 patch: 18 hours (n = 1), 24 hours (n = 3), 30 hours (n = 1) and 42 hours (n = 1) after application of the first patch and 12 hours after the second patch. CONCLUSIONS AND CLINICAL RELEVANCE: A relevant serum concentration obtained with fentanyl TP dosed at 75 µg hour-1 or 100 µg hour-1suggests that TPs could represent an analgesia option for laboratory pigs weighing 25-30 kg. As concentrations of buprenorphine were variable, this study does not support the use of buprenorphine TPs in pigs. Consecutive fentanyl or buprenorphine TPs did not provide reliable serum concentrations. Further pharmacokinetic studies and analgesiometric tests in swine are needed to confirm the clinical adequacy of TPs.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure.

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6.

Detection of busulfan adducts on proteins.

RATIONALE: Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. METHODS: Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides. RESULTS: Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides. CONCLUSIONS: The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.

Aspirin response: Differences in serum thromboxane B2 levels between clinical studies.

Serum thromboxane B2 (TxB2) is a specific marker of platelet inhibition by aspirin. Yet, TxB2 levels differ by up to 10-fold between some aspirin-treated patient cohorts. This study aimed to identify factors responsible for differences in serum TxB2 between cohorts in the ADRIE study (n = 657) and the BOSTON study (n = 678) of aspirin-treated cardiovascular patients originally tested with different ELISA assays. TxB2 levels were assessed in representative subgroups of the two cohorts (34 samples in BOSTON and 39 in ADRIE) by both ELISAs, as well as liquid chromatography and tandem mass spectroscopy (MS). A multivariate analysis was performed on the whole cohort database to identify determinants of the difference of TxB2 levels between cohorts. There was no systematic bias between the original ELISA TxB2 values and the MS values and the median difference was small, 0.12 ng/ml, thus not explaining the difference between median TxB2 levels in the two study populations (7 and 0.6 ng/ml in the ADRIE and BOSTON studies, respectively). In the combined dataset of the ADRIE and BOSTON cohorts (n = 1342), body mass index, age, gender, aspirin dose, time from aspirin intake to blood draw, NSAID intake, platelet count and C-reactive protein were significantly associated with TxB2 levels. After adjustment for patient characteristics, the difference between cohorts did not decrease. Unexplained differences in serum TxB2 levels in different populations of aspirin-treated cardiovascular patients suggest that further studies are needed to confirm the role of serum TxB2 level as a prognostic factor or rather as a marker of therapeutic observance.

Real-time monitoring of exhaled drugs by mass spectrometry.

Future individualized patient treatment will need tools to monitor the dose and effects of administrated drugs. Mass spectrometry may become the method of choice to monitor drugs in real time by analyzing exhaled breath. This review describes the monitoring of exhaled drugs in real time by mass spectrometry. The biological background as well as the relevant physical properties of exhaled drugs are delineated. The feasibility of detecting and monitoring exhaled drugs is discussed in several examples. The mass spectrometric tools that are currently available to analyze breath in real time are reviewed. The technical needs and state of the art for on-site measurements by mass spectrometry are also discussed in detail. Off-line methods, which give support and are an important source of information for real-time measurements, are also discussed. Finally, some examples of drugs that have already been successfully detected in exhaled breath, including propofol, fentanyl, methadone, nicotine, and valproic acid are presented. Real-time monitoring of exhaled drugs by mass spectrometry is a relatively new field, which is still in the early stages of development. New technologies promise substantial benefit for future patient monitoring and treatment.

Prioritization methodology for the monitoring of active pharmaceutical ingredients in hospital effluents.

The important number of active pharmaceutical ingredients (API) available on the market along with their potential adverse effects in the aquatic ecosystems, lead to the development of prioritization methods, which allow choosing priority molecules to monitor based on a set of selected criteria. Due to the large volumes of API used in hospitals, an increasing attention has been recently paid to their effluents as a source of environmental pollution. Based on the consumption data of a Swiss university hospital, about hundred of API has been prioritized following an OPBT approach (Occurrence, Persistence, Bioaccumulation and Toxicity). In addition, an Environmental Risk Assessment (ERA) allowed prioritizing API based on predicted concentrations and environmental toxicity data found in the literature for 71 compounds. Both prioritization approaches were compared. OPBT prioritization results highlight the high concern of some non steroidal anti-inflammatory drugs and antiviral drugs, whereas antibiotics are revealed by ERA as potentially problematic to the aquatic ecosystems. Nevertheless, according to the predicted risk quotient, only the hospital fraction of ciprofloxacin represents a risk to the aquatic organisms. Some compounds were highlighted as high-priority with both methods: ibuprofen, trimethoprim, sulfamethoxazole, ritonavir, gabapentin, amoxicillin, ciprofloxacin, raltegravir, propofol, etc. Analyzing consumption data and building prioritization lists helped choosing about 15 API to be monitored in hospital wastewaters. The API ranking approach adopted in this study can be easily transposed to any other hospitals, which have the will to look at the contamination of their effluents.

Development of an LC-MS/MS Method for the Assessment of Selected Active Pharmaceuticals and Metabolites in Wastewaters of a Swiss University Hospital.

A multi-residue analytical method was developed and validated for the quantification of 11 selected active pharmaceutical ingredients (API) and 2 human metabolites in hospital effluents using solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Targeted analytes belong to different therapeutic classes: non steroidal anti-inflammatory drugs (NSAID), analgesics, antibiotics and psychiatric drugs. Solid-phase extraction recoveries ranged between 21 and 101% for the selected API. Calibration curves were built with 6 standard samples prepared in ultrapure water ranging from 0.05 to 10 µg/L and showed regression coefficients above 0.994. The instrumental detection limits (IDL) varied between 0.05 and 5 µg/L, and the method detection limits (MDL) between 0.1 and 100 ng/L. Precision of the method, evaluated with spiked water samples at four different concentrations, varied between 84 and 117% for all compounds and an overall variability below 20%, with the exception of carbamazepine (71-123%). Except for two compounds, recoveries of spiked hospital wastewaters at four different concentrations (0.1, 1, 10 and 100 µg/L) varied between 44 and 133%, with relative standard deviation (RSD) between 0.6 and 28.5%. The evaluation of the matrix effects showed that diluted samples exhibit lower signal suppression. Analysis of effluent samples from a Swiss university hospital resulted in a mean detection frequency of 92% for the selected compounds, with concentrations up to 1535 µg/L for the analgesic paracetamol.

Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties.

OBJECTIVE: Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation APPROACH AND RESULTS: Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content. CONCLUSIONS: Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Phenotyping of CYP450 in human liver microsomes using the cocktail approach.

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.