Preanalytical Stability of Flucloxacillin, Piperacillin, Tazobactam, Meropenem, Cefalexin, Cefazolin, and Ceftazidime in Therapeutic Drug Monitoring: A Structured Review.

BACKGROUND: Therapeutic drug monitoring is increasingly being used to optimize beta-lactam antibiotic dosing. Because beta-lactams are inherently unstable, confirming preanalytical sample stability is critical for reporting reliable results. This review aimed to summarize the published literature on the preanalytical stability of selected widely prescribed beta-lactams used in therapeutic drug monitoring. METHODS: The published literature (2010-2020) on the preanalytical stability of flucloxacillin, piperacillin, tazobactam, meropenem, cefalexin, cefazolin, and ceftazidime in human plasma, serum, and whole blood was reviewed. Articles examining preanalytical stability at room temperature, refrigerated, or frozen (-20°C) using liquid chromatography with mass spectrometry or ultraviolet detection were included. RESULTS: Summarizing the available data allowed for general observations to be made, although data were conflicting in some cases (piperacillin, tazobactam, ceftazidime, and meropenem at room temperature, refrigerated, or -20°C) or limited (cefalexin, cefazolin, and flucloxacillin at -20°C). Overall, with the exception of the more stable cefazolin, preanalytical instability was observed after 6-12 hours at room temperature, 2-3 days when refrigerated, and 1-3 weeks when frozen at -20°C. In all cases, excellent stability was detected at -70°C. Studies focusing on preanalytical stability reported poorer stability than studies investigating stability as part of method validation. CONCLUSIONS: Based on this review, as general guidance, clinical samples for beta-lactam analysis should be refrigerated and analyzed within 2 days or frozen at -20°C and analyzed within 1 week. For longer storage times, freezing at -70°C was required to ensure sample stability. This review highlights the importance of conducting well-designed preanalytical stability studies on beta-lactams and other potentially unstable drugs under clinically relevant conditions.

Vitamin D status at birth and acute respiratory infection hospitalisation during infancy.

BACKGROUND: Hospital admission for acute respiratory infections (ARIs) during early childhood is a global public health concern. Vitamin D deficiency is prevalent during pregnancy and infancy. Evidence indicates that vitamin D supplementation prevents ARIs. OBJECTIVES: To determine whether vitamin D deficiency at birth is associated with ARI hospitalisations during infancy. METHODS: We performed a nested case-control study in children aged 0-12 months. Cases had ≥1 ARI hospitalisation and 4 controls were individually matched to each case. Newborn 25(OH)D concentration was measured on dried blood spots using two-dimensional liquid chromatography-tandem mass spectrometry. Hospital admissions were measured using health care records. Median serum 25(OH)D concentration in cases and controls was compared, and covariates of ARI hospitalisation during infancy were assessed using conditional logistic regression analysis. RESULTS: Six per cent of the cohort (n = 384) had an ARI hospitalisation during infancy, and 1536 controls were matched to cases. Median DBS [25(OH)D] was lower among ARI cases than controls (46 nmol/l vs. 61 nmol/L). Median 25(OH)D levels were lower for those hospitalised ≥2 times (47, IQR 36, 58) vs. those hospitalised once (52, IQR 42, 62) vs. the controls and also lower for those who stayed in the hospital for ≥3 days (45, IQR 36, 54) vs 1-2 days (48, IQR 38, 59) compared to the controls. After adjustment for season of birth and covariates describing demographic, antenatal, perinatal, and infant characteristics, DBS 25(OH)D concentration (<50 nmol/L) at birth was associated with increased odds of ARI hospitalisation during infancy (odds ratio 2.20, 95% confidence interval 1.48, 2.91). CONCLUSIONS: Vitamin D deficiency at birth is associated with increased odds of ARI hospitalisations in infants. The findings have implications for a developed country like New Zealand where vitamin D supplementation is not routinely recommended and the burden of ARI hospitalisation in young children is high.

New technique to reduce systemic side effects of timolol eye drops: The tissue press method-Cross-over clinical trial.

IMPORTANCE: Nasolacrimal occlusion (NLO) is effective in reducing systemic absorption of eye drop medication but it is difficult and often performed poorly. We propose an alternative easier and equally effective technique. BACKGROUND: To test the effectiveness of systemic absorption, we evaluated plasma concentration and ocular effects after topically administered timolol and compared to NLO. DESIGN: Cross-over trial carried out in Capital Eye Specialist, Wellington. PARTICIPANTS: A total of 21 subjects over 18 years without contraindications for topical beta-blocker medication and not using systemic beta-blockers. METHODS: During three clinic visits separated by at least one week, alternative approaches to reduce systemic eye drop absorption were tested. These were: (a) nasolacrimal (punctal) occlusion for 5 min, (b) tissue press method or (c) no intervention. Timolol plasma levels were measured 1 h after drop application. At each visit, baseline measurement of blood pressure, heart rate and intraocular pressure (IOP) were performed, and repeated 1 h after timolol 0.5% eye drop application. MAIN OUTCOME MEASURES: Comparison of timolol plasma concentration after each intervention. Secondary outcome measurements included effects on blood pressure, heart rate and IOP. RESULTS: Plasma timolol concentrations after tissue press method and NLO were significantly lower than those without intervention. Comparing tissue press method to NLO, there were no significant differences in plasma levels of timolol, blood pressure, heart rate or IOP. CONCLUSION AND RELEVANCE: The tissue press method is equally effective as NLO in reducing systemic absorption of timolol. It is also easier and faster to administer.

Routine Therapeutic Drug Monitoring of Dabigatran: Experience at a Tertiary Center.

BACKGROUND: A liquid chromatography-mass spectrometry assay to determine plasma dabigatran concentrations has been available for routine clinical use at our tertiary institutions since 2017. The aim of the study was to describe (1) the use of the assay over time; (2) the indications for testing; and (3) subsequent dabigatran prescribing decisions. METHODS: Patients for whom dabigatran concentrations were measured were identified using the laboratory database, and clinical data were extracted from the associated electronic health records. RESULTS: There were 233 samples in 24 months. The use of dabigatran increased over time, with a mean (95% confidence interval) increase of +0.5 (0.3-0.7) samples per month. Dabigatran concentrations ranged from <1 to 1060 mcg/L. The main reasons for testing were uncertainty about impact on renal function and drug interactions (39%), to inform prescribing decisions after thromboembolic or bleeding events (21%), and for investigation following dose-adjustment (16%). Dabigatran dose was changed after 30% (68/233) of assay results. CONCLUSIONS: The clinical use of the dabigatran assay has increased, with almost one-third of results associated with a subsequent change in dabigatran prescribing.

Wintertime Vitamin D status and its related risk factors among children living in Auckland, New Zealand.

AIM: To investigate vitamin D status and its determinants in school-aged children living in Auckland, New Zealand. METHODS: Healthy children (n=507) aged 8-11 years were recruited from six primary schools to include a range of ethnicities and sociodemographic characteristics. Finger-prick blood spots were collected and analysed for capillary 25-hydroxyvitamin D (25(OH)D). Weight and percentage of body fat (%BF) were measured using the InBody 230 (Biospace Co. Ltd., Seoul, Korea). Information related to ethnicity, skin colour, physical activity and sun exposure were sought from parents through a questionnaire. RESULTS: Mean±standard deviation (SD) 25(OH)D concentration were 64±21 nmol/L, with 31% of the population presenting with 25(OH)D≥75nmol/L, 41% 50-75nmol/L and 28%<50nmol/L. Capillary 25(OH)D was significantly higher in New Zealand European compared to all other ethnic groups (75±20nmol/L, P<0.001). As expected, children with dark/brown skin colour had lower 25(OH)D levels compared to other skin colour categories (51±18nmol/L, P<0.001). Using multiple logistic regression analysis, determinants of 25(OH)D were %BF and ethnicity. CONCLUSION: Approximately one-third of this population had 25(OH)D<50nmol/L. Determinants of a 25(OH)D<50nmol/L included %BF and ethnicity. Wintertime serum 25(OH)D was highly variable. There are some children at high risk of 25(OH)D<50nmol/L for whom supplementation may be considered.

Transfer of Candesartan Into Human Breast Milk.

BACKGROUND: There are currently no data regarding the transfer of candesartan into human milk. This report provides data on this transfer, an estimation of the amount breastfed infants receive, and plasma concentrations from two breastfed infants. CASES: Three breastfeeding mothers, all stabilized on candesartan (8-32 mg/d), provided milk and plasma samples over one dosing interval (24 hours). Two infant plasma samples were obtained. The amount the infants ingested was estimated to be 0.09% (95% CI 0.07-0.11) of the maternal dose (weight-adjusted). Candesartan was undetectable (less than 0.2 micrograms/L) in infant plasma samples. CONCLUSION: A relative infant dose of 0.09% suggests that maternal benefit from candesartan at standard therapeutic doses may outweigh risk in breastfeeding healthy, term infants.

Preanalytical Stability of Piperacillin, Tazobactam, Meropenem, and Ceftazidime in Plasma and Whole Blood Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring (TDM) is increasingly used to optimize the dosing of beta-lactam antibiotics in critically ill patients. However, beta-lactams are inherently unstable and degrade over time. Hence, patient samples need to be appropriately handled and stored before analysis to generate valid results for TDM. The appropriate handling and storage conditions are not established, with few and conflicting studies on the stability of beta-lactam antibiotics in clinical samples. The aim of this study was to assess the preanalytical stability of piperacillin, tazobactam, meropenem, and ceftazidime in human plasma and whole blood using a liquid chromatography-tandem mass spectrometry method for simultaneous quantification. METHODS: A reverse phase liquid chromatography-tandem mass spectrometry method for the quantification of piperacillin, tazobactam, meropenem, and ceftazidime in plasma after protein precipitation was developed and validated. The preanalytical stability of these beta-lactams was assessed in EDTA- and citrate-anticoagulated plasma at 24, 4, and -20°C. The whole blood stability of the analytes in EDTA-anticoagulated tubes was assessed at 24°C. Stability was determined by nonlinear regression analysis defined by the lower limit of the 95th confidence interval of the time to 15% of degradation. RESULTS: Based on the lower limit of the 95th confidence interval of the time to 15% of degradation, piperacillin, tazobactam, meropenem, and ceftazidime were stable in EDTA-anticoagulated plasma for at least 6 hours at 24°C, 3 days at 4°C, and 4 days at -20°C. Stability in EDTA- and citrate-anticoagulated plasma was similar. Stability in whole blood was similar to plasma at 24°C. CONCLUSIONS: Plasma samples for the TDM of piperacillin, tazobactam, meropenem, and ceftazidime should be processed within 6 hours if kept at room temperature and within 3 days if kept at 4°C. All long-term storage of samples should be at -80°C.

Quantitation of 25-hydroxyvitamin D in dried blood spots by 2D LC-MS/MS without derivatization and correlation with serum in adult and pediatric studies.

BACKGROUND: Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. METHODS: A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d6-25OHD3 in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). RESULTS: The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD3 and 11-91 nmol/L 25OHD2 with a limit of detection of 3 nmol/L. Intra- and inter-day imprecision was <13% CV and bias <12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of ≤13% for 25OHD3. No 25OHD2 was detected. DBS calculated serum 25OHD3 concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). CONCLUSIONS: Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples.

Randomized clinical trial: the impact of gastrointestinal risk factor screening and prophylactic proton pump inhibitor therapy in patients receiving dual antiplatelet therapy.

OBJECTIVE: Dual antiplatelet therapy reduces the risk of ischemic complications after acute coronary syndrome, but increases the risk of bleeding including upper gastrointestinal bleeding (UGIB).The aim of this study was to examine the effect of screening for risk of UGIB and prophylactic proton pump inhibitor (PPI) treatment in dual-antiplatelet-treated patients at risk of UGIB and to assess the significance of dual antiplatelet therapy compliance for cardiovascular events. PATIENTS AND METHODS: In a register-based randomized-controlled trial, 2009 patients were included at the time of first percutaneous coronary intervention and randomized to either screening or control. Screened high-risk patients were prescribed pantoprazole 40 mg during the 1-year after percutaneous coronary intervention. RESULTS: The incidence of UGIB was 0.8 versus 1.3% in screened patients and controls, respectively (P=0.381).Significantly fewer screened patients (5.4%) than controls (8.0%) underwent upper gastrointestinal endoscopy (P=0.026). Screened patients (2.9%) had significantly fewer events of unstable angina pectoris than controls (4.7%) (P=0.036) and a higher compliance to dual antiplatelet therapy (88.3 vs. 85.0%) (P=0.035), but no statistically difference was observed in the incidences of myocardial infarction and all-cause mortality (1.0 vs. 1.5%) (P=0.422). CONCLUSION: Screening for risk factors for UGIB and subsequent prophylactic PPI treatment did not significantly reduce the incidence of UGIB. Prescription of PPI was associated with a higher compliance with dual antiplatelet therapy and decreases the risk of recurrent cardiovascular events.

Development, validation and application of a novel liquid chromatography tandem mass spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, α-fluoro-ß-ureidopropionic acid and α-fluoro-ß-alanine in human plasma.

The plasma 5,6-dihydrouracil/uracil (UH2/U) ratio is a possible phenotypic marker of dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response and toxicity. Studies have re-affirmed the value of 5-FU and 5,6-dihydro-5-fluorouracil (FUH2) for therapeutic drug monitoring (TDM). However, FUH2 has limited stability in plasma, necessitating expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites α-fluoro-ß-ureidopropionic acid (FUPA) and α-fluoro-ß-alanine (FßAL) are stable in plasma and are probable candidates for TDM. This paper describes development, validation and application of an LC-MS/MS assay quantifying U, UH2, 5-FU, FUH2, FUPA and FßAL in human plasma. Extraction was by salt-assisted liquid-liquid extraction (LLE) in two-stages with pH adjustment. The supernatants were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150×2.00mm, 3µ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation in positive polarity. The analytical response was linear (r2≥0.99) in the concentration (ng/mL) ranges: 50-10 000 for FßAL and FUH2, 50-5 000 for FUPA, 50-100 000 for 5-FU, 5-200 for U and 10-400 for UH2. Within- and between-run accuracy and precision were ≤ 10.2% and ≤ 9.8% respectively across the QC range and inclusive of LLOQ. The internal standard (IS) normalised matrix effects were within 93-112% with CV of ≤ 9.7% and normalised recoveries were within 91-107% with CV of ≤ 9.8%. This robust assay was successfully applied to samples from rectal and colorectal cancer patients (n=10) on 5-FU. Deviations ≤ 2.0% from the mean values were observed when incurred samples were reanalysed.

Quantification and clinical application of carboplatin in plasma ultrafiltrate.

Carboplatin is a chemotherapy drug used in a variety of cancers with the primary toxicity being exposure-dependant myelosuppression. We present the development and validation of a simple, robust inductively coupled plasma mass spectrometry (ICP-MS) method to measure carboplatin in plasma ultrafiltrate. Plasma ultrafiltrates samples were prepared using Amicon Ultra 30,000da cut-off filters and then diluted with ammonia EDTA before ICP-MS analysis. The assay was validated in the range 0.19-47.5mg/L carboplatin in ultrafiltrate. The assay was linear (r2>0.9999), accurate (<6% bias, 12% bias at LLOQ) and precise (intra- and inter-day precision of <3% coefficient of variation). No matrix effects were observed between plasma ultrafiltrate and aqueous platinum calibrators and recovery was complete. The assay was applied to 10 clinical samples from patients receiving carboplatin. Incurred sample reanalysis showed reproducible values over 3 analysis days (<6% CV). As plasma stability prior to ultrafiltration has been a major concern in previous clinical studies this was studied extensively at room temperature (22°C) over 24h. Carboplatin was found to be stable in both spiked plasma (n=3) and real patient samples (n=10) at room temperature for up to 8h before ultrafiltration. This makes routine measurement of carboplatin concentrations in clinical settings feasible.

Case series: toxicity from 25B-NBOMe--a cluster of N-bomb cases.

Background A new class of hallucinogens called NBOMes has emerged. This class includes analogues 25I-NBOMe, 25C-NBOMe and 25B-NBOMe. Case reports and judicial seizures indicate that 25I-NBOMe and 25C-NBOMe are more prevalently abused. There have been a few confirmed reports of 25B-NBOMe use or toxicity. Report Observational case series. This report describes a series of 10 patients who suffered adverse effects from 25B-NBOMe. Hallucinations and violent agitation predominate along with serotonergic/stimulant signs such as mydriasis, tachycardia, hypertension and hyperthermia. The majority (7/10) required sedation with benzodiazepines. Analytical method 25B-NBOMe concentrations in plasma and urine were quantified in all patients using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Peak plasma levels were measured between 0.7-10.1 ng/ml. Discussion The NBOMes are desired by users because of their hallucinogenic and stimulant effects. They are often sold as LSD or synthetic LSD. Reported cases of 25B- NBOMe toxicity are reviewed and compared to our series. Seizures and one pharmacological death have been described but neither were observed in our series. Based on our experience with cases of mild to moderate toxicity, we suggest that management should be supportive and focused on preventing further (self) harm. High doses of benzodiazepines may be required to control agitation. Patients who develop significant hyperthermia need to be actively managed. Conclusions Effects from 25B-NBOMe in our series were similar to previous individual case reports. The clinical features were also similar to effects from other analogues in the class (25I-NBOMe, 25C-NBOMe). Violent agitation frequently present along with signs of serotonergic stimulation. Hyperthermia, rhabdomyolysis and kidney injury were also observed.

High prevalence of ulcer bleeding risk factors in dual antiplatelet-treated patients after percutaneous coronary intervention.

INTRODUCTION: Dual antiplatelet therapy is standard treatment following percutaneous coronary intervention (PCI) and stenting. However, such therapy increases the risk of upper gastrointestinal bleeding (UGIB). The risk factors of UGIB are well-documented and proton pump inhibitor (PPI) treatment reduces the risk. The aim was to describe the prevalence of risk factors of UGIB in dual antiplatelet-treated patients. METHODS: A questionnaire was used to assess the prevalence of risk factors of upper gastrointestinal bleeding among dual antiplatelet-treated first-time PCI patients in Western Denmark. The following characteristics were considered risk factors: increasing age (age 60-69 years and ≥ 70 years); dyspepsia; previous peptic ulcer; use of non-steroidal anti-inflammatory drugs (NSAIDs) (weekly or daily), corticosteroids, selective serotonin reuptake inhibitors (SSRIs) and anticoagulants. RESULTS: A total of 1,358 patients with a mean age of 64.1 years (range: 33-92 years) were included. The distribution of risk factors was as follows: dyspepsia: 681 patients (50.1%); previous ulcer: 110 (8.1%; 2.3% with bleeding); use of NSAIDs: 214 (15.8%); corticosteroids (2.9%), SSRIs (5.8%) and anticoagulants (6.3%). Defined high-risk patients: 886 (65.2%). PPI treatment prior to PCI was found in 248 (18.3%), of whom 86% were at high risk of UGIB. CONCLUSION: This study demonstrates a high prevalence of risk factors among PCI patients treated with dual antiplatelet therapy, many of whom were not in PPI treatment.

Quantification of dabigatran and indirect quantification of dabigatran acylglucuronides in human plasma by LC-MS/MS.

BACKGROUND: An assay for the quantification of dabigatran and its active metabolites, dabigatran acylglucuronides, has not previously been described in detail. RESULTS: For the quantification of total dabigatran concentration (free dabigatran and acylglucuronides), samples were subjected to alkaline hydrolysis. For the quantification of free dabigatran, samples were acidified with ammonium formate. Following acetonitrile protein precipitation, the samples were analyzed by LC-MS/MS using gradient elution to ensure separation of dabigatran from dabigatran acylglucuronides. Mean recoveries ≥98% were achieved. The assay was validated over the range 2.5-1000 ng/ml dabigatran, imprecision was <9% CV (<15% at LLOQ) and accuracy was 101-114%. CONCLUSION: An assay for dabigatran with indirect quantification of dabigatran acylglucuronides in plasma was developed, validated and applied.

Correlation between trough plasma dabigatran concentrations and estimates of glomerular filtration rate based on creatinine and cystatin C.

AIMS: Dabigatran is largely cleared by renal excretion. Renal function is thus a major determinant of trough dabigatran concentrations, which correlate with the risk of thromboembolic and haemorrhagic outcomes. Current dabigatran dosing guidelines use the Cockcroft-Gault (CG) equation to gauge renal function, instead of contemporary equations including the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations employing creatinine (CKD-EPI_Cr), cystatin C (CKD-EPI_Cys) and both renal biomarkers (CKD-EPI_CrCys). METHODS: A linear regression model including the dabigatran etexilate maintenance dose rate, relevant interacting drugs and genetic polymorphisms (including CES1), was used to analyse the relationship between the values from each renal function equation and trough steady-state plasma dabigatran concentrations. RESULTS: The median dose-corrected trough steady-state plasma dabigatran concentration in 52 patients (38-94 years) taking dabigatran etexilate was 60 µg/L (range 9-279). The dose-corrected trough concentration in a patient on phenytoin and phenobarbitone was >3 standard deviations below the cohort mean. The CG, CKD-EPI_Cr, CKD-EPI_Cys and CKD-EPI_CrCys equations explained (R (2), 95 % CI) 32 % (9-55), 37 % (12-60), 41 % (16-64) and 47 % (20-69) of the variability in dabigatran concentrations between patients, respectively. One-way analysis of variance (ANOVA) comparing the R (2) values for each equation was not statistically significant (p = 0.74). DISCUSSION: Estimates of renal function using the four equations accounted for 32-47 % of the variability in dabigatran concentrations between patients. We are the first to provide evidence that co-administration of phenytoin/phenobarbitone with dabigatran etexilate is associated with significantly reduced dabigatran exposure.

Coagulation assays and plasma fibrinogen concentrations in real-world patients with atrial fibrillation treated with dabigatran.

AIMS: In patients with atrial fibrillation prescribed dabigatran, the aim was to examine the correlation between plasma dabigatran concentrations and the three screening coagulation assays [international normalized ratio (INR), activated partial thromboplastin time (aPTT) and thrombin time (TT)] as well as the dilute thrombin time (dTT) and to examine the contribution of plasma fibrinogen concentrations to the variability in TT results. METHODS: Plasma from patients with atrial fibrillation on dabigatran were analysed for clotting times and concentrations of fibrinogen and dabigatran. Correlation plots (and associated r(2) values) were generated using these data. The variability in TT results explained by fibrinogen concentrations was quantified using linear regression. RESULTS: Fifty-two patients (38-94 years old) contributed 120 samples, with plasma dabigatran concentrations ranging from 9 to 408 µg l(-1) . The r(2) values of INR, aPTT, TT and dTT against plasma dabigatran concentrations were 0.49, 0.54, 0.70 and 0.95, respectively. Plasma fibrinogen concentrations explained some of the residual variability in TT values after taking plasma dabigatran concentrations into account (r(2) = 0.12, P = 0.02). CONCLUSIONS: Of the screening coagulation assays, the TT correlated best with plasma dabigatran concentrations. Variability in fibrinogen concentrations accounts for some of the variability in the TT.

Transfer of doxazosin into breast milk.

To the best of our knowledge, there have been no published studies of doxazosin transfer into human milk. In rats, milk concentrations twentyfold higher than in plasma have been reported. Based on these animal data, some references advise to avoid breastfeeding during doxazosin therapy. However, the physicochemical properties of doxazosin suggest low transfer into human milk. A 37-year-old breastfeeding woman who was administered doxazosin 4 mg daily for 2 doses was studied. Doxazosin concentrations in milk and plasma were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk/plasma area under the concentration-time curve (AUC0-18 hours) ratio was 0.1. This finding is consistent with what could be predicted based on the physicochemical properties of doxazosin. The average and maximum milk concentrations were 2.9 and 4.2 µg/L. These values correspond to estimated relative infant doses of 0.06% and 0.09%, respectively, assuming standard infant milk intake. These values are well below the generally accepted cutoff of 10% for predicting safety during breastfeeding. A low relative infant dose of < 0.1% suggests that maternal doxazosin therapy may be compatible with breastfeeding after careful individual risk-benefit analysis.

The pharmacokinetics and pharmacodynamics of single dose (R)- and (S)-warfarin administered separately and together: relationship to VKORC1 genotype.

AIMS: 1) To determine the pharmacokinetics and pharmacodynamics of (R)- and (S)-warfarin given alone and in combination and 2) to determine whether the relative potency of (R)- and (S)-warfarin is dependent on VKORC1 genotype. METHODS: A three way crossover study was conducted in which 17 healthy male subjects stratified by VKORC1 1173 C>T genotype and all CYP2C9 1*/1* received (R)-warfarin 80 mg, (S)-warfarin 12.5 mg and rac-warfarin sodium 25 mg. Plasma (R)- and (S)-warfarin unbound and total concentrations and prothrombin time were determined at multiple time points to 168 h. RESULTS: Pharmacokinetic parameters for (R)- and (S)-warfarin were similar to the literature. (R)-warfarin 80 mg alone resulted in a mean AUC(PT) (0,168 h) of 3550 s h (95% CI 3220, 3880). Rac-warfarin sodium 25 mg containing (S)-warfarin 11.7 mg produced a greater effect on AUC(PT) (0,168 h) than (S)-warfarin 12.5 mg (mean difference 250 s.h, 95% CI 110, 380, P < 0.002) given alone. In a mixed effects model the ratio of response between (R)- and (S)-warfarin (AUC(PT((R)-warfarin)) : AUC(PT((S)-warfarin))) was 1.21 fold higher (95% CI 1.05, 1.41, P < 0.02) in subjects of VKORC1 TT genotype compared with the CC genotype. CONCLUSIONS: (R)-warfarin has a clear PD effect and contributes to the hypoprothrombinaemic effect of rac-warfarin. VKORC1 genotype is a covariate of the relative R/S potency relationship. Prediction of drug interactions with warfarin needs to consider effects on (R)-warfarin PK and VKORC1 genotype.

Predicted metabolic drug clearance with increasing adult age.

AIM: To determine the effect of increasing adult age on predicted metabolic drug clearance. METHOD: Predicted metabolic drug clearances (CLPT ) were determined using in vitro-in vivo extrapolation coupled with physiological-based pharmacokinetic modelling and simulation (IVIVE-PBPK) in Simcyp®. Simulations were conducted using CYP-selective 'probe' drugs with subjects in 5 year age groups (20-25 to 90-95 years). CLPT values were compared with human pharmacokinetic data stratified according to age (young = 20-40 years and elderly = 65-85 years) and gender. Age-related changes in the physiological parameters used for IVIVE of CLPT were described. RESULTS: Predicted metabolic drug clearances decreased with increasing adult age to approximately 65-70 years: caffeine from 1.5 to 1.0 ml min(-1) kg(-1) (a 33% decrease), S-warfarin from 0.100 to 0.064 ml min(-1) kg(-1) (36%), S-mephenytoin from 4.1 to 2.5 ml min(-1) kg(-1) (39%), desipramine from 10.6 to 7.3 ml min(-1) kg(-1) (31%) and midazolam from 5.4 to 3.9 ml min(-1) kg(-1) (27%). Except for S-mephenytoin, predictions were within 3.5-fold of clearances from clinical studies when stratified by age and gender. A trend towards higher CLPT was observed in females, but this was only statistically significant in larger virtual trials. Physiological parameters that determine CLPT decreased with increasing adult age: mean microsomal protein g(-1) of liver, liver weight, hepatic blood flow and human serum albumin concentration. CONCLUSION: Decreased metabolic clearance in the elderly was predicted by Simcyp® and was generally consistent with limited clinical data for four out of five drugs studied and the broader literature for drugs metabolized by CYP enzymes. IVIVE-PBPK may be increasingly useful in predicting metabolic drug clearance in the elderly.